Maximising the Value of Marine By-products

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Maximising the value ofmarine by-products

Edited byFereidoon Shahidi

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Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Maximizing the value of marine by-products: an overview . . . . . . . . . . . . . xxi

Part I Marine by-products characterisation, recovery and

processing

1 Physical and chemical properties of protein seafood

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

T. Rustad, Norwegian University of Science and Technology, Norway

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Physical and chemical properties of protein-rich by-products

± seasonal, habitat, species and individual variations . . . . . . . . 11

1.4 Implication for by-products valorisation . . . . . . . . . . . . . . . . . . . . . 16

1.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 17

1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2 Physical and chemical properties of lipid by-products from

seafood waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

J. P. Kerry and S. C. Murphy, National University of Ireland, Cork,

Ireland

2.1 Introduction to fish lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Contents

2.2 Health benefits associated with fish lipids . . . . . . . . . . . . . . . . . . . 24

2.3 Fatty acids found in fish muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.4 Fatty acids found in fish by-products . . . . . . . . . . . . . . . . . . . . . . . . 26

2.5 Factors affecting the fatty acid composition of fish and their

associated by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

2.6 Deterioration of fish lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.7 Implications for fish fat by-product valorization . . . . . . . . . . . . . 32

2.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3 On-board handling of marine by-products to prevent microbial

spoilage, enzymatic reactions and lipid oxidation . . . . . . . . . . . . . . . 47

E. Falch, M. Sandbakk and M. Aursand, SINTEF Fisheries and

Aquaculture, Norway

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3.2 Deterioration of marine biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3.3 Handling and sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

3.4 Conservation and stabilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

3.5 On-board processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

3.6 Utilisation of by-products from gadiform species . . . . . . . . . . . . 59

3.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

4 Recovery of by-products from seafood processing streams . . . . . 65

J. A. Torres, Oregon State University, USA, Y. C. Chen, Chung Shan

Medical University, Taiwan, J. Rodrigo-GarcõÂa, Universidad AutoÂnoma

de Ciudad JuaÂrez, Mexico and J. Jaczynski, West Virginia University,

USA

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

4.2 State of global fisheries and by-products . . . . . . . . . . . . . . . . . . . . . 66

4.3 Basic properties of water, proteins and lipids in aquatic

foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.4 Recovery of functional proteins and lipids from

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4.5 Protein recovery from surimi processing water . . . . . . . . . . . . . . 84

4.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

4.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

5 Increasing processed flesh yield by recovery from marine

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

K. D. A. Taylor and A. Himonides, University of Lincoln, UK and

C. Alasalvar, TUBITAK, Turkey

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

5.2 Recovery of flesh from filleting waste . . . . . . . . . . . . . . . . . . . . . . . 92

vi Contents

5.3 Recovery of flesh from demersal species . . . . . . . . . . . . . . . . . . . . 98

5.4 Quality and improvement of fish mince . . . . . . . . . . . . . . . . . . . . . 99

5.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103


5.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

6 Enzymatic methods for marine by-products recovery . . . . . . . . . . 107

F. Guerard, University of Western Brittany, France

6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

6.2 Overview of by-products extracted by enzymatic methods . . . 108

6.3 Enzymatic extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

6.4 Traceability of by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

6.5 Conclusions and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135


6.7 References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

7 Chemical processing methods for protein recovery from marine

by-products and underutilized fish species . . . . . . . . . . . . . . . . . . . . . . 144

H. G. Kristinsson, A. E. Theodore and B. Ingadottir, University of

Florida, USA

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

7.2 Chemical extraction: fish protein concentrate . . . . . . . . . . . . . . . . 146

7.3 Chemical hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

7.4 Surimi processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

7.5 Fish protein isolates: pH-shift processing . . . . . . . . . . . . . . . . . . . . 152

7.6 Other processes using low or high pH . . . . . . . . . . . . . . . . . . . . . . . 161

7.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162


Part II Food uses of marine by-products

8 By-catch, underutilized species and underutilized fish parts as

food ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

I. Batista, Fish and Sea Research Institute, (IPIMAR), Portugal

8.1 Introduction: by-catch, discards and by-products . . . . . . . . . . . . . 171

8.2 Key drivers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

8.3 Using the by-catch and underutilized species . . . . . . . . . . . . . . . . 174

8.4 Using underutilized fish parts as food and food ingredients . . 179

8.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189


8.7 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

8.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Contents vii

9 Mince from seafood processing by-product and surimi as food

ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

J.-S. Kim, Gyeongsang National University, South Korea and

J. W. Park, Oregon State University, USA

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

9.2 Manufacturing fish mince/surimi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

9.3 Machinery for preparation of fish mince/surimi . . . . . . . . . . . . . . 200

9.4 Mince/surimi processing by-products . . . . . . . . . . . . . . . . . . . . . . . . 204

9.5 Functional properties of fish mince/surimi . . . . . . . . . . . . . . . . . . . 210

9.6 Nutritional characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

9.7 Storage stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

9.8 Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

9.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

10 Aquatic food protein hydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

H. G. Kristinsson, University of Florida, USA

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

10.2 The enzymatic hydrolysis process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

10.3 Properties of fish protein hydrolysates . . . . . . . . . . . . . . . . . . . . . . . 234

10.4 Role in food systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

10.5 Physiological role in humans and animals . . . . . . . . . . . . . . . . . . . 239

10.6 Role in plant growth and propagation . . . . . . . . . . . . . . . . . . . . . . . . 241

10.7 Role as growth media for microorganisms . . . . . . . . . . . . . . . . . . . 242

10.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

10.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

11 Engineering and functional properties of powders from

underutilized marine fish and seafood products . . . . . . . . . . . . . . . . 249

S. Sathivel and P. J. Bechtel, University of Alaska Fairbanks, USA

11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

11.2 Fish protein powder as bio-active ingredients . . . . . . . . . . . . . . . . 250

11.3 Functional properties of fish protein powders . . . . . . . . . . . . . . . . 250

11.4 Flow properties analysis of emulsion containing fish protein

powders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

11.5 Viscoelastic properties of emulsions containing fish protein

powders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

11.6 Thermal properties of fish protein powders . . . . . . . . . . . . . . . . . . 254

11.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

11.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

12 Marine oils from seafood waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

F. Shahidi, Memorial University of Newfoundland, Canada

12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

12.2 Oil from fish processing by-products . . . . . . . . . . . . . . . . . . . . . . . . 261

12.3 Marine mammal oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

viii Contents

12.4 Algal oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

12.5 Marine oil manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

12.6 Health effects of PUFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

12.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

13 Collagen and gelatin from marine by-products . . . . . . . . . . . . . . . . . 279

J. M. Regenstein and P. Zhou, Cornell University, USA

13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

13.2 Key drivers of marine collagen and gelatin . . . . . . . . . . . . . . . . . . 279

13.3 Sources of marine collagen and gelatin . . . . . . . . . . . . . . . . . . . . . . 281

13.4 Manufacture of marine collagen and gelatin . . . . . . . . . . . . . . . . . 281

13.5 Properties of marine collagen and gelatin . . . . . . . . . . . . . . . . . . . . 288

13.6 Food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

13.7 Non-food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

13.8 Improving the quality of collagen and gelatin . . . . . . . . . . . . . . . 298


13.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

14 Seafood flavor from processing by-products . . . . . . . . . . . . . . . . . . . . 304

C. M. Lee, University of Rhode Island, USA

14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

14.2 Aqueous extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

14.3 Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

14.4 Enzymatic hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

14.5 Enzyme-assisted seafood flavors from processing

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

14.6 Flavor-imparting compounds and chemistry . . . . . . . . . . . . . . . . . 319

14.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

14.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

15 Fish and bone as a calcium source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

S.-K. Kim and W.-K. Jung, Pukyong National University, Republic

of Korea

15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

15.2 Biochemical properties of fish bone . . . . . . . . . . . . . . . . . . . . . . . . . 330

15.3 Utilization of fish bone calcium and organic compound . . . . . 331

15.4 In vivo availability of soluble calcium complex from fish bone 335


15.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

16 Chitin and chitosan from marine by-products . . . . . . . . . . . . . . . . . . 340

F. Shahidi, Memorial University of Newfoundland, Canada

16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

16.2 Chemical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

16.3 Applications of chitin, chitosan and their oligomers . . . . . . . . . 353

Contents ix

16.4 Safety and regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364


17 Marine enzymes from seafood by-products . . . . . . . . . . . . . . . . . . . . . 374

M. T. Morrissey and T. Okada, Oregon State University Seafood

Laboratory, USA

17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

17.2 Marine enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

17.3 Producing enzymes from seafood processing by-products . . . . 381

17.4 Marine by-product enzyme utilization . . . . . . . . . . . . . . . . . . . . . . . 384

17.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

17.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

18 Antioxidants from marine by-products . . . . . . . . . . . . . . . . . . . . . . . . . . 397

F. Shahidi and Y. Zhong, Memorial University of Newfoundland,

Canada

18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

18.2 Antioxidants from marine algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398

18.3 Antioxidants from marine animals and their by-products . . . . 404

18.4 Antioxidants from other marine sources . . . . . . . . . . . . . . . . . . . . . 407

18.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408

19 Pigments from by-products of seafood processing . . . . . . . . . . . . . . 413

B. K. Simpson, Department of Food Science and Agricultural

Chemistry, Canada

19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

19.2 Pigment types and sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

19.3 Carotenoid pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

19.4 Other pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422

19.5 Economic, environmental, and safety considerations . . . . . . . . . 426

19.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427


19.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429

Part III Non-food uses of marine by-products

20 By-products from seafood processing for aquaculture and

animal feeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

P. J. Bechtel, University of Alaska, USA

20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

20.2 Driving forces for utilization of by-products . . . . . . . . . . . . . . . . . 436

20.3 By-product components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437

20.4 Overview of different products produced from fish

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

x Contents

20.5 Methods of producing hydrolysates and silage . . . . . . . . . . . . . . . 441

20.6 Nutritional benefits and other properties of fish and animal

feeds made from seafood processing wastes . . . . . . . . . . . . . . . . . 443

20.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444

20.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445

21 Using marine by-products in pharmaceutical, medical, and

cosmetic products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450

J. Losso, Louisiana State University, USA

21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450

21.2 Squalamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451

21.3 Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

21.4 Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

21.5 Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

21.6 Protamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455

21.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

21.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

22 Bio-diesel and bio-gas production from seafood processing

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

R. Zhang and H. M. El-Mashad, University of California Davis, USA

22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

22.2 Quantity and quality of various seafood processing

by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

22.3 Theories and technologies for production of bio-diesel and

bio-gas fuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

22.4 Potential yields and quality of bio-diesel and bio-gas fuels . . 471

22.5 Problems encountered and possible approaches for

overcoming them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

22.6 Future research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

22.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480


22.9 List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481

22.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482

23 Composting of seafood wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

A. M. Martin, Memorial University of Newfoundland, Canada

23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

23.2 Biodegradation of seafood wastes by composting . . . . . . . . . . . . 487

23.3 Composting operational parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 490

23.4 Characteristics of the composting of seafood wastes . . . . . . . . . 494

23.5 Technological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

23.6 Biological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501

23.7 Vermicomposting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503

23.8 Quality considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

Contents xi

23.9 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509


23.11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516

xii Contents

(* = main contact)

Editor

Professor Fereidoon Shahidi

Department of Biochemistry

Memorial University of

Newfoundland

St. John's, Newfoundland

Canada

A1B 3X9

E-mail: [email protected]

Chapter 1

Dr Turid Rustad

Department of Biotechnology

NTNU

7491 Trondheim

Norway


Chapter 2

Dr Joe P. Kerry and Sheila C. Murphy

Department of Food and Nutritional

Sciences

University College Cork

National University of Ireland

Cork

Co. Cork

Ireland

Chapter 3

Dr Eva Falch,* Marit Sandbakk and

Dr Marit Aursand

SINTEF Fisheries and Aquaculture

N-7465 Trondheim

Norway


[email protected]

Contributor contact details

Chapter 4

Dr J. Antonio Torres*

Food Process Engineering Group

Dept. of Food Science & Technology

Oregon State University

Corvallis, OR 97331-6602

USA

E-mail:

[email protected]

Dr Yi-Chen Chen

School of Nutrition

Chung Shan Medical University

Taichung City 402

Taiwan


Professor JoaquõÂn Rodrigo-GarcõÂa

Departamento de Ciencias BaÂsicas

Instituto de Ciencias BiomeÂdicas

Universidad AutoÂnoma de Ciudad

JuaÂrez

Ciudad JuaÂrez

Chih

MeÂxico


Dr Jacek Jaczynski

Dept. of Animal and Veterinary

Sciences

West Virginia University

Morgantown, WV 26506

USA

E-mail:

[email protected]

Chapter 5

Professor K. D. Anthony Taylor* and

Dr Aristotelis Himonides

Faculty of Technology

Food Research Centre

University of Lincoln

Brayford Pool

Lincoln LN6 7TS

UK


Dr Cesarettin Alasalvar

TUBITAK Marmara Research Centre

Food Institute

PO Box 21, 41470

Gebze-Kocaeli

Turkey

E-mail:

[email protected]

Chapter 6

Dr Fabienne Guerard

ANTiOX Laboratory

University of Western Brittany

PoÃle universitaire P.J. Helias

F-29000 Quimper

France


Chapter 7

Dr Hordur G. Kristinsson, Ann E.

Theodore and Bergros Ingadottir

Laboratory of Aquatic Food

Biomolecular Research

Aquatic Foods Product Program

Department of Food Science and

Human Nutrition

University of Florida

Gainesville, FL 32611

USA

xiv Contributors

Chapter 8

Dr Irineu Batista

IPIMAR

Irineu Batista

Av. BrasõÂlia

1449-006

Lisbon

Portugal


Chapter 9

Professor J.-S. Kim*

Gyeongsang National University

South Korea


Professor Jae Park

Oregon State University

2001 Marine Drive #253

Astoria, OR 97103

USA


Chapter 10

Dr Hordur G. Kristinsson

Laboratory of Aquatic Food

Biomolecular Research

Aquatic Foods Product Program


Human Nutrition

University of Florida

Gainesville, FL 32611

USA

Chapter 11

Dr Subramaniam Sathivel*

Fishery Industrial Technology Center

University of Alaska Fairbanks

118 Trident Way

Kodiak, AK 99615

USA

Dr Peter J. Bechtel

USDA/ARS Subarctic Research Unit

245 O'Neill Building


Fairbanks, AK 99775

USA

Chapters 12 and 16

Professor Fereidoon Shahidi



Newfoundland


Canada

A1B 3X9


Chapter 13

Professor Joe M. Regenstein* and

Peng Zhou

Department of Food Science

Stocking Hall

Cornell University

Ithaca, NY 14853-7201

USA


Chapter 14

Professor Chong Lee

Department of Nutrition and Food

Sciences

University of Rhode Island

530 Liberty Lane

West Kingston, RI 02892

USA


Contributors xv

Chapter 15

Dr Se-Kwon Kim* andWon-Kyo Jung

Marine Bioprocess Research Center

Pukyong National University

Busan 608-737

Republic of Korea

Chapter 17

Dr Michael T. Morrissey* and

Tomoko Okada

Professor and Director

Oregon State University Seafood

Laboratory

2001 Marine Drive

Rm. 253

Astoria, OR 97103

USA

E-mail:

[email protected]

Chapter 18

Professor Fereidoon Shahidi* and

Ying Zhong



Newfoundland


Canada

A1B 3X9


Chapter 19

Dr Benjamin K. Simpson


Agricultural Chemistry

514.398.7737 (Office)

Room MS1-034

Macdonald-Stewart Building

21111 Lakeshore Road

Ste. Anne de Bellevue

Quebec, H9X 3V9

Canada


Chapter 20

Dr Peter J. Bechtel

USDA-ARS Subarctic Research Unit

245 O'Neill Building


Fairbanks, AK 99775

USA

Chapter 21

Professor Jack Losso

Department: Food Science

Department

LSU AgCenter

111 Food Science Bldg.

Louisiana State University Campus

LA 70803

USA


[email protected]

Chapter 22

Dr Ruihong Zhang* and

Hamed M. El-Mashad

Biological and Agricultural

Engineering Department

University of California Davis

One Shields Avenue

Davis, CA 95616

USA

E-mail: rhzhang@ucdavis

xvi Contributors

Chapter 23

Dr A. M. Martin

Dept Biochemistry


Newfoundland

St John's

NL, A1A 4A3

Canada


Contributors xvii

Aquatic species provide an important source of nutrients for human

consumption. In addition, beneficial effects of seafoods and marine oils have

contributed greatly to better appreciation of resources and encouraging of full

utilization of the catch. Seafoods originating from the wild or cultivated species

produce a large amount of by-products upon processing or as by-catch of

targetted fishery that may not be put to full use. These by-products could serve

as a rich source of a variety of biomolecules with potential health benefits. Thus,

many niche products can provide a better commercial value and full utilization

of raw materials. In this regard, marine oils, enzymes, hydrolysates, carotenoids

and squalene, chitinous material, N-acetylglucosamine and glucosamine, among

others, are of much importance. Many studies, for example, have shown the

effects of marine oils in cardiovascular diseases, diabetes and rheumatoid

arthritis as well as cancer prevention by different mechanisms at the cellular and

subcellular levels. Therefore, it is imperative to maximize the value of

processing by-products from aquatic species. This book is the first to provide a

comprehensive account of value-added by-products from fisheries processing. It

would serve as an important reference for researchers in academia, industry and

government labs, both in terms of fundamental science and its application.

I am grateful to the experts who have provided state-of-the-art contributions

for inclusion in this book. I am also grateful to Woodhead Publishing personnel

who provided me with much support that was essential for successful

completion of this task.

Fereidoon Shahidi

Preface

The world's annual catch of fish and marine invertebrates has been around 100

million metric tonnes in recent years. However, aquaculture developments have

led to production of high quality products that have also assisted conservation

strategies to be implemented. Of the total amount of harvest, a major portion

remains unused or used for production of fish meal and fish oil. This is due to

the fact that certain species might suffer from small size, high bone, skin and fat

contents as well as unappealing shape. In addition, several species of fish may be

used for their roe and production of caviar. The leftover carcass following roe

extraction as well as those of their male counterparts may be discarded.

Furthermore, processing discards from many species of fish and shellfish could

be successfully processed for production of specialty enzymes, xanthophylls,

chitin/chitosan, glucosamine and other value-added products. Thus, devising of

strategies for full utilization of the catch and processing of discards for

production of novel products is warranted (Shahidi, 2000).

Seafood processing by-products and their use

The seafood processing industry is still producing a large quantity of by-

products and discards. These include heads, tails, viscera and backbone as well

as shells. Utilization of these processing by-products may be exercised in

different ways leading to

1. production of animal and aquaculture feed, similar to that used for whole

fish when producing fish meal and fish oil,

Maximizing the value of marineby-products: an overviewF. Shahidi, Memorial University of Newfoundland, Canada

2. production of food ingredients such as extraction of cheeks and tongue from

cod and production of surimi from frames and

3. production of novel and value-added products for nutraceutical, pharma-

ceutical and fine chemical industries (Table I.1).

Novel and specialty products with potential biological activity and/or

functionality provide a means for value-added utilization of by-products. These

may be used as food ingredients to take advantage of a specific flavour, such as

those from cook water of crab and lobster (Jayarajah and Lee, 1999; Yang and

Lee, 2000), or for rendering a specific functional property such as water-holding,

foaming, emulsifying and gelling properties. The use of by-products as health

food ingredients may be for nutritional purposes; these include proteins, lipids,

mineral and vitamins. Finally, by-products may be employed for nutraceutical

and specialty applications. In this category, protein hydrolyzates, fish oils,

hormones, glucosamine, chitin/chitosan, flavourants and enzymes as well as

other physiologically active ingredients may be included. The following sections

provide a cursory account of current and potential uses of by-products in

different applications and for rendering health benefits.

Proteins from seafoods and their by-products

Seafood by-products are an excellent source of high quality proteins that may

supply a major part of the essential amino acids that are required for a balanced

nutrition. Recovery of proteins from by-products may be carried out by different

processes using mechanical separation from frames, base extraction or hydrolysis.

While hydrolysis of fish proteins by endogenous enzymes prior to or during

primary processing may lead to fish quality deterioration, such processes may be

intentionally carried out to produce specialty products. Thus, production of fish

sauce and silage from fish and processing discards is commonplace. In addition,

enzymes that are commercially available may be used to produce protein

hydrolyzate that could be used in a variety of applications. Protein hydrolyzates

are nearly colourless and appear like milk powder; they may be used in

Table I.1 Physiological components from marine by-products

Ingredient Application area

Proteins/biopeptides Nutraceuticals, immune-enhancersMinerals/calcium Food, nutraceuticalsChitosan, glucosamine Nutraceuticals, agriculture, food, water purificationOmega-3 oils Nutraceuticals, dietary supplements, foodCarotenoids/xanthophylls Nutraceuticals, fish feedChordprotein sulphate Supplements, arthritic pain reliefSqualene Skin careSpecialty chemicals Miscellaneous

xxii Maximizing the value of marine by-products: an overview

applications where water solubility and water-holding capacity are important.

Protein hydrolyzates may possess biological activity in enhancing immune

response and may also render antioxidant as well as angiotensin converting

enzyme (ACE) inhibitory activity (Je et al., 2004) among others.

Carotenoids (C40H56) and their oxygenated derivatives (xanthophylls) are

another group of bioactives that are present in salmonid fish, crustaceans and

their processing by-products, among others (Shahidi et al., 1998). These are

often present in combination with proteins, known as carotenoproteins. Extrac-

tion and isolation of carotenoproteins as ingredients for potential use in salmonid

fish aquaculture has been reported (Cremades et al., 2003).

Digestive proteases from fish and shellfish processing discards may be used

as industrial processing aids (Shahidi and Kamil, 2001). Suggested uses of

digestive proteases from fish include acids for cheese making, herring fermenta-

tion, fish skinning, roe processing and production of specialty kits, as well as

medical applications.

Lipids from processing by-products

Seafood lipids provide unique health benefits to consumers, but also present a

challenge to scientists and technologists for delivering their highly unsaturated

fatty acids (HUFA) in an odour-free and appealing form. The oils originate from

the body of fatty fish as a by-product of fish meal industry, liver of white lean

fish such as those of cod and halibut, and finally blubber of marine mammals

such as seals and whales. Viscera from fish also provide for a rich source of

lipids. These lipids include a high proportion of long-chain polyunsaturated fatty

acids (LC PUFA) belonging to the omega-3 family, namely eicosapentaenoic

acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).

There is a rapidly growing body of literature illustrating the health benefits of

HUFA. These effects include protection against cardiovascular disease, auto-

immune and mental disorders, diabetes, arthritis and arrhythmia, among others

(Shahidi and Finley, 2001; Shahidi and Miraliakbari, 2004, 2005).

Marine lipids are highly prone to oxidation, hence their processing under

relatively mild conditions and stabilization following refining, bleaching and

deodorization is recommended. This is partly due to the fact that the refining

process leads to the removal of endogenous antioxidants from the oil and hence

replenishment with antioxidants, particularly those from natural sources is

important. In addition, microencapsulation of the oils may prove useful when

such oils are to be used in fortification of food and beverages.

Minerals and chitinous materials

Seafood processing discards contain a large proportion of frames as well as

shells that are primarily composed of calcium salts. Thus, the resultant calcium

Maximizing the value of marine by-products: an overview xxiii

may be solubilized and potentially used for addressing concerns about bone

health due to insufficient intake of calcium. Jung and co-workers (2006) have

clearly demonstrated the solubilization of calcium from fish frames and their

benefits.

Most shellfish, especially those from shrimp, crab, lobster and crayfish con-

tain a large amount of chitin that may be recovered following deproteinization

and demineralization. The recovered chitin may be used for chitosan production

using concentrated base or render pressure, glucosamine preparation or chitosan

oligmers, short-chain copolymers of glucosamine and N-acetylglucosamine and

derivatives thereof.

Glucosamine, the monomer of chitosan, has been reported to possess benefits

for joint health and build up as well in wound-healing, among others. The

product is generally sold as glucosamine sulphate, but this is often a mixture of

glucosamine hydrochloride and sodium or potassium sulphate. Furthermore,

glucosamine may be sold in combination with chondroitin 4- and 6-sulphates.

Chondroitins are mucopolysaccharides (MPs) with molecular weights of up to

50,000 Da and could be prepared from connective tissues of slaughtered animals

and seafoods (Jo et al., 2005). In combination, while glucosamine helps to form

proteoglycans that sit within the space in the cartilage, chondroitin sulphate acts

as a liquid magnet (Shahidi and Kim, 2002).

Future trends and prospects

Dwindling supply of seafoods from the wild dictates full utilization of the

harvest. In addition, the advent of aquaculture allows a better control over the

harvest time and hence better quality of products, including processing by-

products. A stricter environmental restriction on dumping of discards also serves

as a further incentive to explore novel uses of products that might otherwise be

considered uneconomical. Low temperature activity of enzymes as well as

unique characteristics of products from processing discards might also catalyze

new developments in value-added utilization of specialty products from

processing lines.

References

CREMADES, O., PARRADO, J., ALVAREZ-OSSORIO, M.C., JOVER, M., COLLANTES-DE-TERAN, L.,

GUTIERREZ, J.F. and BAUTISTA, J. 2003. Isolation and characterization of

carotenoproteins from crayfish (Procambarus clarkii). Food Chem. 82, 559±566.

JAYARAJAH, C.N. and LEE, C.M. 1999. Ultrafiltration/reverse osmosis concentration of

lobster extract. J. Food Sci. 64, 93±98.

JE, J.Y., PARK, P.J., KWEN, J.Y. and KIM, S.K. 2004. A novel angiotensin converting enzyme

inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein

hydrolysate. J. Agric. Food Chem. 52, 7842±7845.

xxiv Maximizing the value of marine by-products: an overview

JO, J.H., DO, J.R., KIM, Y.M., KIM, D.S., LEE, T.K., KIM, S.B., CHO, S.M., KANG, S.V. and PARK, D.C.

2005. Optimization of shark (squatina oculata) cartilage hydrolysis for the

preparation of chondroitin sulfate. Food Sci. Biotechnol. 14, 651±655.

JUNG, W.K., LEE, B.J. and KIM, S.K. 2006. Fish-bone peptide increases calcium solubility and

bioavailability in ovariectomised rats. British J. Nutr. 95, 124±128.

SHAHIDI, F. 2000. Seafood in Health and Nutrition ± Transformation in Fisheries and

Aquaculture: Global Perspectives. ScienceTech Publishing Co., St. John's, NL,

Canada.

SHAHIDI, F. and FINLEY, J.W. 2001. Omega-3 Fatty Acids: Chemistry, Nutrition, and Health

Effects. ACS Symposium Series 788, p. 330. American Chemical Society,

Washington, D.C.

SHAHIDI, F. and KAMIL, Y.V.A.J. 2001. Enzymes from fish and aquatic invertebrates and

their application in the food industry. Trends Food Sci. Technol. 12, 435±464.

SHAHIDI, F. and KIM, S-K. 2002. Quality management of marine nutraceuticals. In C-T. Ho

and Q.T. Zheng (eds), Quality Management of Nutraceuticals. ACS Symposium

Series 803, pp. 76±87. American Chemical Society. Washington, DC.

SHAHIDI, F. and MIRALIAKBARI, H. 2004. Omega-3 (n-3) fatty acids in health and disease:

Part 1 ± Cardiovascular diseases and cancer. J. Med. Food 7, 387±401.

SHAHIDI, F. and MIRALIAKBARI, H. 2005. Omega-3 fatty acids in health and disease: Part 2 ±

Health effects of omega-3 fatty acids in autoimmune diseases, mental health and

gene expression. J. Med. Food 8, 133±150.

SHAHIDI, F., METUSALACH and BROWN, J.A. 1998. Carotenoid pigments in seafoods and

aquaculture. Crit. Rev. Food Sci. Nutr. 38, 1±67.

YANG, Y. and LEE, C.M. 2000. Enzyme-assisted bioproduction of lobster flavor from the

process by-product and its chemical and sensory properties. In Shahidi, F. (ed.),

Seafood in Health and Nutrition ± Transformation in Fisheries and Aquaculture:

Global Perspectives. ScienceTech Publishing Co., St. John's, NL, Canada, pp.

169±194.

Maximizing the value of marine by-products: an overview xxv

Part I

Marine by-products characterisation,recovery and processing

1.1 Introduction

Overexploitation of fish resources is a major problem as only 50±60% of the

catch is used for human consumption. Globally, more than 91 million tonnes of

fish and shellfish are caught each year. Some of the by-products are utilised but

huge amounts are wasted. Annual discard from the world fisheries has been

estimated to be 25% of the catch. Only a small amount of the by-products is used

for human consumption, the rest is used for production of fishmeal, silage and

animal feed. A list of valuable components in fish by-products is given in Table

1.1. Fish provides about 14% of the world's need for animal proteins and 4±5%

of the total protein requirement (Venugopal, 1995). The amino acid composition

and digestibility of fish proteins is excellent. It is a challenge both to increase the

utilisation of the protein fractions from marine by-products and to use more of

these valuable proteins as food ingredients.

Use of by-products is not new. In the Nordic countries a lot of the by-

products have been and are still being used for various purposes. For instance

fish skin has earlier been used to cover window openings, to make clothes,

shoes, carrier packs and sacks. Some fish by-products that are used for human

consumption include roe (canned, salted/marinated or as cod roe emulsion), liver

(Eastern Europe), cleaned stomach, fried fish milt (a snack) and head products

from Iceland (cheeks, tongues, dried heads).

In 2000 a total of 251 000 metric tonnes of by-products were created by the

Norwegian cod fisheries alone, of this 114 000 tonnes were dumped while

137 000 tonnes were utilised. Only 33 000 tonnes of the by-products were used

for human consumption which amounts to about 13% of the total (RUBIN,

1

Physical and chemical properties ofprotein seafood by-productsT. Rustad, Norwegian University of Science and Technology,Norway

2001). The rest is used for production of fishmeal, silage and animal feed. A

large part of the by-products that are dumped at sea are made up by heads

(Stoknes and Hellevik, 2000). In Norway only about 20% of the cod heads are

exploited. Of this 9000 tonnes goes to human consumption, mostly as dried

heads to Nigeria. In some fisheries the tongues and cheeks are cut out. The rest

is minced and used as feed for fur animals.

Fisheries and fish industries are the single most important industry in Iceland.

In 2001 the total catch was around 2 million tonnes, accounting for 62% of the

value of exported products and around 48% of the foreign currency earnings that

year. Fish meal and oil constitute the bulk of the volume of products from

fisheries in Iceland or 63% of total, but their value is far less or only about 14%

of the total value of exported seafood products. In 2001, Iceland exported about

45 500 tonnes of by-products with a value of US$73.5 million.

Of a total available UK fish and shellfish resource of approximately 850 000

tonnes, only 43% end up as products for human consumption. The rest is

categorised as `waste'. About 300 000 tonnes of this is produced on shore,

whereas the rest is produced at sea (145 000 tonnes discarded fish and 43 000

tonnes processing waste). For cod-fish these numbers can be broken down to

154 000 tonnes on shore and 37 000 tonnes at sea. In the UK the major outlet for

this raw material is fishmeal and oil production, only small quantities are used

for other purposes (pet food, animal feed, fishing bait, etc.). In some areas of the

UK the by-products primarily end up in landfill sites. Much of the waste

production is concentrated in regional processing centres. Only very few fishing

vessels utilise the by-products. The non-utilised by-products generated onboard

are dumped at sea.

The annual harvest of seafood in Alaska is over 2 million tonnes and yields

more than 1 million tonnes of by-products, some of this is produced into

fishmeal and oil but the majority is discarded (Crapo and Bechtel, 2003; Sathivel

et al., 2004).

1.2 Overview

The protein-rich by-product fractions include cut-offs, backbones, heads, skin,

roe, milt, stomachs, viscera and blood. The proportion of different by-products

Table 1.1 Valuable components of fish by-products

Lipids Proteins Other components

Oils Hydrolysates Nucleic acidsOmega-3: EPA, DHA Surimi CalciumPhospholipids Thermostable dispersions Bioactive compoundsSqualen Peptides, amino acids ColoursVitamins Gelatine, collagenCholesterols Protamines

4 Maximising the value of marine by-products

from different species is given in Table 1.2. For the industry to be able to utilise

more of the by-products, data on the available amounts as well as on the

chemical composition and the properties of both the protein and the lipid

fractions as a function of species, season and fishing ground are needed.

On average, production of cod fillets will generate two-thirds of the whole

body weight as by-products. The data of Falch et al. (2006a) show that the

viscera (all inner fractions) makes up 12±15% of the whole body weight of four

Gadidae species (cod, saithe, haddock and tusk), caught in the Barents sea, the

head 15±20% and the backbone and trimmings (cut-offs) make up 18±30%. In a

study including 750 specimens from five different Gadidae species caught at

three different fishing grounds in Europe, the viscera was found to constitute 3±

7% of the round weight of the fish. The highest proportion was found in the

Norwegian Gadidaes and the lowest in the Irish (Falch et al., 2006b).

For Gadidae species caught in Icelandic water, the proportion of intestines in

tusk and ling was lower than in the other cod species (Thorarinsdottir et al.,

2004). The proportion of liver, roe and milt were positively correlated, but

negatively correlated to viscera and CF (condition factor). The proportion of

head was similar for all cod groups (different sizes of cod), ranging from 27 to

33%. The proportion of head of tusk, ling, saithe and haddock was lower than

that of cod. The proportion of backbone varied from 14.9 to 16.3% for cod and

from 14.9 to 18.7% for haddock. The size of the cod did not affect the proportion

of the backbone. The proportion of skin from cod fillets was in the range 5.1 to

7.7%. It was highest for ling (8.7 to 10.8%) and tusk (10.6 to 12.4%) at all

seasons. Cut-offs in cod groups were in the range 9.5 to 12.0%, in haddock in the

range of 7.7 to 11.7%, in saithe 10.5 to 15.6%, ling 12 to 15.4% and in tusk 11.5

to 13%. Seasonal effects were not significant.

The global production of farmed Atlantic salmon was estimated to be

1025 000 tonnes in 2002 (Globefish, 2002) and more than 50% of this weight is

regarded as by-products or waste. The largest fractions constitute the cut-offs

(including backbone) (14%), viscera (13%) and head (10%), and these fractions

might serve as a source of valuable marine lipids and proteins. Blood makes up

2% of the total weight of salmon.

The composition of different by-product fractions from different species is

given in Table 1.3. In different sources reporting protein content in various by-

product fractions, the protein content is usually given as crude protein, Nx6.25,

this value also includes the NPN (non-protein-nitrogen). In some fractions this

may constitute up to 25% of total N (Sikorski, 1994). In the meat of white fish,

the NPN content is usually from 9 to 15% of total N. About 95% of NPN is

made up of free amino acids, dipeptides, trimethylamine oxide (TMAO) and

degradation products, urea, guanidine, nucleotides and degradation products of

nucleotides.

Liaset et al. (2000, 2002) reported that the protein content of cod frames was

16.9% while the content in salmon frames was 17.4%. In another study, the

protein content in salmon frames was found to be 18.2% (Michelsen et al., 2004)

and the protein content in salmon viscera was found to be 10.6%.

Physical and chemical properties of protein seafood by-products 5

Table 1.2 Amount of different by-product fractions

Species By-product fraction amount of total fish weight (%)

Head Backbone/ Cut-offs Skin Roe Milt Viscera Referenceframes

Cod 20.2 9.7 8.2 4.2 0.7 1.3 5.6 Falch et al. (2006a)Saithe 15.3 9.9 8.8 4.8 0.3 0.2 7.2 Falch et al. (2006a)Haddock 18.9 10.6 9.3 4.5 0.7 0.1 6.2 Falch et al. (2006a)Tusk 17.9 8.4 21.2 6.4 2.0 0.0 9.9 Falch et al. (2006a)Ling 18.6 1.7 3.3 Falch et al. (2006b)Atlantic salmon 10.0 10.0 5.01 14.0 Sandnes et al. (2003)Carp, wild 21±25 5±9 6±8 3±4 Bukovskaya and Blokhin (2004)Carp, cultured 20±21 6±8 8±11 4±5 Bukovskaya and Blokhin (2004)

1Includes bellyflap

Table 1.3 Protein content in different by-product fractions

Protein content % of wet weight

Species Head Backbone/ Cut-offs Skin Roe Milt Viscera Referenceframes

Cod 13±23 9±13 Sùvik (2005)Saithe 15±19 12±19 Sùvik (2005)Haddock 15±18 7±11 Sùvik (2005)Tusk 17±23 3±12 Sùvik (2005)Ling 15±20 8±12 Sùvik (2005)Carp, wild 14±22 14±27 15±23 Bukovskaya and Blokhin (2004)Carp cultured 12±17 19±25 26 Bukovskaya and Blokhin (2004)Atlantic salmon 11±13 10±15 8±121 5±7 Sandnes et al. (2003)Herring 13.1 18.0 Sathivel et al. (2004)

1 Includes bellyflap

Sikorski et al. (1984) have reported protein content in different fish skins to

be between 18 and 35%, with a collagen content between 10.6 and 28.8%.

Sikorski (1994) reported the crude protein content in different fractions of fish

from New Zealand waters. The protein content in viscera varied from 7.5 to

23.9% while the content in skin varied between 11.9 and 29.6% and in frames

the protein content was between 13.1 and 25.3%.

Fish roe has high concentrations of proteins and lipids (Bledsoe et al., 2003).

In general fish roe products are high in protein (16±30%). Crude lipid content

can vary from less than 5% to 20% with an average of 10% in salmon roe. The

protein quality of fish roe is high, either methionine/cystine or tryptophan/

tyrosine are the limiting amino acids.

The backbone is one of the major by-product fractions, yielding about 15% of

the fish weight (Gildberg et al., 2002). About 15% of the wet weight is pure

bone, the rest is white muscle (Jeon et al., 2000). The bone makes up about one-

third of the dry weight and consists of minerals (60±70%) and proteins (30%).

The protein is mainly collagen (Lall, 1995; Nagai and Suzuki, 2000). Based on

these values, it can be calculated that 80±85% of the protein in cod backbone

fraction is muscle protein and the rest is collagen. Gildberg et al. (2002) found

that the muscle proteins from the backbone fraction could be extracted using

hydrolysis with commercial enzymes and the resulting bone could be used to

recover gelatine using heat extraction. The gelatine extracted had a low mole-

cular weight, but could be suitable for technical applications and nutraceuticals.

Flesh from backbones and cut-offs may be a suitable raw material for pro-

duction of fish mince, surimi and surimi-based products. Surimi is mechanically

deboned fish flesh that has been washed with water or dilute salt solutions and to

which cryoprotectants have been added. Surimi is used as the raw material for

preparation of seafood analogues, such as shrimp, crab legs, scallop and lobster

tail. In addition the surimi industry has the potential to develop new products.

éines et al. (1995) found that the yield of fish mince that could be separated from

salmon and white fish cut-offs and backbones was between 48 and 56% of the

weight of the by-product fractions with a protein content ranging from 13 to 17%.

The demand for collagen and gelatine from the industry throughout the world

is considerable and still rising. By-products from fish processing are a potential

source of collagen. In fish the largest collagen concentrations are found in the

skeleton, fins and the skin (Norland, 1989; Sikorski and Borderias, 1994). Fish

gelatine and collagen have been much less studied than mammalian gelatine and

collagen (Norland, 1989; Gudmunsson and Hafsteinson, 1997). Mammalian

collagen in its purified form has found a number of pharmaceutical and cosmetic

applications. Similarly, gelatin, the hydrolysed form of collagen, is an ingredient

extensively used in the food industry. Gelatin is used as a food additive to

improve the texture, the water-holding capacity and stability of several food

products (Borderias et al., 1994). Some documents also suggest that fish

collagens were used in ancient times. Plinius (AD 23±79) speaks of ìchtyocolla'

(fish glue) from Pondus (The Black Sea), and Plinius the Elder reports use of fish

glue as a medicine against headaches and cramps (Solstad and Muniz, 2001).


The quality and specific application of the extracted collagen and gelatine is

highly related to their functional properties and to purity. Known problems with

the extraction of collagen from fish skins are the abundance of pigments and the

presence of fish odours, which would restrict its potential use. The uniqueness of

fish collagen from cold water fish lies in the lower content of amino acids,

proline and hydroxyproline (Haard et al., 1994). Although fish gelatine does not

form particularly strong gels, it is well suited for certain industrial applications,

as, for example, micro-encapsulations, light-sensitive coatings, and low-set-time

glues. The extraction of native collagen, as described by van de Vis et al. (1996),

instead of gelatine, which is the hydrolysed form of collagen, is strongly

preferred, because native collagen provides more and better opportunities to

modify the functional properties as well as the possible applications of collagen

in the food ingredient industry.

The skeleton, fins and skin constitute the main part of whitefish òffal'. The

Norwegian fish industry produces approximately 600 000 metric tonnes of

`waste' per year. This includes 10 000±12 000 tonnes of skins from white fish

(cod, pollack, haddock, etc.) which could be used to produce at least 1500±2000

tonnes of fish gelatine (RUBIN, 2001). The search for new gelling agents to

replace mammalian gelatine has led to patents for fish gelatine production

(Grossman and Bergman, 1992; Holzer, 1996) as well as several published

methods (Gudmunsson and Hafsteinsson, 1997; Nagai and Suzuki, 2000;

Gomez-Guillen and Montero, 2001). Collagen from fish has just recently been

identified as a potential allergen and this may be a potential problem for the use

of fish gelatine in commercial products (Sakaguchi et al., 1999; Hamada et al.,

2001).

Fish milt is a product that is often wasted, even though canned milt from cod

and herring is a traditional food product in England (Gildberg, 1999). The milt

contains a high amount of protamine and nucleic acids and is used for industrial

production of nucleic acids. The products are used in health food and cosmetics

and the remainder used as feed supplement in aquaculture. Studies have shown

that supplementation of cod milt cationic proteins to the feed of juvenile fish

may improve their resistance to V. anguillarum infection (Pedersen et al., 2004).

Real caviar is made from sturgeon, but a wide variety of other fish roes are

consumed in their own right, as well as products sold as substitutes for sturgeon

roe. Important commercial fish roes include salmon roe (Ikura), lumpfish roe,

flying fish roe, whitefish roe, cod roe, mullet roe, herring roe (Kazunoko),

pollack roe (Bledsoe et al., 2003).

A wide variety of by-products have been used for making fish silage

(Gildberg, 2002). Fish silage can be produced from all kinds of low-value fish

and fish by-products and is almost entirely used for feed. Fish silage is normally

made by mixing 2±3% formic acid into the minced raw material and storing at

ambient temperatures till endogenous enzymes have dissolved the fish tissue. A

well-preserved fish silage will normally have a pH of 3±4 which is the optimum

pH for fish pepsins. The process usually takes a few days, provided that the raw

material has a sufficiently high content of pepsins and other acid proteases


(cathepsins). The silage may be used directly in feed or processed further by

separation of the oil and evaporation to give a protein concentrate. The

advantage of producing fish silage is the low capital investment and simple

processing equipment. The disadvantage is the high transport costs due to the

high water content. Norway is the major fish silage producer ± producing about

140 000 tonnes per year, mainly from aquaculture by-products (salmon). Fish

silage is a low price product, but is a good alternative for utilising by-products

that might otherwise have been wasted.

A large proportion of the catch (~ 30%) is used for fish meal and animal feed

because of its poor functional properties. One of the approaches for effective

protein recovery from by-products is enzymatic hydrolysis, which can be

applied to improve and upgrade the functional and nutritional properties of

proteins (Gildberg, 1993; Liaset et al., 2000). Proteolytic modification of food

proteins to improve palatability and storage stability of available protein

resources is an ancient technology (Adler-Nissen, 1986). Hydrolysates can be

defined as proteins that are chemically or enzymatically broken down to

peptides of varying sizes. Today much of the fish flavour, fish soup and fish

paste products available on the market are prepared by enzymatic hydrolysis

(Shoji, 1990). Protein hydrolysates can be used as emulsifying agents in a

number of applications such as salads dressing, spreads, and emulsified meat

and fish products like sausages or luncheon meat (Badal and Kiyoshi, 2001).

They can also be used in bacterial growth media (Poeronomo and Buckle, 2002).

A large variety of different fish protein hydrolysates are being produced. The

oldest is fish sauce which has long traditions in South-East Asia. Fish sauce, which

is the major fermented fish product, was known in Ancient Greece and Rome

(Corcoran, 1963). Fish sauce is produced in a quantity of about 250 000 tonnes per

year. It is made by mixing three parts of fish raw material with one part of salt and

storing at ambient tropical temperatures for 6±12 months. Both endogenous and

microbial enzymes contribute to the degradation of the proteins in the fish and the

resulting fish sauce is an amber liquid with 8±14% digested proteins and about

25% salt. The production is simple and requires little sophisticated equipment, but

there is a need for a large storage space (Gildberg, 2002).

Fish protein hydrolysates can be made with enzymes, acids or alkali.

Enzymatic hydrolysis is strongly preferred over strictly chemical methods for

producing hydrolysates for use in nutritional applications. Enzymatic hydrolysis

can produce hydrolysates with well defined peptide profiles (Lahl and Braun,

1994). This approach gives an effective recovery of proteins, in addition to

upgrading the functional and nutritional properties of the by-product proteins

(Shahidi et al., 1995; Liaset et al., 2000; Slizyte et al., 2005). Enzymatic

hydrolysis of fish by-products may be accomplished by an autolytic process,

using the digestive enzymes of the fish itself or using added commercial

enzymes, or by a combination of these (semiautolytic). The autolytic process

lasts from a few days to several months. There are no enzyme costs involved and

it is a simple operation. However, prolonged digestion may adversely affect the

functional properties of the resulting hydrolysate, and such products are gener-


ally used in feed formulations. Semiautolytic processes including the endog-

enous enzymes as well as commercial enzymes may be the most interesting, but

require knowledge about the raw material composition and the activity, stability

and specificity of the endogenous proteolytic enzymes regarding variations

according to species, season and fishing ground (Slizyte et al., 2005; Dauksas et

al., 2004).

The development of fish protein concentrates (FPC) was one of the earliest

attempts to recover fish protein from processing wastes. FPC has been produced

using solvent extraction, resulting in a concentration of the proteins by removal

of the water and oil from the raw material (Kristinsson and Rasco, 2000).

By-products from cod species are a potential source for bioactive compounds.

This may be both secondary metabolites as well as enzymes, lipids and hetero-

polysaccharides (Gudbjarnason, 1999). Protamine is a basic peptide containing

more than 80% arginine. Protamine has been found in the testicles of more than

50 fish species. Protamine has the ability to prevent growth of Bacillus spores

and may be used as an antibacterial agent in food processing and preservation.

It has been reported that many proteins possess antioxidative activities, and

fish protein hydrolysates have been found to be antioxidative (Amarowicz and

Shahidi, 1997; Kim et al., 2001).

Proteolytic enzymes from cold-adapted fish have received a lot of interest in

recent decades. These enzymes have been found to be more catalytically active

at relatively low temperatures compared to corresponding enzymes from

mammals, thermophilic organisms and plant sources (De Vecchi and Coppes,

1996; Gudbjarnason, 1999). Enzymes with unique properties for industrial

utilisation can be recovered from fish by-products (Haard et al., 1994). The

Icelandic Fisheries laboratory has developed a process to recover trypsin-like

enzymes from cod viscera (Stefansson and Steingrimsdottir, 1990). In Norway

industrial processes for recovery of pepsin, trypsin, chymotrypsin, alkaline

phosphatase and hyaluronidase from fish viscera have also been developed

(AlmaÊs, 1990). Alkaline phosphatase is recovered from the thaw water from

frozen shrimp (Olsen et al., 1990). This is used in diagnostic kits.

1.3 Physical and chemical properties of protein-richby-products ± seasonal, habitat, species and individualvariations

It is a challenge to utilise more of the protein fractions from fish by-products as

food ingredients. Many protein-rich marine by-products have a range of

dynamic properties and can potentially be used in foods as binders, emulsifiers

and gelling agents (Sathivel et al., 2004). Fish proteins have unique functional

properties such as capacity to bind water, lipids, rheological properties, etc. but,

due to lack of a suitable purification process to preserve protein functionality,

fish protein has been lacking in the rapidly growing protein ingredient and health

markets. Retaining the functional properties through preservation and processing


operations is therefore of great importance. The functional properties of proteins

are defined as `those physical and chemical properties which affect the

behaviour of proteins in food systems during processing, storage, preparation

and consumption' (Kinsella, 1976). The sensory properties of foods result from

interactions between several functional ingredients. The physical and chemical

properties that determine protein functionality include the size and the shape of

the proteins, the charge and the distribution of charge and the flexibility as well

as the ratio between the hydrophobicity and the hydrophilicity. Handling,

processing and storage of the raw materials will all affect the functional

properties and it is therefore important to both characterise the functional

properties of the raw material and find out how the different processing steps

will affect these properties.

Fish is regarded as an excellent source of high-quality protein, particularly

the essential amino acids lysine and methionine. Comparison of PER (protein

efficiency ratio) from cod muscle and cod by-products shows that fish by-

products have a high content of essential amino acids and can be used to produce

products with high nutritional value (Shahidi, 1994). Hydrolysates also have a

high chemical score and the amino acid composition is generally similar to that

of the raw material, except for content of sulphur amino acids, histidine and

tryptophan which are affected during hydrolysis (Quaglia and Orban, 1987;

Shahidi et al., 1995; Kim et al., 1997). The content and properties of the proteins

in marine by-products vary with regard to species, fishing ground and season. In

order to increase the utilisation of the protein fractions, knowledge about these

variations is necessary.

Seasonal differences in the amount of different by-product fractions were

found, with a higher proportion of viscera during autumn and spring (Falch et

al., 2006b). The Gadidae species caught in these waters spawn in the first six

months of the year (January to June) and the proportion of gonads will therefore

be highest during these months. The proportions of roe were, as expected,

significantly higher in the autumn and spring. Higher proportions of roe were

generally found in Gadidaes from Iceland and Ireland compared to Gadidaes

from Norway. During the spawning season, the roe made up 2.2% of the round

weight of fish on average. The lowest ratio of skin was found in spring and the

highest in autumn for cod species from Icelandic waters (Thorarinsdottir et al.,

2004).

Also the chemical composition of the by-products varies according to

species, season, fishing grounds and growth conditions. The protein content in

by-product fractions from five cod species collected at three different fishing

grounds and three different seasons was analysed. Total protein content was

highest in the cut-off fraction and lowest in the viscera. All fractions showed

variation in protein content with fishing ground, species and season. Amount of

water-soluble protein was highest in liver and cut-offs and lowest in the viscera;

the low content in viscera is due to the high degree of degradation in this

fraction. The amount of free amino acids was generally lowest in the liver and

highest in the viscera. The degree of hydrolysis was lowest in the cut-offs and


highest in the viscera. In some liver samples a high degree of hydrolysis was

also found (Sùvik, 2005). For cut-offs an inverse relationship was found between

water and protein content (Thorarinsdottir et al., 2004); a similar relationship

existed for roe.

In carp by-products seasonal differences in both the amount and properties of

the proteins were found for both wild and farmed carp. The amount and

properties of the proteins also varied between wild and farmed carp by-products

(Bukovskaya, unpublished results). The protein content in cut-offs and gonads

were higher in the wild than in the cultured carp. During summer, the content of

water-soluble proteins did not differ significantly between cultured and wild

carp. In winter a substantial increase of water-soluble proteins content in

intestine of wild carp was observed. Liver had the highest content of free amino

acids followed by viscera and cut-offs, a substantially higher content of free

amino acids was found in the liver and viscera of cultured carp compared to wild

carp. This reflects the influence of the feeding on the composition not only in the

fish fillets but also in the by-products.

For products such as fish mince and surimi, the water-holding capacity and

the gelling properties which determine the textural attributes of the products are

important quality parameters (Venugopal and Shahidi, 1995). Knowledge of

how to retain these properties during storage and processing is therefore

important. Seasonal variations in water-holding capacity in cut-offs from cod

species was noted, with a lower water-holding capacity found in autumn, except

for small cod, tusk and haddock. Minced meat is less stable than intact muscle.

By-products from filleting have the same good quality as the main products

(fillets), but unfortunately are not always treated in the same way, resulting in a

rapid loss in quality. If the by-products are intended for use in food, the frames

and cut-offs should be stored at 0ëC (éines et al., 1995). Freezing of the raw

material will generally lead to loss of both water-holding capacity and gel

forming ability, but freezing of the cut-offs/frames may result in smaller

reduction in these parameters than freezing of mince. Both the freezing tem-

perature and time as well as thawing conditions may influence the loss of

functional properties.

Gildberg (1993) found that the sauce fraction develops much faster if fresh

intestines are used than if the material has been freeze-stored. The reason for this

is not clear, but it is well known that freeze-storage of fish material implies a

tougher tissue structure that may be less susceptible to digestion by enzymes.

Another possible explanation is that bursting of lipid cells during freezing and

thawing involves formation of a stable lipid-protein, emulsion which hinders a

fast phase separation.

The quality of by-products limits the possibilities for utilisation of the raw

material, and enzymatic activities along with microbial degradation are the most

important factors determining raw material quality. Variations in enzymatic

activities and therefore quality are important when finding possible uses for the

different by-product fractions. In processes utilising by-products, such as pro-

duction of fish protein hydrolysates (FPH), minced and surimi-based products,


extraction of lipids, enzymes and/or other bioactive compounds, the activity of

the endogenous enzymes in the raw material needs to be controlled and

knowledge about how these activities change according to temperature is

important.

Significant variations in quality parameters and enzymatic activities in by-

products from cod according to species, seasons and fishing grounds have been

demonstrated (Sùvik and Rustad, 2004, 2005a, 2005b). The highest overall

proteolytic activity in the by-product fractions was found in viscera at pH 3

(35ëC). Cut off and liver fractions also showed maximum activity at pH 3, 35ëC

and 50ëC, respectively. Maximum median proteolytic activity in viscera is

approximately 20 times higher than that in liver and 250 times higher than that

in cut-off. Species affected the general proteolytic activity (pH 5 and 7), and

activity of trypsin and chymotrypsin in viscera from cod species. Trypsin and

chymotrypsin are the major proteolytic enzymes active at neutral pH in the

viscera of cod species. Viscera from Atlantic cod, saithe and haddock had a

higher proteolytic activity compared to tusk and ling. In cut-off and liver,

general proteolytic activity (pH 5 and 7), activity of cathepsin B and collagenase

varied according to species. Variations according to season were found in the

activity of trypsin, chymotrypsin, elastase, cathepsin B, collagenase and lipase

(pH 7) in the viscera from Atlantic cod. The results clearly indicated that viscera

samples from the Icelandic sea had lower enzymatic activities in April±June

compared to the other seasons. Cut-off samples from Icelandic Sea also had

lower cathepsin B and collagenase activity in April±June compared to February±

March; fishing ground influenced general proteolytic activity as well as activity

of trypsin, chymotrypsin, elastase, cathepsin B, collagenase and lipase. Heat

stability of the enzymes is also important and it has been shown that trypsin,

chymotrypsin and cathepsin B in crude extracts from viscera from cod species

lost 50% of initial activity after incubation for 10 min at 60ëC, while elastase,

collagenase and lipase lost 50% of their initial activity after incubation for 10

min at 50ëC. The ratio between proteolytic activities in different by-products are

similar in carp and cod and can be described as: INTESTINE > LIVER > CUT-

OFFS (Sùvik et al., 2004). In carp the difference in the levels of proteolytic

activity in intestine, liver and cut-offs are less significant than in cod. Both in

carp and cod, the total proteolytic activity in the intestine was highest at pH3

followed by pH 7 and lowest at pH 5. For cut-offs this correlation was similar

between species: pH3 > pH5 > pH7 (Sùvik et al., 2004).

The presence of proteolytic enzymes in the viscera of fish had a significant

influence on the production of hydrolysates (Shahidi et al., 1995; Slizyte et al.,

2005, 2006). When producing hydrolysates from capelin, endogenous enzymes

alone gave a protein recovery of approximately 23% after 4 h at pH 3.0 (Shahidi

et al., 1995). The hydrolysis of ground capelin by endogenous enzymes

enhanced the overall extraction of fish protein at both acid and alkaline pH,

since both acid and alkaline proteases are present in fish muscles and viscera.

Pre-digestion of fish mince prior to the addition of exogenous enzymes might

enhance the yield of protein extraction, however, autolytic enzymes may also


bring about undesirable changes in the products, as it may be difficult to control

the degree of hydrolysis during storage and processing. Furthermore, autolytic

protease activity varies from species to species and depends on the season of

harvest. Therefore, properties of functional protein hydrolysates may vary

greatly under the same processing conditions. The chain length of the peptides

formed during the hydrolysis process is important: this is one of the parameters

determining both the functional and the organoleptic properties of the

hydrolysate (Slizyte et al., 2005, 2006; Dauksas et al., 2004).

The high collagen content in skeleton, fins and skin which constitute the main

part of whitefish òffal' can become a problem when collagen turns into sticky

fish glue in the production of fishmeal (Sikorski et al., 1984). In the production

of fishmeal and oil from fish òffal' problems can arise due to gelatinisation of

collagen into fish glue causing problems in concentration of stick water and

drying of fishmeal. The content of collagen is thought to contribute significantly

to the viscosity of the stickwater.

Common problems connected with fish gelatin from cold water species are

low gelling and melting temperatures and low gel modulus. The differences in

physical and rheological properties between mammalian gelatine and gelatine

from cold water species are due to a lower content of the amino acids proline

and hydroxyproline (Sikorski et al., 1984). The quality and specific application

of the extracted collagen and or gelatine is highly related to their functional

properties and purity. There is also a market for non-gelling gelatine, which has

a potential in the cosmetic industry as an active ingredient (i.e., shampoo with

protein). Using fish collagen and gelatine generates new applications as a food

ingredient, because it has properties different from mammalian collagen and

because it can be used in food where mammalian gelatine from a cultural or

safety point of view is not wanted.

Generally, the enzymatic activities in by-products are high and it is therefore

important that they are rapidly and continuously stored at cold temperatures. It is

also important that the by-products are treated as valuable raw material in the

same way as the main product, starting from the time the fish is caught.

Separating the different by-product fractions, and fractions from the Lotidae

family from the Gadidae family, will ensure that fractions with low enzymatic

activity are not contaminated by fractions with higher enzymatic activities.

Viscera from Atlantic cod caught in the Icelandic sea and from fish from the

Gadidae family may be better raw materials for protein hydrolysates, especially

if autolytic or semi autolytic processes are used or for extraction of proteolytic

enzymes. Viscera samples from Atlantic cod caught in the Barents Sea have

lower lipase activity and may therefore be a better raw material for extraction of

marine oils compared to samples from the Icelandic Sea. Cut-off from ling and

saithe, Atlantic cod caught in the Barents Sea (April±June and October±

December) and Icelandic Sea (April±June) may be used for minced products or

surimi-based products. Cut-off from haddock, Atlantic cod caught at the south

coast of Ireland and in the Icelandic Sea (February±March) is a poor raw

material for these kinds of products due to the high activity of cathepsin B


(Sùvik, 2005). Results from this latter study also suggest that viscera should be

kept separately. Contamination of liver and cut-off fractions with viscera

fractions should be avoided.

1.4 Implications for by-products valorisation

To be able to produce value-added products from fish by-products for human

consumption, it is necessary that the by-products are treated as a valuable raw

material and the name should perhaps be ùpgraded' to rest of raw material.

Efficient on-board handling and sorting of specific by-products are important to

reduce the rate of enzymatic degradation and microbial spoilage. The enzymatic

activities in by-product fractions are high and it is important that they are rapidly

and continuously stored at low temperatures. Rapid sorting and separation of

different by-product fractions so that fractions with high enzymatic activity do

not contaminate the fractions of low activity is also important.

It is also possible to take the fish onshore ungutted and gut the fish in efficient

processing lines onshore. In a Norwegian study, it was found that cod could be

landed ungutted and gutted within 12 hours after catch without any negative

effects on the quality of the fish or the by-products. Spawning cod with a low

degree of filling in the stomach/intestines could be kept ungutted for up to 48

hours. Heavily feeding cod should not be stored ungutted for more than 12

hours. The quality of by-products (liver, gonads and stomach/intestine) from fish

landed ungutted was found to be better compared to controls gutted immediately

after catch and stored in bags on ice for up to 48 hours after catch (Akse et al.,

2002).

There is a need to develop methods to preserve different by-product fractions.

Methods of preservation and utilisation depend upon the application of the by-

product. It is important to specify the shelf life at different levels of processing

and product quality changes during storage. Potential preservation methods may

include chilling, freezing, drying, fermentation and use of preservatives such as

natural antioxidants. These methods should be tested for bulk- and final products

and the different fractions (fat, protein/gelatine) from the most valuable by-

products. The final products should be evaluated for their usage in food

applications.

More research is also needed to develop processing methods to extract the

fractions/biomolecules of interest. The processes developed should be able to

handle variations on the basis of season, habitat and species. The processing

steps should be optimised both regarding yield and product quality, but also

about processing costs. Products should have as high quality as possible at the

lowest possible price.

In order to evaluate possible applications for products and conservation

techniques of the rest of raw material, it is important that the raw material is

characterised based on its chemical composition and enzymatic activity.

Characterisation of variation in chemical compositions has mainly been carried


out on fish muscle. Limited work has also been done on the chemical charac-

terisation of some fishery by-products. To obtain a total picture of the

applications and potential for new use, detailed characterisation including

seasonal, species and habitat variations is still needed.

For achieving profitable utilisation of by-products from the fish industry the

final products require market interest. Knowledge about quality and composition

is a necessity. The products and the processes to produce these must be

economically viable. There is still great potential in utilisation of these fractions

and a need for further investigations. There is also a need for environmental

restrictions and economic incentives to increase the utilisation of marine by-

products.

1.5 Future trends

The available catch from marine fisheries is not expected to rise in the future.

Increase in quantity of fish will probably come from aquaculture. The increase

in aquaculture will result in a demand for more feed. This will have to come

from agriculture, from harvesting at lower trophic levels (krill and plankton) and

also from use of marine by-products, especially as a source of marine oil.

Increasing amounts of farmed fish will be a source for fresh by-products with

better possibilities for sorting and preservation and utilisation for value-added

products/products for human consumption.

The majority of by-products utilised today are used for feed (fishmeal and oil,

fish silage). The largest potential for value addition is in increasing the amount of

by-products used for human consumption, either as food ingredients or as

nutraceuticals. The need to use more of the by-products for human consumption

will demand that this raw material is treated as a valuable raw material starting

onboard the fishing boat or at the processing plant, resulting in rapid sorting and

storage/preservation or processing into bulk products for later processing. There

is also a need for national and international authorities to provide stronger

regulations so that wasting of this valuable raw material is not possible. Enzymes

or other bioactive molecules found in by-products will usually be found in very

low concentrations and production of these molecules will therefore most likely

be through the use of microorganisms via recombinant technology.

1.6 Sources of further information and advice

BREMNER A H ed (2002), Safety and quality issues in fish processing, Boca Raton, FL,

CRC Press.

SIKORSKI Z E, SUN PAN B and SHAHIDI F eds (1995), Seafood proteins, New York, Chapman

& Hall.

VANNUCCINI S (2003), Overview of fish production, utilisation, consumption and trade

(based on 2001 data). FAO, Fisheries Information, Data and Statistics Unit.


VOIGT M N and BOTTA J R eds (1990), Advances in fisheries technology and biotechnology

for increased profitability, Lancaster, PA, Technomic Publishing.

Websites:

http://www.rubin.no/eng/ (visited 15 June 2005).

http://www.dced.state.ak.us/oed/seafood/by_products.htm (visited 15 June 2005).

http://www.Fishbase.org (visited 15 June 2005).

http://www.intrafish.com/laws-and-regulations/report_bc/vol3-d.htm (visited 15 June

2005).

http://www.globefish.org (visited 15 June 2005).

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2.1 Introduction to fish lipids

Lipids constitute between 10 and 40% of the total human diet. They play a

pivotal role in terms of flavour and palatability of food products and, in addition,

their presence affects general physical properties of foods. Furthermore, lipids

are an important source of essential fatty acids and serve as carriers of fat

soluble vitamins. Renewable marine resources have always been important

origins of the human food supply. In recent times, these resources have been,

and continue to be, overexploited. This has resulted in global regulatory

organizations restricting numerous fishing practices, while at the same time,

emphasizing a requirement to do more with fishery by-products in order to

maximize product utilization. Recent research on the beneficial health effects

associated with the long chain polyunsaturated omega-3 fatty acids and more

specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has

led to an increase in seafood consumption and a renewed interest in fish lipids,

particularly those present in the under-utilized by-product landing.

Lipids are soluble in organic solvents such as acetone, ethanol and

chloroform but insoluble in water. They include fatty acids, fats, oils, waxes,

phospholipids, glycolipids, steroids and some vitamins. Lipids are defined on the

basis of a specific physical property ± namely, solubility. They do not have any

common structural feature (Freemantle, 1995). Naturally occurring fats and oils

are the esters formed by propane-1,2,3-triol (also known as glycerol or

glycerine) and fatty acids. They are known as triacylglycerols. Fatty acid is a

general name for a monobasic aliphatic carboxylic acid, often abbreviated as

RCOOH. Fatty acids may be loosely divided into three categories: saturated,

2

Physical and chemical properties of lipidby-products from seafood wasteJ. P. Kerry and S. C. Murphy, National University of Ireland,Cork, Ireland

unsaturated and branched or cyclic (Freemantle, 1995). Naturally occurring

branched or cyclic fatty acids are rarely found in foods, whereas, saturated and

unsaturated fatty acids are found in abundance (Freemantle, 1995; Gunstone and

Norris, 1982). The major fatty acid classes in animal fats and fish oils are

saturated and unsaturated fatty acids (Gunstone and Norris, 1982).

Saturated fatty acids have the general formula CH3 (CH2)nCOOH, where n

may range from 2 to over 20 carbon atoms. When n is low the acid is known as a

short chain fatty acid and when n is high the acid is known as a long chain fatty

acid (Freemantle, 1995). Long chain palmitic acid CH3(CH2)14COOH (C16:0)

has been shown to be a predominant saturated fatty acid in fish species

(Freemantle, 1995; Moreira et al., 2001; Mendez and Gonzalez, 1997; Nettleton

et al., 1990; Watanabe et al., 1995).

Unsaturated fatty acids contain at least one double bond in the chain and may

range in length from short chain to long chain fatty acids (Freemantle, 1995).

The unsaturated fatty acids may be subdivided into monounsaturated or

polyunsaturated fatty acids (PUFA). Monounsaturated fatty acids (MUFA)

contain one double bond and PUFA contain more than one double bond

(Gunstone and Norris, 1982). One of the most abundant fatty acids in all plants

and land animals is oleic acid (Freemantle, 1995; Gottenbos, 1985). Oleic acid

CH3(CH2)7CH=CH(CH2)7COOH (C18:1) has been shown to be a predominant

MUFA in fish species (Freemantle, 1995; Moreira et al., 2001; Mendez and

Gonzalez, 1997; Nettleton et al., 1990; Watanabe et al., 1995). Most PUFA

contain two or more double bonds (Gunstone and Norris, 1982). PUFA,

especially the n-3 and n-6 fatty acids have been shown to be essential for healthy

human development and since they cannot be synthesized by the body, they

must therefore be supplemented in the diet (Bjerve et al., 1989). The essential

omega-6 fatty acid, from which other omega-6 fatty acids like arachidonic acid

can be synthesized, is called linolenic acid. The corresponding essential omega-

3 fatty acid, from which other omega-3 fatty acids such as eicosapentaenoic acid

(EPA) and docosahexaenoic acid (DHA) are made, is referred to as �-linolenic.Arachidonic acid and EPA are responsible for the further production of the

metabolites called eicosanoids, which play a very important role in different

reactions in the vascular system and other bodily functions such as the immune

system (Leaf and Weber, 1988; Simopoulos, 1991). PUFA particularly EPA and

DHA, have been shown to be abundant in fish lipids (Alasalvar et al., 2002;

Aursand et al., 1994; Hardy and Keay, 1972; Mendez and Gonzalez, 1997;

Nettleton et al., 1990; Njinkoue et al., 2002; Osman et al., 2001; Saglik and

Imre, 2001; Trigari et al., 1992; Watanabe et al., 1995).

Although, it is generally recognized that PUFA composition may vary

among species of fish, when choosing fish for human consumption little

attention has been paid to the PUFA composition. All fish are considered to be

of similar nutritional value, and selection is chiefly based on availability,

freshness, flavour, colour, odour and texture (Hearn et al., 1987). Research has

shown that freshwater fish have lower contents of PUFAs than equivalent

marine fish species (Vlieg and Body, 1988). Osman et al. (2001) suggested that

Physical and chemical properties of lipid by-products from seafood waste 23

the difference in PUFA content may be attributed to the fact that freshwater fish

feed largely on vegetation and plant materials, whereas marine fish staple diets

are mainly zooplankton rich in PUFAs. The omega-3 fatty acids of marine

origin are formed in the chloroplast of marine plants that form part of the

phytoplankton or algae consumed by fish. The main source for novo synthesis

of n-3 fatty acids are marine autotrophic bacteria, algae and protozoa which

constitute the phytoplankton and the zooplankton (Iwamoto and Sato, 1986;

Seto et al., 1984). Fish, higher in the marine food chain, incorporate the n-3

PUFA and further elongate them to 20 and 22 carbon atom fatty acids

countering four, five and up to six double bonds by the action of specific

desaturases. Thus fish will concentrate EPA and DHA as triglycerides, mainly

in adipose tissue and in the fat of muscle and visceral organs (Brockenhorff et

al., 1963). Therefore, the greater the fat content of fish, the higher the content

of n-3 fatty acids in the fish.

2.2 Health benefits associated with fish lipids

Fish lipids are known to be rich in polyunsaturated fatty acids (PUFA), espe-

cially the n-3 PUFA family of linolenic acid (C18:3) and its derivatives such as

eicosapentaenoic acid (EPA/C20:5), docosapentaenoic acid (DPA/C22:5) and

docosahexaenoic acid (DHA/C22:6) (Sargent et al., 1989, 1999). Interest in the

high content of these long chain polyunsaturated omega-3 fatty acids (EPA and

DHA) grew during the 1970s and 1980s with the observation that the mortality

of coronary heart disease was low in Eskimos who consume a large amount of

seafood. This was first proposed by Bang and Dyerberg (Bang et al., 1971;

Dyerberg and Bang, 1979) who studied the diet of Greenland Eskimos who are

known to have an exceptionally low frequency of such diseases. Medical

investigations have shown that the intake of oil containing high contents of these

acids have beneficial effects on human health and, more particularly, lower

susceptibility to coronary heart disease (Abeywardena and Head, 2001; Baybutt

et al., 2002; Chagan et al., 2002; Fantoni et al., 1996; Herrera, 2002; Love,

1997; Uauy and Valenzuela, 2000; Rafflenbeul, 2001).

These fatty acids play a vital role in human nutrition, disease prevention and

health promotion (Horrocks and Yeo, 1999; Kinsella, 1986; Simopoulos, 1991;

Ulbricht and Southgate, 1991). Long chain n-3 PUFA cannot be synthesized by

humans and must be obtained from the diet. The consumption of fish has been

linked to increased health benefits, such as reduced risk of coronary heart

disease attributable to the PUFAs in fish oils (Burr, 1992; Gordon and Ratliff,

1992; Hearn et al., 1987; Kinsella, 1986; Morris and Culkin, 1989; Paige et al.,

1996). A preventative and/or curative effect also linked to the PUFAs in fish oils

has been reported for arterial hypertension (Millar and Waal-Manning, 1992),

human breast cancer (Rose and Connolly, 1993), inflammatory diseases (Belluzi

et al., 1993: James and Cleland, 1996), asthma (Dry and Vincent, 1991; Hodge

et al., 1996) and disorders of the immune system (Kenneth, 1986; Levine and


Labuza, 1990). Links between these PUFAs and health benefits have stemmed

the recent increased interest in fish and fish oils arising from such PUFA

content. Fish liver has long been a source of oils (e.g., cod liver oil) for the

prevention of disorders associated with vision and growth (Njinkoue et al.,

2002). Results of clinical and epidemiological research suggest that EPA and

DHA acids, found primarily in fish and seafoods, have extremely beneficial

properties for the prevention of human coronary artery disease (Leaf and Weber,

1988). The beneficial effects of such lipids have been attributed to an increased

ratio of omega-3 and omega-6 PUFAs in human blood lipids and cell mem-

branes. Extracted fish fractions with high omega-3/omega-6 ratios have been

shown to have the greatest health benefits (Hearn et al., 1987).

Adults are recommended a daily intake of 350mg of EPA/DHA, which

corresponds to approximately 30 g fish/day of mixed fish species (The Danish

Ministry of Health). Generally, marine organisms are very rich in EPA and

DHA, especially oily fish like herring, mackerel and salmon. For those who

prefer not to eat fish, they may use dietary supplementation of marine oil in the

pure oil form, oil enriched food or nutritional products, or capsules (Andersen,

1994). White bread enriched in n-3 fatty acids in the form of gelatine-coated fish

oil and marketed in Denmark since 1990 under the name of Òmega Bread', was

found to be a reliable and significant source of higher n-3 PUFAs such as EPA

and DHA (Nielsen, 1992). Kolanowski et al. (1999) suggested that it may be

possible to increase the nutritional quality of food products by the addition of n-

3 PUFA, resulting in increased EPA and DHA content in the diet. More recent

research indicates that a high intake of monounsaturated fatty acids from marine

mammals may also contribute to protection against coronary heart diseases

(Elvevoli et al., 1990).

2.3 Fatty acids found in fish muscle

Lipids are basic components of marine organisms. Although present in all

tissues, they are concentrated mostly in the subcutaneous fatty layer of marine

mammals and fatty fish, in the liver of lean fish, in muscle tissue and in mature

gonads (Sikorski et al., 1990).

Kiessling et al. (2001) measured the fatty acid composition in the red and

white muscle, viscera and tissue from the abdominal wall of rainbow trout and

reported that the most important factor governing fatty acid composition in fish

tissue, pending changes in feeding, is total lipid content. The distribution of

lipids in fish muscle has shown to vary with species, type of muscle and muscle

location (Ackman, 1967). Therefore, when comparing population and samples,

it is necessary to specify the exact area of sampling when determining the

muscle lipid content of fish species (Bell et al., 1998). The considerable

variation in lipid content among the fish population reported in many studies

may be attributed to factors such as season, temperature, diet, age, size and sex

(Alasalvar et al. 2002; Aursand et al., 1994; Chen et al., 1995; Jahncke et al.,


1988; Kherunnisa et al., 1996; Suzuki et al., 1986; Waagbo et al., 1993). Arts et

al. (2001) and Shirai et al. (2002) reported that the percentages of PUFA such as

DHA and EPA in fish muscle are dependent on diet.

Many authors have determined the fatty acid composition in the fillets of fish

species. Palmitic acid (C16:0) and oleic acid (C18:1) have been shown to be the

predominant saturated and monounsaturated fatty acids, respectively, in the

fillets of southwest Atlantic hake (Mendez and Gonzalez, 1997), catfish

(Nettleton et al., 1990), Brazilian brycon freshwater fish (Moreira et al., 2001)

and in bonitio, caught off the Japanese coast (Watanabe et al., 1995), whereas,

EPA and DHA were shown to be the predominant PUFAs.

Njinkoue et al. (2002) reported C16:0 as the main fatty acid in Sardinella

maderensis, Sardinella aurita and Cephalopholis taeniops, species from the

Senegalese coast, and they also found high concentrations of PUFAs, including

EPA and DHA. Njinkoue et al. (2002) reported that the percentages of PUFA

found in such fish species were very similar to those species used commercially

as sources of PUFA such as herring, sardines and menhaden. Osman et al.

(2001) determined the fatty acid composition of selected marine fish caught in

Malaysian waters. They reported that all fish samples showed a much higher

content of n-3 PUFA when compared to standard menhaden oil. European

anchovy and European pilchard fish species from Turkey were discovered to be

good dietary sources of n-3 PUFA (Saglik and Imre, 2001).

Commercial fish oil is usually composed of over 90% triacylglycerols, each

usually containing three different fatty acids (Hjaltason, 1990). An additional

8% consists of mono- and di-acylglycerols and other lipids such as phospho-

lipids. The unsaponifiable portion that accounts for an additional 1.0% to 2.0%

consists principally of sterols, glyceryl ethers, hydrocarbons and fatty alcohols,

along with the fat-soluble vitamins A, D and E (Hjaltason, 1990).

Marine oils are produced from the body of fatty fish, livers of lean fish, as

well as from blubber of marine mammals. Marine oils form a significant

proportion (2±3%) of the world's edible oil production. The relative amount of

EPA and DHA varies from 5±20% and 3±26%, respectively, of fatty acids

(Kamal-Eldin and Yanishlieva, 2002).

2.4 Fatty acids found in fish by-products

Renewable marine resources are important origins of human food supply

(Shahidi et al., 1991). Annually, over 100 million tonnes of fish are harvested

worldwide (Venugopal, 1992), however, up to 25% of marine catches are dis-

carded (Valdimarsson and James, 2001). Fish is a rich source of easily digestible

protein that also provides polyunsaturated fatty acids, vitamins and minerals for

human nutrition. Increasing concern worldwide about the use and over

exploitation of natural seafood resources and in particular, the dwindling stocks

of commercially important fish species has led to greater utilization of fish

landings, and waste use has become an important issue for the seafood industry


(Morrissey et al., 2000; Venugopal, 1997). Processing discards from fisheries

account for as much as 70±80% of the total weight of catch after filleting, and

these discards offer a myriad of possibilities in extracting valuable components

and have a huge potential for further use.

To date, many studies have measured the fatty acid composition of fish

muscle from species such as hake (Mendez and Gonzalez, 1997), blue whiting

(Dapkevicius et al., 1998), sea bass (Trigari et al., 1992), mackerel (Hardy and

Keay, 1972), turbot (Ruff et al., 2002a,b), halibut (Ruff et al., 2002b,c) and

salmon (Ackman and Takeuchi, 1986; Aursand et al., 1994; Katikou et al.,

2001), however, little work has focused on extracting these lipids from by-

products for use as potential functional ingredients. Watanabe et al. (1995)

measured the fatty acid composition in the gills, spleen, heart, pyloric caecum,

stomach and gonads in bonitio, caught off the Japanese coast. They reported that

C16:0, C16:1, C18:1, C20:5 and C22:6 were the predominant fatty acids

determined and that DHA was the overall dominant fatty acid accounting for

25% or more of the total fatty acids. Njinkoue et al. (2002) recorded the fatty

acid composition in the liver and skin of three fish from the Senegalese coast, in

addition to that of the muscle. They determined that C16:0 was the dominant

fatty acid in the skin of all samples and C18:1 was dominant in the liver of

Sardinella maderensis and Sardinella aurita. They also reported high PUFA

content in these by-products as well as in the muscle of the three fish species

studied. Aursand et al. (1994) reported EPA and DHA levels were higher in liver

than in the edible belly flap region of Atlantic salmon. Sun et al. (2002) reported

that viscera from salmon was a good source of EPA and DHA and by using

microbial lipases the concentration of these omega-3 PUFAs could be doubled.

Brockenhorff et al. (1963) reported that EPA and DHA are stored as

triacylglycerols, mainly in the adipose tissue and in the fat of the visceral

organs. Satoh et al. (1989) suggested that catfish can accumulate n-3 fatty acids

in liver lipids. Chantachum et al. (2000) recovered oil from precooked and non-

precooked skipjack tuna heads by wet reduction. The study found that oil

obtained from non-precooked tuna heads gave superior yield and higher quality

compared to the oil from precooked tuna heads. However, DHA content was

higher in the crude oil extracted from precooked tuna heads. Dauksas et al.

(2005) recovered oil fractions from enzymatic hydrolysis of combinations of

viscera, backbone and digestive tract of cod.

While many of the studies described above are limited in scope and magni-

tude, they all share the important commonality of demonstrating the potential

that exists in terms of extracting valuable lipid contents, and possibly other and

more significant components, from the by-product remains of fish landings.

Clearly, more concerted research is required in order to fully elucidate the true

potential that fish wastes, in the form of fishery by-products, have in terms of

their contribution of extractable bulk and fine chemicals for commercial

utilization.


2.5 Factors affecting the fatty acid composition of fish andtheir associated by-products

It has been reported that the type and amount of fatty acids in fish tissue are

influenced by diet, and other factors such as size, age, reproductive status,

geographical location and season have been shown to affect fat content and

composition of fish muscle (Ackman, 1989; Nettleton 1985; Saito et al., 1999).

Fishing region has been shown to affect the condition, diet and composition of

fish species (Du Buit, 1995; Gieseg et al., 2000; Ratz and Lloret, 2002; Shirai et

al., 2002). Ratz and Lloret (2002) suggested that the condition of cod stock in

the North Atlantic may be affected by the different temperature regimes of their

habitats.

Mendez and Gonzalez (1997) reported seasonal variation in fatty acid

composition in hake. Similarly, Malone et al. (2004) also reported variation in

fatty acid composition of by-products liver, viscera and cut-off of cod, ling,

saithe and haddock due to season. Lipid content of fish is dependent on breed,

food supply and other factors beside season (Hardy and Keay, 1972). According

to Viarengo et al. (1998) and Kiessling et al. (2001), dietary intake is the most

important factor governing fatty acid composition and �-tocopherol levels.

Thererfore, it is necessary to review how fishing region influences the dietary

intake of fish species.

Du Buit (1995) suggested that variations in the diet of cod in the main stocks

of the North Atlantic may be a function of temporal or geographic changes, and

the diversity of prey increases as the cod move from northern to southern

regions. Prey such as pelagic species including krill, capelin and herring are

mostly dominant in the cold waters of the Arctic Ocean (Mehl, 1986; Zamarro,

1985), off Iceland (Palsson, 1983), and in the fjords of northern Norway (Dos

Santos and Falk-Petersen, 1989; Klemetsen, 1982). Trisopterus, and to a lesser

extent, blue whiting, are the most important prey in more temperate waters off

the Faroe Isles, the British Isles and the Norwegian Deep (Bergstad, 1991; Du

Buit, 1995; Rae, 1967) whereas, such species are in less abundance in the North

Sea (Daan, 1973). Other prey, such as benthic species are in maximum

abundance in the southern North Sea (Cramer and Daan, 1986; Daan, 1973), the

Irish Sea (Armstrong, 1982; Brander, 1981) and the Celtic Sea (Du Buit, 1995).

In addition to both pelagic- and benthic-based diets, all proportions of nektonic

and bottom-dwelling or even burrowing prey are available for feeding by cod

species, depending on location.

Few authors have compared the fatty acid profiles of wild fish species caught

in different sea fishing regions. Many authors have compared the fatty acid

compositions of wild and cultured fish species such as catfish (Shirai et al.,

2002), sea bass (Alasalvar et al., 2002; Orban et al., 2002) and yellowtail

(Arakawa et al., 2002) and generally the wild fish species tended to have higher

n-3 PUFA content than their cultured counterparts. Many of these authors

attributed the differences in PUFA content to the dietary intake of the fish

species. Trigari et al. (1992) compared the fatty acid compositions of warm and


cold adapted sea bass and overall the fatty acid composition did not indicate any

increase in unsaturation in response to temperature. Shirai et al. (2002)

compared the lipid content between cultured Thai and Japanese catfish and

reported higher lipid content in the dorsal muscle of Thai catfish when compared

with Japanese catfish. They suggested that lipid content may be affected by

species and/or dietary intake. They also suggested that, in general, high lipid

contents in muscle may result in an increase in the percentage of triacylglycerol,

the storage lipid.

Dey et al. (1993) and Wodtke (1981) have suggested that saturated and

monounsaturated fatty acids are generally abundant in fish from warm and

temperate regions, whereas PUFAs are more abundant in fish from colder

regions. Olsen and Skjervold (1991) studied the effect of geographical location

on n-3 fatty acids in wild Atlantic salmon and suggested that the habitat and

season of capture may produce broad changes in the omega-3 content of fish oil,

even among the same fish species. As the temperature of the water decreased, as

in the polar regions, fish showed an increase in PUFAs in their tissues, in order

to compensate for the reduction in the fluidity of their membranes due to low

temperature. When capture occurred in temperate regions where the seawater

was over 12ëC, the oil obtained after fish processing showed significant reduc-

tions in omega-3 content. By similar mechanism, the season of capture may have

influenced the omega-3 content of the same species (Olsen and Skjervold,

1991).

Czesny et al. (2000) compared the fatty acid composition between wild and

domestic sturgeon eggs. They showed that fatty acids C18:0 and C18:1 n-9 were

origin specific rather than species specific. They reported lower levels of C18:0

in wild fish eggs than in domesticated fish ova, whereas, C18:1 n-9 showed the

opposite trend. Czesny et al. (2000) demonstrated that sturgeon environment

played an important role and markedly influenced fatty acid composition of their

eggs.

Research into measuring natural levels of �-tocopherol in wild fish species

and the effect fishing region has on these levels is only beginning to emerge in

the literature. Ruff et al. (2002c) measured the natural �-tocopherol levels in the

fillets of wild turbot caught off the south coast of Ireland and in Atlantic halibut

caught off the north-west coast of Iceland. They reported fillet �-tocopherolcontent was nearly twice as high in Atlantic halibut than in turbot, and suggested

this could be due to differences in the natural diet since they came from different

waters. Gieseg et al. (2000) compared the �-tocopherol levels in the plasma of

two Antarctic and two temperate water fish species. They showed that the

plasma from both Antarctic fish species had �-tocopherol concentrations 5±6

times higher than those found in the two temperate water fish species. They

attributed these varying levels of �-tocopherol to the diet obtained by the fish

species in their specific habitats. Yamamoto et al. (1999) isolated a new vitamin

E, �-tocomonoenol from salmon eggs having an unusual methylene unsaturation

at the isoprenoid-chain terminus. This new vitamin E was designated `marine-

derived tocopherol' (MDT) for its exclusive occurrence in marine organisms.


High concentrations of MDT were found in fish inhabiting cold-water environ-

ments suggesting a specific metabolic function in low temperature adaptation

(Yamamoto et al., 2001). Dunlap et al. (2002) examined the vitamin E content

of tissues from Antarctic notothenioid fish and extracts of Antarctic krill and

reported very high levels of �-tocopherol in these species, which were accom-

panied, by high levels of MDT. They suggested that these high levels of �-tocopherol in Antarctic fish species might be attributed to the cold-water

environment.

Seasonality of fish capture has been shown to affect nutritional composition

including fatty acid profiles and �-tocopherol levels. Mendez and Gonzalez

(1997) reported seasonal variation in the condition factor, the total non-volatile

nitrogen and the lipid contents in hake species. These values decreased during

the spawning period for hake species and they attributed this to the use of

muscle lipids and protein as energy reserves during these reproductive months.

Seasonal variation in the fatty acid composition and total fat content has been

reported for Cornish mackerel (Hardy and Keay, 1972), south-west Atlantic

hake (Mendez and Gonzalez, 1997), and in Japanese and Thai catfish (Shirai et

al., 2002). These authors have reported a decline in the total fat content and

percentage total fatty acids during the spawning season of these fish species.

Cordier et al. (2002) reported seasonal effects on the fatty acid composition of

tissue phospholipids in farmed sea bass. They observed major changes in

percentage phosphatidylethanolamine and phosphatidylcholine in all tissues

between February and March, and the phosphatidylethanolamine and

phosphatidylcholine ratio was drastically reduced at this time. They found that

these changes corresponded to the beginning of the spawning period of sea bass.

Both Refsgaard et al. (1998) and Hamre and Lie (1995) reported that the �-tocopherol levels of Atlantic salmon were affected by season. Hamre and Lie

(1995) reported that the �-tocopherol levels in the whole body of Atlantic

salmon fed diets unsupplemented with vitamin E, showed significantly higher

levels of �-tocopherol in May compared with January (Hamre and Lie, 1995).

These results were consistent with those recorded by Refsgaard et al. (1998) and

they suggested seasonally varying factors such as water temperature may have

influenced the �-tocopherol content. Nettleton et al. (1990) reported very low

levels of �-tocopherol in the fillets of Channel catfish and seasonal variation

was not detected, with little deviation in �-tocopherol levels recorded in

February, April and August. To date little research into the effect of season on

the natural �-tocopherol levels in wild fish species has been carried out and the

seasonal variation reported above is that for cultured fish species fed diets

supplemented with dietary �-tocopheryl acetate.

2.6 Deterioration of fish lipids

Oxidation of lipids in biological food systems adversely affects nutritional

quality, wholesomeness, safety, colour, flavour, and texture. Major negative


quality changes that occur during processing, distribution, and final preparation

of lipid-rich foods may be attributable to oxidation effects (Shahidi and

Wanansudara, 1992). Fish lipids are known to be rich in PUFAs, however, these

are highly vulnerable to oxidative deterioration, especially, during and after

processing of fish products (Aidos et al., 2002; Kamal-Eldin and Yanishlieva,

2002; Kulas et al., 2002; Jensen et al., 1998; Sargent et al., 1989; Yanishlieva

and Marinova, 2001). Lipid oxidation is a process by which molecular oxygen

reacts with unsaturated lipids to form lipid peroxides (Hawrysh, 1990; Lawson,

1995), and is catalysed by factors such as temperature, water activity, pH and

chemical environment (Ashie et al., 1996). Oxidation results in the production

of off-flavours and off-odours, as well as colour and texture deterioration. Gu

and Weng (2001) reported that oxidation of fatty acids found in oils may induce

aging and carcinogenesis. Other workers reported that oxidation may also

generate toxic compounds, which have the potential to affect health (Gurr,

1984).

In the living animal the ingestion and regeneration of antioxidants prevents

excessive oxidative deterioration of important biological components. Post

mortem, the protective systems become depleted and are unable to regenerate

and thus, oxygen may react with any of the biochemical components when

exposed to air (Ashton, 2002).

Post mortem lipid degradation proceeds mainly due to enzymatic hydrolysis.

Phospholipids are hydrolyzed most readily, followed by triacylglycerols, to

produce free fatty acids (lipolysis) (Sikorski et al., 1990; Ashton, 2002). Free

fatty acids oxidize readily, especially in the presence of enzymes. The develop-

ment of free fatty acids in different marine systems has been well documented

(Harris and Tall, 1994; Hwang and Regenstein, 1993; Ingemansson et al., 1995;

Kaneniwa et al., 2000; Miyashita and Takagi, 1986; Refsgaard et al., 1998).

Free fatty acids are not only important from the point of view of oxidation

products, but they have also been reported to have a direct sensory impact

(Refsgaard et al., 1998). Lipid oxidation is an autocatalytic chain reaction,

which takes place through four main stages: initiation, propagation, chain

branching and termination (Allen and Foegeding, 1981; Hultin, 1992). The

primary products of lipid oxidation, lipid hydroperoxides, are generally con-

sidered not to have a flavour impact. The volatile secondary oxidation com-

pounds, aldehydes and ketones, derived from breakdown of primary oxidation

compounds are responsible for rancid flavours and aromas. Some aldehydes and

ketones react further with compounds containing free amino groups, resulting in

oxidation products affecting fish colour and texture (Aubourg et al., 1997; Milo

and Grosh, 1993).

The level of lipid in fish varies depending on species, and it is well

established that oily fish are particularly susceptible to lipid oxidation and

spoilage due to their high content of PUFA in their lipid. As well as lipid level

and fatty acid composition of the lipids, levels of endogenous antioxidants and

endogenous oxidative catalysts will also affect development of rancidity.

External factors, such as heat, light, processing procedures and handling will


also change the equilibrium of tissue compounds and thus play a large part in the

development of rancidity.

Susceptibility to rancidity does not only depend on the amount of lipid

present, but the lipid composition and location in the fish matrix (Khayat and

Schwall, 1983; Ingemansson et al., 1993; Icekson et al., 1998). Oxidative

rancidity problems appear differentially disposed among specific tissue regions.

Undeland et al. (1998, 1999) reported that compositional analysis showed the

highest levels of pro-oxidants in dark muscle and the highest level of polar lipids

in light muscle in herring fillets. Ablett and Gould (1986) found that in cooked

mussels, oxidation occurred especially in tissue from the digestive gland region

yielding highest oxidative rancidity.

To reduce the formation of volatile compounds associated with off-flavour,

the oxidation process has to be stopped or slowed down. The most widely

studied methods for reducing oxidation are the direct application of antioxidants.

There are several categories of antioxidants that may be used and, in general,

antioxidants must be effective at low concentrations, not have a sensory impact

and must be non-toxic (Ashton, 2002). Antioxidants work by a number of

methods including sequestration of catalytic metal ions preventing propagation,

decreasing oxygen concentration, quenching singlet oxygen, decomposing

primary oxidation products to non-volatile compounds, preventing first chain

initiation by scavenging initial radicals and chain breaking or free radical

interceptor antioxidants (Ashton, 2002). Vitamin E has been shown to be an

effective antioxidant in fish systems (Ohshima et al., 1998) and is regarded as

nature's most effective lipid soluble scavenger of free radicals.

2.7 Implications for fish fat by-product valorization

As previously stated, oxidation results in the production of off-flavours and

odours, as well as colour and texture deterioration. Gu and Weng (2001)

reported that oxidation of fatty acids found in oils may induce aging and

carcinogenesis. Other workers reported that oxidation may also generate toxic

compounds, which have the potential to damage health (Gurr, 1984). Oxidation

can be controlled to an extent by limiting the factors that influence the process,

such as light, temperature, heat and metals, processing procedure and oxygen.

As previously outlined, season can have a large effect on properties of lipids

present. The most significant seasonal effect is that of spawning when as a

consequence much of the lipid is converted to either eggs or sperm. Rough

handling may also cause loss of desirable properties, and bruising may increase

the rate of rancidity development on frozen storage (Hedges, 2002) and thus

careful handling and storage of fish onboard is essential.

Diet is a pre-slaughter factor that can have a major effect on the quality of

fish. From an aquacultural perspective, modification of the fish diet can

significantly and positively affect the quality and stability of the final product,

both at a primary and secondary product level. Dietary supplementation with


�-tocopherol has been reported to improve the stability of tissue lipids to

oxidation in trout (Frigg et al., 1990), Atlantic Salmon (Waagbo et al., 1993),

turbot (Ruff et al., 2002a; Stephan et al., 1995), sea bass (Messager et al., 1992;

Pirini et al., 2000) and halibut (Ruff et al., 2002b, 2004a,b). A study by Ruff et

al. (2002a) examined the effect of slaughtering method as well as dietary

supplementation on the quality of turbot fillets. The authors showed that fish fed

supplemented diets of �-tocopheryl acetate had reduced levels of lipid oxidation

post mortem in comparison to fish fed unsupplemented diets. Slaughtering

method was also shown to have an impact on the final quality of the fish.

To date, many studies have measured the fatty acid composition of fish fillets

such as hake (Mendez and Gonzalez, 1997) blue whiting (Dapkevicius et al.,

1998), sea bass (Trigari et al., 1992), mackerel (Hardy and Keay, 1972) turbot

(Ruff et al., 2002a,b), halibut (Ruff et al., 2002b,c, 2004a,b) and salmon

(Ackman and Takeuchi, 1986; Aursand et al., 1994; Katikou et al., 2001), but

little work has focused on extracting these lipids from by-products for use as

potential functional ingredients.

Increasing research has been conducted on extraction, concentration and

stability of fish oil. Fish oil can be produced by several methods, including

physical fractionation (Hirata et al., 1993), low temperature solvent fraction-

ation (Moffat et al., 1993), supercritical fluid extraction (Dunford et al., 1997)

and wet reduction (Bimbo and Crowther, 1990; Chantachum et al., 2000). Many

approaches using enzymatic methods have also been carried out to increase the

concentration of n-3 fatty acids in fish oil (Moore and McNeill, 1996; Shimada

et al., 1997).

Optimum freezing practices may reduce rancidity during storage by reducing

ice crystal damage, particularly to membrane lipids where the initial onset of

lipid oxidation may occur. It is possible that the formation and even distribution

of small ice crystals via a rapid freezing process could reduce initial damage

generally associated with freezing. The use of cryoprotectants to reduce freezing

damage could also be a potential route to prevention of off-flavour development

(Ashton, 2002; Toama, 1990). Cryoprotectants are compounds that have the

ability to extend the shelf life of frozen foods. A wide variety of compounds will

cryoprotect during freezing, including sugars, amino acids, polyols, methyl

amines, carbohydrate polymers, synthetic polymers and inorganic salts

(MacDonald and Lanier, 1991; Matsumoto and Noguchi, 1992; Toyoda et al.,

1992; Park, 1994; Park et al, 1997; MacDonald et al., 2000).

Modified atmosphere and vacuum packaging are designed to extend shelf life

by reducing or eliminating oxygen and thereby reducing oxidative deterioration

during storage. Modified atmosphere packaging (MAP) has been used to extend

the shelf life and maintain high quality of salmon (Amanatidou et al., 2000: Stier

et al., 1981), whiting and mackerel (Fagan et al., 2004; Hong et al., 1996),

channel catfish (Silva et al., 1993; Silva and White, 1994), haddock and herring

(Dhananjaya and Stroud, 1994; Ozogul et al., 2000), tilapia (Reedy et al., 1994)

and halibut (Ruff et al., 2004a). Vacuum packaging may also be used effectively

to inhibit lipid oxidation, but should be accompanied by low chill temperatures,


as the growth of anaerobic bacteria is favoured under these conditions (Sikorski

et al., 1990).

Antioxidants are compounds which have been shown to maintain acceptable

levels of food quality by neutralizing the free radicals produced during the

oxidative process (Haard and Simpson, 2000) and subsequently delaying the

deterioration of food quality. Traditional methods to reduce lipid oxidation

utilize synthetic phenolic antioxidants such as butylated hydroxyanisole (BHA),

butylated hydroxytoluene (BHT) and propyl gallate (Shahidi and Wanasundara,

2001). Ito et al. (1986) reported a possible link between the dose of synthetic

antioxidants used in food products and the development of cancer in rats. This

study and other research (Ito and Hirose, 1989; Clayson et al., 1993; Botterweck

et al., 2000) have resulted in pressure from consumer groups to reduce the

amount of synthetic additives in foods and substitute with natural alternatives

(Marshell, 1974).

Many authors (Chang et al. 1977; Stoick et al. 1991; Frankel et al. 1997)

have highlighted the antioxidant potential of both tea and rosemary in fish oil

systems. Tsimidou et al. (1995) reported a decline in oxidation when rosemary

was added to mackerel oil at 0.5% (w/w) concentration and Roedig-Penman and

Gordon (1997) further reported that tea extract (0.03%, w/w) produced an equal

antioxidant activity to that of BHT (0.02%, w/w) in an oil-in-water emulsion.

Tang et al. (2001) also reported a significant decrease in oxidation when tea

catechins were incorporated into cooked fish patties.

�-Tocopherol (Ohshima et al., 1998; Kulas et al., 2002), tea (Wanasundara

and Shahidi, 1998), rosemary (Xin and Shun, 1993; Frankel et al., 1996) and

oregano (Tsimidou et al., 1995) have been shown to possess antioxidant

properties in fish systems. Other natural alternatives such as clove (Beddows et

al., 2000) and mustard (McCarthy et al., 2001a,b) have exhibited antioxidative

effects when added to food systems. A recent study by O'Sullivan et al. (2005)

examined the effect of a variety of antioxidants in oil extracted from cod and

pollack. The authors found that cod liver oil samples containing rosemary and

tea catechins had lower levels of lipid oxidation in comparison to the samples

containing the other test antioxidants (BHT, mustard, carvacrol, white clove oil,

black clove oil, natural vitamin E and synthetic vitamin E). White pollack liver

oil samples containing tea catechins had lower levels of lipid oxidation in

comparison to the oil samples containing the other test antioxidants (rosemary,

BHT, mustard, carvacrol, white clove oil, black clove oil, natural vitamin E and

synthetic vitamin E). Black clove oil, white clove oil, mustard and carvacol had

no significant effect whereas the addition of synthetic or natural vitamin E had a

negative effect on the oxidative stability of the extracted oil samples.

2.8 Future trends

No growth is expected in marine fisheries in the foreseeable future. Any increase

in total quantity of raw fish materials will come as a result of increased


aquaculture production. In the by-product sector, however, there is still a large

potential for improved recovery both from fisheries and aquaculture. With an

increasing awareness among consumers of marine foods as a source of healthy

polyunsaturated omega fatty acids, the fisheries industry must focus on the huge

potential of recovering valuable marine lipids from by-products. By-products

could provide us with a constant supply of valuable marine lipids and by

applying appropriate measures and technologies for lipid recovery, it may be

possible to achieve more efficient utilization of total catch as well as a potential

ingredient source for the ever increasing demand for omega fatty acids. This

would utilize both fishery by-products and secondary raw materials and, in

addition, underutilized species that would otherwise be discarded.

Rancidity is a clear and evident problem associated with marine lipids.

Measures must be taken to prevent or slow the oxidation process and prevent the

deterioration of flavour and aroma. Therefore, the control of lipid oxidation in

such products is of vital importance. The use of dietary antioxidants or anti-

oxidants used as processing aids are important areas of research which warrant

further investigation, particularly, in relation to the use of natural and potent

forms of antioxidants to stabilize fish oil systems.

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3.1 Introduction

Globally, more than 100 million tonnes of fish and shellfish are caught annually

with 75% being utilised for human consumption (FAO, 2004). Deterioration of

the biomass begins immediately post mortem leading to quality loss and

limitations in the possible end products generated from the raw material. Optimal

handling procedures, from the catching to the product being conserved, are

necessary for reducing the rate of the spoilage and thereby maintaining the fresh

quality that is harvested from the sea. The quality of the raw material will be

crucial for pricing of the end products. Fish are caught by different fishing vessels

operating in different areas of the sea, such as ocean and coastal trawlers, Danish

seine, and net fishing vessels. While the coast fleet generally deliver on shore on a

daily basis, the ocean trawlers may be out for weeks before unloading the catch.

These vessels are generally freezing the fish round or gutted; however, some of

the vessels contain an on-board processing plant producing fish fillet as the main

product. These two groups of vessels, divided into the coastal fleet and the ocean

trawlers, will bring up different possibilities and require different solutions.

3.2 Deterioration of marine biomass

Microbial safety is crucial for all fish product intended for human or animal

consumption. Fish as a raw material is highly perishable and the action of

3

On-board handling of marine by-products to prevent microbial spoilage,enzymatic reactions and lipid oxidationE. Falch, M. Sandbakk and M. Aursand, SINTEF Fisheries andAquaculture, Norway

microbes might spoil and make it into waste, if it is not satisfactorily preserved.

Deterioration of the biomass is also due to lipid oxidation and reactions

catalysed by enzymes. The acceptable levels of lipid and protein hydrolysis are

highly product specific, depending on the end use of the products.

3.2.1 Microbial activity

The fish biomass is generally a good substrate for microbial growth. While the

muscle tissue of fresh and healthy fish is sterile (Liston, 1980), some of the by-

products such as intestines and gills contain large numbers of bacteria (Sikorski

et al., 1990). It is therefore important to take some precautions such as to

inactivate these microbes or to separate these fractions from the more valuable

parts. The raw materials and processed products might also be contaminated by

the microbial flora from people, equipment or other environmental conditions

which also involve a risk of introducing harmful pathogens. By-products for

consumption should be produced hygienically after a gentle first handling of the

fish. Bacteria may produce degradation enzymes such as lipases, proteases,

peptidases and reductases (e.g. TMAO reductase) resulting in spoilage of the by-

products. Furthermore, microbes are responsible for the formation of certain

volatile bases and biogenic amines. Microbial spoilage of fish is excellently

reviewed by Gram and Huss (1996).

3.2.2 Enzymatic spoilage

In live fish the enzymes are regulated by different biochemical systems, but

immediately post mortem, these systems are inactivated and the deterioration

begins, leading to the development of off-flavour, off-taste and release of

nutrients enabling microbial growth. The hydrolysis of fish by-products is

mainly caused by proteases, peptidases and lipases and the activity of these

enzymes is reported to be organ- and species-specific (Hidalgo et al., 1999). The

activity also varies among different organs with higher activity generally found

in the viscera and the intestinal tract in particular (Hildago et al., 1999; Sùvik

and Rustad, 2005a,b). The digestive processes in fish are less known than in

mammals, however, the digestive enzymes are reported to be qualitatively

similar (Hildago et al., 1999). Post-mortem enzymatic reactions can be catalysed

by both endogenous and bacterial enzymes, however, it is difficult to precisely

distinguish between the origin of the enzymes (Sikorski et al., 1990). The major

enzymes of fish gut are proteases (pepsin, trypsin and chymotrypsin), lipases

and carbohydrases (Mukundan et al., 1986). The proteolysis degrades proteins

into smaller peptides and amino acids. The molecular weight is important for

proteins to act as functional ingredients in food or feed applications. Minimising

or controlling the proteolysis is therefore important whether the end products are

intended for food or feed (Falch et al., 2000). The uncontrolled proteolysis of

fish viscera may also lead to the formation of bitter peptides that affect the

sensory properties of the end products (Dauksas et al., 2004). Lipolytic activity


leads to formation of free fatty acids which affect the sensory attributes and the

oxidative shelf life of the lipids. Phospholipids are hydrolysed most rapidly

followed by triacylglycerols, cholesterol esters and wax esters (Sikorski et al.,

1990). Fish lipases are also shown to specifically hydrolyse polyunsaturated

fatty acids (Lie et al., 1987) and, since free fatty acids are shown to oxidise at a

faster rate compared to their esterified counterparts (Labuza, 1971; Shewfelt,

1981) it also affects the nutritional value of the marine lipids. The level of free

fatty acids is one of the main parameters classifying the quality of marine oils.

Other lipolytic activity in fish by-products caused by hydrolases (wax ester

hydrolases and cholesteryl ester hydrolases) and esterases are not widely

studied; however, such activity has been demonstrated in different fish species

(Tocher, 2003). Esterification of cholesterol into cholesterol esters has been

reported during chilled storage of cod gonads (Falch et al., 2005). Lipases in

teleost fishes are discussed in a comprehensive review on lipid metabolism in

fish in general by Tocher (2003) while the literature describing the proteolytic

activity in fish is generally divided into papers on individual species or families

of fish.

3.2.3 Lipid oxidation

The rate of lipid oxidation in fish biomass is generally high due to the levels of

polyunsaturated fatty acids. Lipid oxidation leads to the formation of secondary

products such as aldehydes, ketones and acids that affect the sensory and

nutritional properties of lipids. Reaction products from oxidation of fish oil are

reported (Olsen et al., 2005) and some of these reaction products are detectable

by sensory analysis at levels as low as 10ÿ5±10ÿ2�g/g oil (KulaÊs et al., 2003).

With no conservation, fish by-products will generally be rejected based on

spoilage by bacteria or enzymes long before lipid oxidation reaches an

unacceptable level. Lipid oxidation is, however, the main cause of shelf-life

limitation of marine oil, so the lipid oxidation should therefore be taken into

account when producing marine lipids and products with high lipid content.

3.3 Handling and sorting

The coast fleet generally deliver the catch with or without refrigeration on-

board. The fishermen deliver the fish round or gutted, depending on factors such

as fish species, statutory regulations, temperature and season and whether the

by-products should be utilised or not. The limited area on-board vessels, is in

many cases reducing the possibilities for handling and utilisation of by-products.

A system has been developed for small coastal vessels in order to easily sort the

individual by-products. This recently commercialised system is illustrated in

Fig. 3.1 and enables sorting of head, liver and roe, among others, from whitefish

into thick plastic bags placed in slurry ice. Loading of round fish, both fresh and

frozen, has received growing interest and is under investigation as a possible

Preventing microbial spoilage, enzymatic reactions and lipid oxidation 49

solution (Akse, 2002), where the whole processing can be done on-shore, under

controlled conditions. Furthermore, it will be possible to process raw material

from several vessels at the same place, and thereby enable a more economic and

better utilised production facility.

The ocean trawlers are generally freezing the fish round or gutted; however,

some of the vessels contain on-board processing plants producing fish fillet as

the main product. By-products are generated during a typical filleting opera-

tion and suggested preservation technology is shown in Fig. 3.2. Whitefish

such as gadiform species deliver a wide range of possible products that can be

sorted out, depending on the processing; these are head, liver, roe, milt,

stomach, skin, bones, and trimmings. The main processing steps are bleeding,

head cutting, gutting and filleting and the technology chosen to preserve the

by-products is highly dependent on the end product. Some of the fractions are

more valuable than others and need particular care. These are, for example,

liver, roe and milt from white fish since they may be used non-processed as

consumer products.

In vessels utilising specific by-products such as liver, roe, milt, and stomach,

the gutting operation has traditionally been done manually in order to keep the

inner fractions intact. Focus has recently been put into the development of more

gentle automatic processing machines (Fig, 3.3) and the visceral fractions are

now successfully recovered automatically from whitefish (Baader 444) and

Fig. 3.1 A system for sorting and refrigerating by-products in a coastal vessel. Thefishermen (one or two) are handling cod by-products and sort them into head, liver,

roe, milt, stomachs, etc. Source: Reprinted with the permission from Rubin(www.rubin.no).


salmon (Baader 142) (Einarsson, 2004). The new and gentle gutting operations

now available enable differentiation between quality levels and end uses and

thereby yield individual optimal handling and preservation of the different

fractions. Gentle and non-destructive gutting also makes it possible to get

reasonable prices for the most valuable parts. An automatic process to utilise

individual by-products should satisfy the following three stages: 1) gentle

removal of viscera enabling preservation of each fraction undamaged; 2) recog-

nition and automatic separation of specific fractions; and 3) automatic sorting of

the separated fractions.

Fig. 3.3 A recently developed gutting machine with gentle removal of visceralcomponents. Source: Reprinted with permission from BAADER.

Fig. 3.2 By-products generated during filleting of gadiform species with suggestedmethods of preservation and bulk production.


The visceral fractions obtained from the gentle gutting machine might be

automatically sorted using imaging technology. This technology is under

development. Tests on images from cod by-products indicate that the proposed

method has the potential to automatically recognise cod viscera components.

The combination of automatic visual recognition and robotic technology to

facilitate subsequent separation and sorting, have a lot of possibilities for use in

the fish processing industry (Mathiassen et al., 2002). The system has to perform

the separation at a speed that satisfies the need of processing. This work is

brought forward in new projects with industrial partners, with an objective of a

commercialised solution. However, it is necessary to get profitability in such

concepts and the possibilities for obtaining relatively high prices for the end

products are essential to defend this still expensive technology.

Independent of fishing vessels, gentle handling during and after catch will

retain the biological membranes and act as a physical barrier to biochemical

deterioration until the products are conserved. Furthermore, the time of bleeding

will affect the quality since the blood, in fish that are not bled, will diffuse into

the muscle and visceral fractions and promote biochemical degradation such as

lipid oxidation.

3.4 Conservation and stabilisation

Traditional methods of preservation might be divided into four different

categories: refrigeration technology, heat preservation, dehydration (drying) and

chemical preservation (Table 3.1). Based on the end purpose of the raw material;

human consumption or feed, several different techniques of conservation and

stabilisation can be employed, alone or in combination. Quality loss is an

irreversible process and is also cumulative. This means that a quality loss from

one stage of the line cannot be regained at later stages. It is crucial to reduce the

possible quality losses to a minimum in all stages, from the very beginning.

3.4.1 Refrigeration technology

Refrigeration technology including freezing and chilling are basic technologies

offering stabilisation of the products, particularly on the matter of microbial

growth but also autolysis, and lipid oxidation processes are reduced at lower

temperatures. As early as 1797, natural ice was used for chilling of fish that was

transported to the UK (Dellacasa, 1987). Later, various mechanical systems

were developed, using several different approaches.

For raw material that can be used directly as consumer products, such as roe,

liver and stomach, proper chilling and freezing capacity are essential. Chilling is

an operation where the temperature is lowered down to values between ÿ1 and

�8ëC. The lower the storage temperature one can achieve, the longer the

possible storage time. For fresh fish products the suitable temperature range is

ÿ1 to �1ëC (Fellows, 2000). It is appropriate to consider these numbers as


relevant for the by-products as well. Storage life of fish products stored at

different chilling temperatures has shown an increase in shelf life of up to

several days if temperature is reduced by 2ëC (Rùyrvik, 1979), and the storage

temperature during the first 24 hours has a major influence on the subsequent

shelf life. As for freezing, a temperature of ÿ18ëC is considered to be the normal

value (Fellows, 2000), but lower temperatures will add shelf life to the product.

Some products are more demanding than others. In a Norwegian report from

1995 (Hardarson, 1995) frozen storage of cod liver, a product with extremely

high lipid content, has been reported to be of little use to prevent lipolysis unless

the temperature could be reduced to ÿ45ëC (Fig. 3.4). Normal storage

temperatures, like ÿ18ëC gave the same level of free fatty acids as chilled

storage.

It is crucial that the catch is adequately chilled from when it is taken on board

and that backbones and/or trimmings are chilled and kept under hygienic

conditions throughout the processing, if the by-products are sources for minced

products. For bulk products, like silage, oil or meal, the correct combination of

temperature treatment (chilling of the catch, preheating of the raw material to

the correct temperature) and processing aid (e.g., organic acid, commercial

proteases, etc.) will give the possible directions for the end product and decide

Table 3.1 Effect of different preservation techniques on the main deteriorationprocesses in by-products. A positive effect of the technique is marked

Main deterioration processes

Microbial Autolysis Lipidgrowth oxidation

REFRIGERATIONChilling and freezing X X X

HEAT PROCESSING X1 X2

DEHYDRATIONDrying X X

CHEMICAL PRESERVATIONLowering of pHUse of antioxidants X XSalting X X

PROCESSINGSorting (prevent contamination) X X XSeparation of oil X3 X3 X3

Use of inert gaseous or replacement of O2 X XPhysical membranes intact X X X

1 Reduced growth.2 Inactivation suggested for cod by-products: 10 min, 70ëC (Sùvik, 2005).3 Production of oil as soon as possible after catch will prevent microbial growth in the oil fraction(there is generally no microbial growth in pure oil).


whether the quality is acceptable for applications for human consumption, or

used in low value applications (feed).

There are several possibilities for chilling onboard the vessels: use of ice,

RSW (mechanically refrigerated sea water), CSW (ice-chilled sea water), ice-

slurry, cold air and others. Achieving refrigeration demands electricity and this

is usually produced with diesel engines. The horsepower of the engine will be

the limitation for the possibility to produce refrigerating capacity. A major effort

has been put into developing refrigeration technology for fishing vessels that

demands less space and power consumption than traditional solutions (Wang

and Wang, 2005). For smaller vessels in the coastal fleet, carrying ice produced

on shore with them out to the fishing grounds is a possibility but for the trawlers,

suitable equipment has to be placed on board.

Using chilled sea water for the chilling of the catch offers several advantages

over the use of ice (Kelman, 2001): 1) due to better heat transfer, the catch is

cooled more rapidly; 2) less effort is required to stow and unload it; 3) less

likelihood of fish being crushed or losing weight; and 4) in addition, sea water

can be safely reduced in temperature to about ÿ1ëC without freezing the fish

contained in it.

This method is commonly in use within the aquaculture slaughtering industry

as a suitable pre-chilling before further processing, and will offer good

possibilities also for vessels that process on board.

Fig. 3.4 Free fatty acids development during frozen storage of cod liver at differenttemperatures. Source: Reprinted with permission from Hardarson (1995).


A cold chain on board can include an initial stage of RSW or CSW for

chilling of the catch (round fish), then the different products will follow different

lines through and after sorting and processing. Chilled water may not be

adequate for the sorted fractions of by-products. Unless the fractions are packed

in water-resistant packages (e.g., vacuum-packed), direct contact with water is

not recommended. Use of ice and storage in chilled rooms is the best choice.

Freezing and frozen storage is a possible choice for some products.

3.4.2 Heat preservation

High temperature treatment is commonly used in processing of fish by-products.

Both enzymes and microbes might be inactivated at high temperatures. Heat

treatment reduces the number of microbes and prolongs the shelf life of the

product. Microbes generally do not grow at temperatures above 90ëC; however,

a full heat sterilisation requires stronger thermal treatment.

Enzymatic deterioration is arrested by inactivation of enzymes as soon as

possible post mortem and might be obtained by heat treatment. Unfortunately,

high temperatures lead to unwanted side reactions such as denaturation of

proteins, which will affect the physical properties of the proteins generated. The

inactivation conditions for enzymes are temperature and time dependent;

however, factors such as pH and type of enzyme are important influences

(Sùvik, 2005). Overall, a temperature of 85ëC for 10 minutes is found to

inactivate proteases and lipases in visceral fractions of cod (unpublished data).

In some products, such as silage, the enzyme activity is wanted and high

temperature treatment should be avoided.

3.4.3 Dehydration (drying)

The shelf life of fish by-products is increased by reducing the moisture content

and thereby the water activity to levels (aw < 0.6) preventing growth of bacteria

and moulds. Also, enzymes are affected by water activity and most enzymatic

reactions are reduced at water activities below 0.8. The rate of lipid oxidation,

however, is reduced at aw levels between 0.2 and 0.6, but is higher above and

below these levels (Labuza, 1971). aw in oils is generally too low to permit any

microbial growth.

3.4.4 Chemical treatment

It has been reported that chemicals are, in some countries, used to add shelf life

to fresh fish, but these applications are usually subject to local or national

restrictions (FAO). Addition of acid, however, is widely used to reduce the pH

and liquefy by-products.

In order to protect the by-product from microbial spoilage, hurdle technology

might be used. This technology is based on the fact that a combination of

different preservation methods results in a synergistic effect and therefore rough


treatment might be avoided. The principles and uses of hurdle technology have

been reviewed by Leistner (1995).

The major factors necessary to control microbial activity, enzymatic activity

and lipid oxidation are explained below (see also Table 3.1). Microbial activity is

reduced by refrigeration and heat treatment, low water activity, adjustment of pH

and gentle and hygienic handling. To prevent formation of microbial enzymes,

such as TMAO-reductase, the recommended practice is to reduce the amount of

microbes and handle the by-products with sufficient hygiene practice. Enzyme

activity is reduced by refrigeration and heat treatment, low water activity, adjust-

ment of pH, chemicals which can inhibit enzyme action, alteration of substrates,

alteration of products and pre-processing control. Lipid oxidation is reduced by:

reduction of temperature, minimising oxygen availability and light and retaining

the biological membranes intact. Lipid oxidation may also be reduced by

antioxidants and it has become more common to use antioxidants in combination

with an appropriate acid during bulk processing such as silage production.

3.5 On-board processing

Fresh raw material will always be an advantage, and offers a wider range of

possibilities compared to frozen-thawed raw material or material that has been

stored for several days or weeks under varying conditions. On board the fishing

vessels there is a unique possibility for supply of completely fresh raw material,

and, it is an advantage to be able to utilise as much as possible of the catch on

board. This adds quality to the end product that cannot be achieved otherwise.

3.5.1 Process technology

Fish processing traditionally takes place at on-shore processing facilities, and

the processing of by-products on-board fishing vessels has declined during the

last decades. This is especially due to the decrease of on-board fishmeal and

fish-oil processing plants. Three of the most important bulk processes utilising

by-products are shown in Fig. 3.5, these are fish silage, fish-oil and fish protein

hydrolysate (FPH).

Fish silage (A) is produced by acidification of fish material (offal and

viscera) using an organic acid such as formic acid to reduce the pH to around 4.

This pH will prevent microbial growth, but endogenous enzymes achieve

optimal conditions to hydrolyse proteins and thereby liquefy the material. An

excellent review of the technology used to produce silage is written by Raa and

Gildberg (1992). Lipid oxidation in the silage during storage might be prevented

by using antioxidants along with the acid. Much research has stated the fact that

the acidic treatment deteriorates the lipids by increasing the level of free fatty

acids (Johnsen and Skrede, 1981; Tatterson and Windsor, 1974).

Fish protein hydrolysates (FPH). In recent years, the fishing industry and

research, has addressed the use of commercial proteases (Fig. 3.5(B)) to produce


Fig. 3.5 Illustration of three alternative production lines for on-board processing of by-products. (A) Silage production where an organic acid isadded to preserve the raw material by lowering the pH. (B) Controlled hydrolysis of proteins by a reaction catalysed by added enzymes. Bulk

products are oil, fish protein hydrolysates (FPH) or fish powder. The FPH are perishable and need to be preserved by reducing pH, for example.(C) Traditional production of cod liver oil. Steaming and decanting. Rest material from oil production has a water activity > 0.6 and therefore

needs to be conserved (Beuchat, 1981). This illustration is made in Microsoft Visio 2000 SR1 by E. Falch.

fish protein hydrolysate from marine raw material (Gildberg, 1992, 1994; Liaset

et al., 2002; Mohr, 1977, 1980; Slizyte et al., 2003, 2005a,b; Dauksas et al.,

2005) under controlled conditions. During this process the stick water, and

alternatively parts of the sludge, are evaporated. This pumpable liquid demands

conservation, such as acid addition (lowering of pH), in order to be storage

stable on-board. Alternatively, the evaporated FPH can be dried to storage stable

powders.

Fish oil and fish meal. Process technology where the marine lipids are the

main product generated from the raw material, has traditionally been based on

thermal treatment followed by centrifugal separation (Aure, 1967) for liver oils

(Fig. 3.5(C)) and cooking, pressing and centrifugal separation (mechanical

expression) (Windsor and Barlow, 1981) of fish raw material. This mechanical

expression is the traditional process converting anchovies, menhaden, sardines

and Atlantic herring into fishmeal and oil. Today, these species are diverted for

human consumption and the resulting reduced availability of raw material for

feed applications has led to the use of other raw material in the processes, such

as fish by-products. Fishmeal is produced through thermal treatment. Focus on

increased protein digestibility of feed has led to the development of gentle heat

treatment (LT quality) in order to prevent protein denaturation. The digestibility

of fishmeal is also affected by the freshness of the raw material (Aksnes and

Mundheim, 1997) and effort has been put into the early conservation and

production of meal. Fishmeal plants are offered to be individually designed for

on-board operation for fishmeal (BAADER Food Processing Machinery). In

Norway, less than 5% of the fishmeal is currently produced on on-board

processing facilities.

Drying the raw material on-board is a solution that has an answer to two

different challenges: 1) Dry matter weighs less than wet matter; this reduces the

weight of the vessel and thereby also reduces the fuel-costs and 2) Dry matter

takes less space/volume than wet matter.

Other possibilities. The cut-offs and trimmings, mainly generated during the

filleting process of white fish might be used in fish mince or washed to produce

surimi. The surimi process is currently located on-board some fishing trawlers.

Fish sauce, which is a popular product in south-east Asia (Huang and Huang,

1999) is another product possible to produce from fish by-products (Gildberg,

2001). The raw material is conserved by using large amounts of salt followed by

a fermentation period. Both of these process steps might be done on-board the

vessel.

Gelatine and dried heads are usually not produced on-board, but heads, skin

and bones are, in some vessels, frozen for later processing. In Iceland, dried cod

heads are a major export product (Arason, 2002). Heads are generally dried at on

shore processing facilities.

One of the main challenges on-board is the possibility to handle, store and

transport the bulk products ± which are factors limiting the processing and

utilisation. This is a matter of available space and also a matter of weight. Figure

3.6 illustrates parts of an on-board processing plant for producing hydrolysed


by-products. Compact and efficient process equipment is necessary due to the

limited available area on board.

3.6 Utilisation of by-products from gadiform species

It is a great potential for the fishing industry to land and utilise a greater part of

the total catch of gadiform species for higher value products. Utilisation of by-

products requires a predictable delivery quantity and also raw material with

potential for producing relatively standardised products to be able to satisfy the

customers.

To utilise more than the fish fillet for consumable products, data on weight

and composition of different by-products are needed (Froese and Pauly, 2000).

Fishbase (www.fishbase.org), which is a comprehensive database, has included

a processing table for each species. These tables show the weight distribution of

by-products along with some approximate composition data. Such data on

different gadiform species are not completely established at this stage. However,

a major study on the available by-products, available lipids and their composi-

tion has been reported for five different gadiform species (Falch et al., 2006a,b).

These data show that, from an average daily production of fillet (10 tonnes), the

by-products contain approximately one tonne of the health beneficial marine

lipids with 30% n-3 fatty acids. The data show that, regardless of species and

fraction, the n-3 content is high and close to the specifications for medicinal oil

(European Pharmacopoeia, 1999). However, the lipid classes such as phospho-

lipids and triacylglycerols are unequally distributed among different fractions

Fig. 3.6 Parts of an on-board processing plant for hydrolysation of by-productsfollowed by a three phase separation into oil, glue water and sludge. Photo: Eva Falch

(Falch et al., 2000).


and processing technology such as the enzymatic processing (Fig. 3.5, process

(B)) generally differentiate among the lipid classes (Dauksas et al., 2005). These

experiments showed that the phospholipids are generally following the protein-

rich fractions during centrifugal separation. Gadiform species such as cod,

saithe, haddock, tusk and ling, are also reported to be good sources for produc-

tion of fish protein hydrolysates and protein-rich fractions (Slizyte et al., 2003,

2004a,b) with specific functional properties applicable in food. The processing

challenges are discussed in the above-mentioned papers.

In order to explore the possible profit from processing by-products and to

decide on where to do the processing; on the vessel or on shore, profitability

analyses are needed. As assistance to such an analysis, a program called

MaxFish has been developed to help calculate the possible output and

profitability from a given catch. For the time being, the database is built on

data from cod species and the weights and fractions from these. Chemical

composition data and their variations may be added and increase the usefulness

even more. It can also easily be extended with any species of interest. An

example of the use of the program is shown in Fig. 3.7. Basically it utilises

knowledge of the different end products and the possible income these can give,

and the program enables calculation of the investments that can be justified.

Studies of biochemical reactions in the visceral fractions of cod show that

these organs are highly perishable during chilled storage. One week of storage

(4ëC) shows minor lipid oxidation, but major changes due to lipolysis

(unpublished data). In our studies of roe and milt from cod we found limitations

Fig. 3.7 An example showing applications for the MaxFish program to plan theutilisation of by-products.


in using free fatty acid assessment as a measure of lipolysis in such raw material

since fatty acids hydrolysed from phospholipids and triacylglycerols are

esterified to cholesterol and thereby not detected by using free fatty acids as

an indicator of lipolysis (Falch et al., 2005).

3.7 Future trends

The growing awareness of the health benefits of marine products is turning the

focus from waste or bulk products into higher value end products such as food

ingredients and bioactive compounds. This will be a challenge for the fishing

vessels, demanding more advanced equipment on board and more competence

from the people handling the raw material and the equipment. The end product

will not necessarily be produced on the vessel, but preservation or alternative pre-

processing will be recommended on board to retain the quality. There will be a

need for technology and knowledge which necessitate investment in hardware and

in education of the personnel. The consumer trends show preference for more

freshness and higher quality levels, but also milder processing, minimal preserva-

tion and replacement of synthetic additives with naturally occurring compounds.

Knowledge about the available biomass and its chemical composition will be

required in order to secure a delivery of a specified quality and also use such

data to find optimal combinations of raw material available. Several vessels

have quotas for different fish species and finding optimal combinations of raw

material will therefore be an advantage. Furthermore, the sorting operation,

which is now a manual operation, will have to be more efficient and more

automated solutions are currently being investigated.

New technologies are currently put into more gentle catching methods.

Catching the fish into lock bay is one of these new technologies, which prevent

physical damage of the fish and thereby a more gentle treatment of the by-

products. It will try to ensure that the fish is alive when reaching the slaughter line.

Overall, the health aspect is becoming more important and fish species

traditionally used for production of fishmeal into aquaculture feed are now

turned into consumer products. As global fish farming increases, the aquaculture

industry will continue to request marine feed ingredients, and higher utilisation

of by-products is therefore an alternative. The marine resources, however, are

limited and an optimal utilisation of all the available raw material is essential.

3.8 Acknowledgements

Norwegian Research Council (project: Increased value adding from by-products

and by-catches and project: Technology for production of ultra stable marine

oils and functional food applications) and EU (QLK1-CT2000-01017)

Utilisation and stabilisation of by-products from cod species are thanked for

funding the majority of the research presented from our research group.


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4.1 Introduction

The transformation of raw materials into foods inevitably generates some type of

by-products and the processing of aquatic foods is no exception. Therefore,

developing new technologies for the full utilization of these by-products is of

critical importance to the future economic viability of this industry (Gildberg,

2002). In traditional and non-industrialized fisheries, where most of the labour is

provided by personnel with skills often passed down by generations, the fish is

almost completely utilized for human consumption, animal feed, or as plant

fertilizer. The economy-driven industrialization of fisheries brought incredible

advances, but at the same time, the amounts of by-products generated during

harvesting and processing increased dramatically (Gildberg, 2002). Typical

examples are commercial shrimp trawling, krill processing and mechanized fish

filleting. In shrimp trawling, sometimes 90% of the total catch volume corres-

ponds to species with no commercial value and this by-catch is therefore most

often discarded (Raa and Gildberg, 1982). The meat recovery yield during the

commercial processing of whole krill (Euphausia superba) is extremely low,

fluctuating between 10 and 15% by weight (Suzuki, 1981). Finally, when fish

are mechanically processed for fillets, the recovery yields are typically 30±40%

of fillets and the by-products account for 60±70% by weight of the whole fish

(Gildberg, 2002). While it is not uncommon to just grind-and-discard this 60±

70% of fish by-products, this practice should be considered an irresponsible

utilization of natural resources and should be used instead to fulfil human

nutritional needs.

4

Recovery of by-products from seafoodprocessing streamsJ. A. Torres, Oregon State University, USA, Y.-C. Chen, ChungShan Medical University, Taiwan, J. Rodrigo-GarcõÂa, UniversidadAutoÂnoma de Ciudad JuaÂrez, Mexico and J. Jaczynski, WestVirginia University, USA

In the fisheries industry and the scientific literature, there is a common

misunderstanding regarding how processing `by-products' are defined. Three

terms are frequently and interchangeably used to describe the same materials:

(1) òffal', (2) `waste', and (3) `by-products'. The first two terms imply that

these materials must be discarded, because they cannot be used for any

application, and therefore, have a negative connotation. The third term suggests

that they may be valuable if an appropriate technology is available, and is

therefore positive. Currently, the preferred and most common definition of the

term `by-products' is to refer to all edible or inedible materials remaining

following processing of the main product. A typical example is fish filleting

where the fillets are the main product and the frames, heads, and guts are the

`by-products'. It would be misleading to name these by-products as `waste',

because the fish meat and oil left on the frame and heads have value and could

be recovered if their quality is not compromised by poor handling (Strom and

Eggum, 1981). With an appropriate technology, the meat reclaimed represents

additional revenue for the processor, and at the same time, its recovery decreases

environmental pollution.

By-products can be converted into four major product groups: (1) plant

fertilizers, (2) livestock feeds, (3) value-added foods, and (4) specialty

ingredients. In general, conversion of by-products into fertilizers results in the

lowest value addition, while it is highest for value-added foods and specialty

ingredients. The future is promising as it has been estimated that as a result of

new technologies, the value addition to by-products will increase fivefold within

the next decade (Gildberg, 2002).

4.2 State of global fisheries and by-products

Global fish catch and aquaculture production information is readily available

from the Food and Agricultural Organization (FAO) Fisheries Department

(Anonymous, 2004). However, the amounts of by-products generated by these

activities can only be estimated (Table 4.1). While the global capture fisheries

have remained fairly stable for the past twenty years and have been forecasted as

unlikely to increase in the future, the aquaculture sector increased its production

by 27% for the same period and currently contributes nearly 50% of the global

annual catch (Vannuccini, 2004). The Food and Agriculture Organization (FAO)

predicts that the future demand for aquatic foods will have to be met by

increasing aquaculture production. Then again, in 2002, about 76% of world

fisheries production was used for direct human consumption and the remaining

24% was converted into fishmeal and oil (i.e., reduction fisheries), yielding

essentially no by-products. However, while on a weight basis this industry

accounted for a quarter of total fish utilization, it contributed a low 3.8% to the

fisheries total economic value (Anonymous, 2004).

About 100 million tonnes of the global fisheries production is processed for

direct human consumption. Commercial processing of fish such as cod, salmon,


trout, tilapia, seabream and pollack typically yields about 30±40% of fillets as

products, and while meat and oil left on the remaining by-products range widely,

they account typically for 20±30% and 5±15% of whole fish weight, respec-

tively. Fish oil is highly polyunsaturated (omega-3 fatty acids), and therefore,

very susceptible to lipid oxidation, resulting in rancidity development. If fish oil

were to be efficiently recovered from by-products, the use of fat-free fish by-

products for animal feed and pet food could be expanded.

The 100 million tonnes processed for direct human consumption do not

include the fish by-catch and discards estimated to account for an additional 30

million tonnes of available catch not yet utilized (Zugarramurdi et al., 1995).

Another aquatic resource that is scarcely utilized for human consumption is krill

(Fig. 4.1). At present, krill is commercially utilized mostly by the reduction

fisheries to manufacture fish feed. The development of an appropriate tech-

nology to efficiently convert this resource into food could contribute signifi-

cantly to fulfil nutritional needs for proteins and help alleviate over-fishing and

stock depletion problems affecting several aquatic species. This vast resource

has been estimated at 400±1550 million tonnes with a sustainable annual harvest

of about 70±200 million tonnes (Suzuki and Shibata, 1990). The krill biomass

potentially available for human food is comparable to that of all of the other

aquatic species currently under commercial exploitation and is probably the

largest of any multicellular animal species on the planet (Nicol and Endo, 1999).

Krill, small crustaceans that resemble shrimp, are not fully utilized for human

consumption owing to the lack of efficient meat recovery technology. Krill meat

is literally liquefied at high rates by extremely active proteolytic enzymes

released during harvest (Kolakowski and Lachowicz, 1982).

The biomass of aquatic by-products and underutilized species is staggering. At

the same time, over-fishing, stock depletion, and other environmental issues asso-

ciated with aquatic food production are increasingly more emphasized in popular

media. Also, the world population is increasing and it is becoming more difficult to

meet nutritional needs for proteins and lipids from aquatic resources. Although

aquaculture can alleviate these problems, technologies that increase recovery

yields and reduce the amount of by-products will have to be developed and

Table 4.1 Global fisheries production and utilization in million tonnes

1998 1999 2000 2001 2002 2003

Capture 87.7 93.8 95.5 92.9 93.2 90.3Aquaculture 30.6 33.4 35.5 37.8 39.8 41.9Total fisheries 118.2 127.2 131.0 130.7 133.0 132.2Human consumption 93.6 95.4 96.8 99.5 100.7 103.0Non-food uses 24.6 31.8 34.2 31.1 32.2 29.2Population (billions) 5.9 6.0 6.1 6.1 6.2 6.3Per capita fish food supply 15.8 15.9 15.9 16.2 16.2 16.3

Source: Adapted from Anonymous (2004). The state of world fisheries and aquaculture. Food andAgriculture Organization. Rome (Italy).

Recovery of by-products from seafood processing streams 67

implemented. A challenge will be to find applications for the materials recovered,

to ensure that the process is not only environmentally sustainable but also

economically viable. This industrial development effort must be based on a full

understanding of fundamental properties of the raw materials. Only then, will it be

possible to manipulate the behaviour of the aquatic biomolecules and in turn

increase recovery yields. By definition, meat is the edible portion of aquatic

animals derived from muscle tissue and is mostly a mixture of water, proteins, and

intramuscular lipids. Therefore, to develop and implement new recovery techno-

logies, it will be necessary to understand the behaviour of these three components.

4.3 Basic properties of water, proteins and lipids in aquaticfoods

4.3.1 Water properties

The moisture content of meat depends on the species and may range widely,

reaching values up to 90%. In a final food product, not only does water control

product weight, and therefore, the revenue, but also the sensory attributes

perceived by customers. From a nutrition standpoint, water does not contribute

calories and therefore, the higher the water content, the lower the caloric

contribution of the product. However, when water is added to the product, water-

binding compounds may need to be used to prevent `drip' losses. Meat (muscle)

proteins have good water holding capacity (WHC) if they have not been abused,

Fig. 4.1 Body composition of krill whose annual sustainable biomass for humanconsumption has been estimated as larger than the global consumption of all other aquatic

species combined.


particularly from a thermal point of view. For example, krill meat proteins have

good WHC; however, they are particularly sensitive to temperature abuse

(Suzuki, 1981).

In meat systems, water provides a reaction medium in which all other

compounds such as proteins and lipids may be dissolved or suspended. There-

fore, it is critical to be familiar with how water interacts with them. A water

molecule is a dipole due to the slight negative and positive charge of its oxygen

and hydrogen atoms, respectively. These charges allow an interaction called

hydrogen bond between water dipoles and also with other charged molecules

(Fig. 4.2). This property is very important in food technology, because important

molecules such as proteins may also be charged, and therefore, have an ability to

interact with water dipoles via electrostatic interactions.

4.3.2 Fish proteins

The biological value (BV) of a dietary protein measures its efficiency in sup-

porting physiological needs reflecting its digestibility and content of essential

amino acids. Egg proteins are used as a reference protein source and have a BV

of 100, while milk, beef, corn, and rice proteins have BV of 93, 75, 72, and 59,

respectively. The BV of fish meat is 75 while the value for krill meat proteins is

slightly higher than that of milk proteins (Whitney and Rolfes, 2005; Murano,

2003; Suzuki and Shibata, 1990).

Two muscle types are present in aquatic foods, striated muscle characterized

by transverse stripes and smooth muscle that lacks them. Striated muscle is the

Fig. 4.2 Electrostatically charged water dipoles are the basis for the solubility ofproteins in water. In the water dipole, the oxygen atom is negatively charged (2�ÿ) withrespect to the two positive (��) hydrogens. In a meat system, dipoles form hydrogen

bonds between each other and with charged molecules such as proteins.


main component in fish meat while smooth muscle is typical of meat from

molluscans. Fish muscle is divided further into white and dark muscle. The dark

muscle lies alongside the fish body and under the skin. White muscle proteins

can be further classified into (www.fao.org/documents/show_cdr.asp?url_file=/

docrep/v7180e/v7180e05.htm):

(1) Myofibrillar proteins. These are proteins organized into myofibrils that are

soluble in concentrated saline solutions. The myofibrils are made of two

major ultramicroscopic myofilaments, a thick and a thin filament containing

the major muscle proteins myosin and actin, respectively, the former being

largely responsible for the functional properties of meat. In the process of

muscle contraction, tropomyosin and troponin act as regulatory proteins,

initiating and terminating the contraction process of myosin and actin.

These contractile agents are arranged to form the myofilaments of a

sarcomere, continuing with the formation of myofibrils from many myo-

filaments kept together by connective tissue or stroma proteins. However,

the amount of connective tissue in fish meat (3±5%) is low when compared

to other meat sources (e.g., 16±28% in beef) (Suzuki, 1981).

(2) Sarcoplasmic proteins. These proteins are soluble in dilute saline solutions

and correspond to myoalbumins, globulins and various enzymes. In the

muscle, myoglobin has a role similar to that of blood haemoglobin. Both

form a complex with oxygen, but myoglobin is essentially an oxygen

storage mechanism in the cell. It is a conjugated protein with a peptide

chain bound to the heme containing an iron atom and a large heterocyclic

organic ring called a porphyrin.

(3) Connective tissue or stroma proteins. These proteins are insoluble in

concentrated saline solutions, and when heated to 60 or 70ëC the collagen

fibres contract to one third or one quarter of the original length. At 80ëC, the

collagen becomes water soluble gelatine.

Ionic strength (IS) is an important factor in the water solubility of meat

proteins. The IS represents the status of electrolytes (i.e., ionized salts) present in

a system. The two major myofibrillar proteins, myosin and actin are water

insoluble at the fish physiological pH (about 0.05 for rainbow trout), but they are

soluble at extremely low IS or above 0.6. While sarcoplasmic proteins are quite

water soluble, stroma proteins are completely water insoluble. The water

solubility of sarcoplasmic proteins decreases with the increase of IS (Lanier et

al., 2005; Suzuki, 1981).

In an aqueous protein solution, the side chain and the amino and acid

(carboxyl) groups bound to the central carbon atom of an amino acid can be

electrostatically charged, and therefore, participate in protein-water interactions

via weak hydrogen bonds (Fig. 4.3). Although the bonding energy of the

hydrogen bond is low, however when present in high numbers, these bonds

efficiently stabilize the complex three dimensional structure of food proteins.

Depending on the side chain attached to the central carbon atom of an amino

acid, a protein will have different properties. While hydrophobic side chains


limit its water solubility, polar (charged) side chains may result in considerable

protein-water interaction via the hydrogen bonds (Fig. 4.3). The protein-water

interaction is essential for protein solubility as well as water holding capacity

(WHC). On the other hand, the weak hydrophobic interactions may result in

protein-protein interactions causing their aggregation and subsequent precipita-

tion. Finally, the side chain of cysteine contains a sulphydryl group (±SH) that

when oxidized (i.e., during heat treatment) will create a covalent disulphide

bond (±SS±). This strong bond combined with weak interactions between

hydrophobic side chains are important in the gelation of fish proteins and are

Fig. 4.3 Proteins are chains of amino acids containing an amino (�NH3) group, an acid(COO±) group and a side chain (R) all bonded to a central carbon atom. The side chainallows proteins to participate in protein-protein hydrophobic interactions and can also

form protein-water hydrogen bonds.


Fig. 4.4 A protein at its isoelectric point (pI) has a zero net electrostatic charge. At its pI, protein-water interactions are at its minimum, whileprotein-protein interactions via weak hydrophobic bonds are at its maximum, causing protein precipitation. Protein-water interactions prevail under

acidic or basic conditions far from the pI, resulting in protein solubility.

essential for the proper texture development of value-added foods. The side

chains of amino acids can be chemically modified, and even though chemically

modified proteins may be unsuitable for human consumption, unconventional

applications may be developed for proteins recovered from by-products.

The side chains of the amino acids can assume different electrostatic charges

depending on the conditions of the fish protein solution (Fig. 4.4). Acid added to

a protein solution dissociates forming the positive hydronium ion (H3O+)

neutralizing negative charges on the protein and making it more positively

charged. By adding a base to a solution, the protein will increase negative

charges on its surface. As the protein becomes more positively or negatively

charged, electrostatic interactions with water dipoles increase while hydrophobic

protein-protein interactions decrease. Therefore, as a protein becomes more

charged (polar), its solubility increases and the protein becomes water soluble.

The pH at which the overall electrostatic charge of a protein is zero is called the

isoelectric point (pI).

4.3.3 Fish lipids

The fat content in fish muscle is highly variable, varying with species, age,

spawning season, fish diet, and body part. While the protein content in fish muscle

is relatively stable, the fat content is in general inversely correlated to the moisture

content. Although, fat deposits are found over the entire muscle mass, the

concentration of fatty cells seems to be higher near the myocommata and in the

region between the white and dark muscle (Kiesling et al., 1991). Dark muscle

contains some triacylglycerols in the muscle cells, even in lean fish, because this

muscle can metabolize lipids directly to generate energy. This is why the dark

muscle contains more fat and more blood supply to metabolize lipids; as well as a

higher concentration of pro-oxidative myoglobin and mitochondria (organelle that

oxidize energy-yielding substrates) than white muscle. Therefore, the dark muscle

is more prone to lipid oxidation and the development of rancidity (Hultin et al.,

2005). For these reasons, the white flesh fish is preferred by customers, and hence,

it would be economically beneficial to develop a technology capable of fish oil

removal from the dark fish meat to add value to lower grade products.

The properties of the triacylglycerols in fish oils depend on the composition

of the fatty acid (FA) chains attached to glycerol (Fig. 4.5). Lipids interact with

the hydrophobic side chains of amino acids forming weak lipid-protein

hydrophobic bonds.

Probably more important to fish technologists than triacylglycerols are

phospholipids, an integral component of cell membranes. Phospholipids are

amphiphilic compounds, that is, they have lipo- and hydrophilic properties.

Phospholipids can create hydrophobic bonds with other apolar substances (for

example, fish oil) and also interact with water and charged proteins (Fig. 4.6).

Therefore, when processing aquatic foods such as minced fish meat, the

separation of fish oil from the water and proteins is difficult and requires the use

of additives that can act as emulsion breakers.


Fig. 4.5 Triglycerides are a major component of meat lipids.

Fig. 4.6 Phospholipids, another major component of meat lipids, are water soluble andcan interact with charged proteins and water dipoles.


Fish oil is liquid at ambient temperature, but becomes significantly more

viscous at the colder temperatures used in its production. To facilitate the

separation of fish oil from the fish meat, producers could elevate slightly the

processing temperature so as to solve this viscosity problem. This strategy is

used in the dairy industry to separate cream from milk, although it should be

pointed out that fatty acids in milk are more saturated, and therefore, less

susceptible to lipid oxidation.

Membrane phospholipids have a larger surface area than muscle triacyl-

glycerols, tend to include fatty acid side chains with a higher level of unsatura-

tion, and are often associated with pro-oxidative processes such as those

occurring in the mitochondria. They are also important components in semi-

permeable membranes, which require certain fluidity for proper functioning, and

this property is a direct function of the unsaturation degree of the fatty acid side

chains of phospholipids. For all these reasons, membrane phospholipids are

more susceptible to oxidation than intramuscular triacylglycerols, and even

though their content is lower than that of triacylglycerols, they contribute more

to rancidity development in fish meat (Hultin et al., 2005). The problem is

complex, because phospholipids are difficult to separate from minced fish owing

to their amphiphilic characteristics.

Although the higher levels of polyunsaturated fatty acids (PUFA) in fish oil

have been correlated with cardiovascular health improvement, they are highly

susceptible to lipid oxidation. Omega-3 (!-3) fatty acids are considered

èssential' diet components, because they cannot be synthesized by humans. The

!-3 nomenclature refers to the third carbon atom in the fatty acid chain where

the first double bond (C=C) occurs counting from the methyl end (Fig. 4.5). The

health benefits of fish oils have been ascribed to their eicosapentaenoic (EPA,

20:5!-3) and docosahexaenoic (DHA, 22:6!-3) content. Although these oils are

highly valued by consumers, if fish products are not properly processed and

stored, their contribution to rapid rancidity can be offensive.

4.4 Recovery of functional proteins and lipids fromby-products

4.4.1 Isoelectric precipitation and solubilization of fish muscle proteins

The pH adjustment of a protein solution to its isoelectric point (pI) is used in

manufacturing cheese and soy protein isolates. In cheese making, pH is lowered

to pH 4.6 by direct acidification or the action of lactic acid bacteria, which

precipitates casein at its pI and forms the curd.

The pI of fish muscle proteins is at pH 5.5 and the proteins precipitate at this

pH while becoming gradually soluble as the pH is more acidic or basic than this

value. The isoelectric precipitation and solubilization of fish muscle proteins

with concurrent separation of fish oil was recently patented (Hultin and Kelleher

1999, 2000, 2001, 2002) by food scientists from the University of Massachusetts

and is under investigation in several laboratories. The temperature during the


following five processing steps is controlled at 1±8ëC to minimize protein and

lipid degradation (Fig. 4.7):

(1) Homogenization of by-products in a meat homogenizer (for example MCH-

10, Stephan Machinery, Columbus, OH) reducing particle size to below

0.2mm to increase surface area for the protein solubilization in the next step.

(2) Solubilization of fish muscle proteins at either acidic or basic pH. This pH

shift results in weaker protein-protein hydrophobic interactions, increased

electrostatic protein-protein repulsion and more predominant protein-water

interaction. As proteins start interacting with water, viscosity increases

drastically, but drops when the proteins become water soluble. This viscosity

increase may cause mixing problems, poor pH control and foam formation

unless the solution is maintained at the desired pH, which is facilitated by a

recovery system working in a continuous mode instead of batch.

(3) Centrifugation to separate the solution into a light, medium and heavy fraction

corresponding to fish oil, solubilized muscle proteins, and fat-free impurities

(bones, skin, scale, skin and insoluble proteins), respectively. While the

hydrophobic triacylglycerols are relatively easy to separate from the solution,

the membrane phospholipids are relatively persistent due to their amphiphilic

characteristics. The crude fish oil, rich in !-3 PUFA such as DHA, EPA, and

ALA, can be further processed into numerous food and other applications.

Although the relatively extreme pH values used in this technology could cause

degradation of fatty acids, it appears to be minimal (Fig. 4.8). This is likely due

Fig. 4.7 Diagram of the isoelectric precipitation and solubilization technology withconcurrent oil separation proposed for processing fish by-products. Materials in boxes are

fractions to be further processed into food and other applications.


Fig. 4.8 Effect of the solubilization pH used in the solubilization and isoelectric precipitation technology on the recovery !-3 and !-6 fatty acids.

to the low temperature and short exposure time during the solubilization and

first separation (Steps 2 and 3). The heavy fraction is essentially fat-free and

rich in minerals and therefore, can be used in formulating animal feed and pet

foods (Table 4.2). Since the fish oil has been removed from this fraction,

unlike typical fishmeal, it should not impart a fishy odour to the meat of

animals fed this fraction.

(4) Precipitation of proteins in the medium fraction by pH adjustment to the pI

of the fish muscle proteins (5.5). Similarly to the pH adjustment in the

second step, as proteins gradually stop interacting with water dipoles, the

viscosity increases significantly. This viscosity problem can be solved by

maintaining the pH at 5.5, which is again facilitated by a continuous instead

of a batch recovery system.

(5) Centrifugation to recover the precipitated fish muscle proteins, which can

be used as a food ingredient. In continuous systems, the water recovered is

reused in Step 1.

The solubility curves of fish muscle proteins show that myofibrillar proteins

are soluble at acidic and basic pH (Fig. 4.9). Minimum water solubility, and

therefore, maximum protein precipitation, occurs at pH 5±6. However, when IS

is adjusted to 0.2, the minimum solubility shifts by ~1 pH unit and myofibrillar

proteins precipitate at more acidic conditions. If acid or base is used in Step 2,

then a base or acid, respectively, is required to adjust pH to 5.5. In either case,

the salt formed increases the IS, and if the water is recycled as in a continuous

recovery system, salt build up will occur, changing the pH at which myofibrillar

proteins precipitate. Finally, sarcoplasmic proteins are soluble at both acidic and

basic pH and do not precipitate with myofibrillar proteins (Fig. 4.8). However,

as the IS increases, they begin to precipitate at pH 5.5. Therefore, sarcoplasmic

proteins will be recovered in continuous systems, due to salt build up.

The isoelectric solubilization and precipitation technology results in rela-

tively high protein recovery even when using a batch mode system consisting of

beakers and a typical laboratory centrifuge (Table 4.3). It is likely that a

Table 4.2 Ash contents (dry basis) of fractions recovered from krill and trout framesusing the isoelectric solubilization/precipitation technology compared to krill tail meatand boneless skinless trout fillets, respectively

% Ash % AshKrill tail meat 11.1 Boneless skinless trout fillet 5.5Whole krill 17.4 Trout frames (by-products) 13.9

2.0 6.0Recovered fat-free impurities 41.1

Protein 2.5 4.3 2.5 2.1solubilization 3.0 4.0 Protein 3.0 1.6pH 12.0 4.9 solubilization 12.0 0.9

12.5 5.7 pH 12.5 1.413.0 5.7 13.0 2.1


continuous process would result in even higher recovery yields. In general,

recovery yields are slightly higher for acidic solubilization (Table 4.3); however,

protein gels made from proteins recovered at basic pH have higher texture

quality (Fig. 4.10) and are described as firmer and whiter when compared to

those obtained from proteins recovered at acidic pH (Hultin et al., 2005).

Fig. 4.9 Solubility of myofibrillar and sarcoplasmic fish proteins as a function of pHand ionic strength (IS).

Table 4.3 Protein recovery yield from processing trout by-products by the isoelectricsolubilization/precipitation technology

pH %(solubilization/precipitation) protein recovery1

2.5/5.5 89.02.5/5.0 81.92.5/6.0 85.92.0/5.5 91.33.0/5.5 86.212.5/5.5 84.412.5/5.0 77.712.5/6.0 83.412.0/5.5 82.913.0/5.5 88.1

1 Calculated as (protein concentration in by-products/protein concentration in the recovered proteins)� 100, all on a dry basis.


The materials recovered from fish by-products in Step 3 include fish-oil rich

in !-3 PUFA and the fat-free fraction rich in minerals such as Ca, Mg and P, and

in Step 5 proteins recovered along with water to be reused in Step 1 (Fig. 4.11).

If the proteins recovered are for use in foods, knowing their nutritional value is

essential. The proteins recovered from trout frames and whole krill contain all

essential amino acids (EAA), and even though they fall short when compared to

the ideal pattern established by the Food and Nutrition Board, they are an

Fig. 4.10 Effect of solubilization pH on the texture of gels prepared from proteinsrecovered from fish by-products.

Fig. 4.11 Major materials recovered from processing fish by-products using the newisoelectric precipitation and solubilization of proteins technology: (a) Fish oil recoveredin Step 3. (b) Fat-free fraction containing bones, skin, scale, fin, insoluble proteins andothers recovered in Step 3. (c) Proteins recovered in Step 5. (d) Gel-forming ability of

recovered proteins.


Table 4.4 Essential amino acid composition of the proteins recovered from krill and trout frames using the isoelectric solubilization/precipitationtechnology compared with that of a soy protein isolate and the high-quality protein pattern established by the Food and Nutrition Board Research Council

Essential amino acids

Thr Val Met Ile Leu Phe His Lys Trp Total Average

trout frames 1.8 2.2 1.4 1.8 3.1 1.6 1.2 3.5 0.5 17.2 17.2

Trout proteins 2.0 3.7 4.6 2.6 3.9 6.6 3.4 2.1 7.4 1.0 35.3solubilized at pH 2.5 3.4 4.3 2.2 3.6 6.0 3.1 1.9 6.7 0.9 32.3 34.3

3.0 3.7 4.7 2.6 4.0 6.6 3.4 2.1 7.3 0.9 35.2

12.0 3.8 5.0 2.6 4.2 6.9 3.5 2.3 7.6 1.1 37.212.5 3.9 4.9 2.6 4.1 6.9 3.5 2.2 7.6 1.1 36.9 37.413.0 4.1 5.1 2.6 4.3 7.1 3.7 2.3 7.8 1.2 38.2

whole krill 2.2 2.6 1.5 2.5 4.0 2.2 1.1 4.4 0.7 21.2 21.2

Krill proteins 2.0 4.8 6.0 2.9 5.5 9.0 5.0 2.6 9.2 1.5 46.6solubilized at pH 2.5 4.5 5.8 3.2 5.7 8.9 4.9 2.5 9.2 1.6 46.3 47.0

3.0 4.8 5.9 3.3 5.9 9.2 5.2 2.6 9.6 1.6 48.1

12.0 4.6 5.8 3.4 5.7 8.8 5.1 2.7 9.2 1.7 47.012.5 4.5 5.6 3.2 5.5 8.6 5.0 2.5 8.9 1.5 45.3 45.513.0 4.4 5.5 3.1 5.5 8.4 4.8 2.5 8.7 1.5 44.3

soybean 3.9 4.6 1.1 4.6 7.8 5.0 2.6 6.4 1.4 37.4 37.4

FNB 3.5 4.8 2.6 4.2 7.0 7.3 1.7 5.1 1.1 37.3 37.3

Source: Adapted from Hui, Y.H. 1999. Soybean and soybean processing. In: Wiley Encyclopedia of Food Science and Technology, 2nd edn. Francis, F.J. (ed.). JohnWiley & Sons. Hoboken (United States). Abbreviations: Thr threonine, Val valine, Met methionine, Ile isoleucine, Leu leucine, Phe phenylalanine, His histidine, Lyslysine, Trp tryptophan.

excellent source of methionine and lysine (Table 4.4). Lysine concentration is

also critical for some non-food applications. For example, lysine-rich proteins

can be chemically modified to make biodegradable super-absorbent hydrogel

(SAH) trapping 400 g water/g SAH and are potential substitute for non-

biodegradable hydrocarbon-based SAH used in diapers, paper towels and others

(Damodaran, 2004).

Krill has extremely potent endogenous proteolytic enzymes limiting the

development of food products from krill (Suzuki, 1999; Kolakowski and

Lachowicz, 1982). Beef plasma protein (BPP) has been used as a protease

inhibitor for surimi produced from fish species prone to enzymatic proteolysis

such as Pacific whiting (Choi et al., 2005). When krill protein paste was

formulated without BPP and slowly heated in a dynamic rheometer, extensive

proteolysis occurred up to 60ëC and the proteins failed to form a gel. However,

when BPP was added to the krill protein paste and subjected to the same heat

treatment, the recovered proteins gelled, suggesting that krill proteases are

present in the proteins recovered (Fig. 4.12). There are several protease

inhibitors commercially available besides BPP.

4.4.2 Some equipment considerations

Following homogenization (Step 1), the homogenate is pumped to the first bio-

reactor for a 10-min solubilization reaction (Step 2). The bioreactor is equipped

for continuous pH control, and because the incoming homogenate pH is close to

neutrality (~6.6±7.0), a base will be rapidly pumped into the vessel to adjust its

pH to 11. Bioreactors are also equipped with mixing baffles to prevent pH

Fig. 4.12 Improvement of the viscoelastic properties of krill protein gels using beefplasma proteins (BPP).


gradients and excessive foaming. A refrigerant is used to maintain constant

temperature while small pumps are used to inject food-grade emulsion breakers

and antifoam agents. The experimental recovery system shown works at 300 L/h

and can process ~43 kg/h of fish by-products (Fig. 4.13). Although these small

scale bioreactors are manufactured from glass and stainless steel components,

industrial strength polyethylene can be used in a fish processing plant. Based on

the experimental system (Fig. 4.7), a modular 600-L bioreactor has been designed

to process 12 tonne/day of fish by-products.

Following the 10-min pH adjustment in Step 2, the solution is pumped to a

decanter centrifuge (Fig. 4.14) working typically below 4000 g and commonly

used in surimi processing plants. However, surimi technology does not work

under acidic or basic pH, therefore, the assessment of an available decanter

should be performed prior to use with this protein recovery technology. There

are no pH issues when separating proteins (Step 5); however, this can be

relatively slow unless the particle size of the precipitated muscle proteins is

increased by promoting protein-protein hydrophobic interactions using an

extended precipitation time (~24 h) in Step 4.

The particle settling velocity under the centrifugal force (g) depends on the

density differential between phases (��), viscosity (�), and particle size

Fig. 4.13 Bioreactors equipped with automatic pH and temperature controls, continuouspumping of feed and treated stream, and dosing of food-grade additives such as emulsionbreakers, protein flocculants and antifoaming agents. Bioreactor A is used for proteinsolubilization (Step 2) while Bioreactor B is used for isoelectric precipitation (Step 4). A

control box is placed between both bioreactors. This configuration is working in acontinuous mode at flow rate of 300L/h.


expressed as equivalent diameter (D), which is the only variable that a processor

can modify in this protein recovery technology:

S � �� g � D2

��4:1�

Using only 10-min in Step 4, protein particle size can be increased by adding

commercially available flocculants approved by local authorities (Figs 4.15 and

4.16).

4.5 Protein recovery from surimi processing water

Surimi is minced fish meat repeatedly washed and dewatered that is used as raw

material to produce seafood analogues such as crabmeat substitutes. In the year

2002, the annual frozen surimi production in the USAwas over 95 thousand tonnes.

In the Pacific Northwest, the most utilized fish species are Pacific whiting and

Alaska Pollock. However, processing fish using surimi technology recovers only

myofibrillar proteins (Lee, 1999), while the isoelectric solubilization/precipitation

allows the recovery of 78 to 91% of all by-product proteins (Table 4.3).

The relatively low protein recovery by the surimi technology means that

proteins accumulate in the surimi wash water. The effluent water is high in

biological oxygen demand (BOD), and therefore, should be treated before

Fig. 4.14 Decanter centrifuges, typical separating equipment used in fish processing,could be utilized in the new isoelectric solubilization/precipitation technology. (a)Commercial unit shown by courtesy of Alfa Laval. (b) Cross-sectional view of the

decanter centrifuge bowl.


Fig. 4.15 Reduction of the supernatant optical density by 10-min incubation of the pH precipitated fish proteins with an anionic flocculant of highmolecular weight.

discharging it into local watercourses. Even more important than the low

recovery yield is the use of large amounts of freshwater, about 20 times the

weight of the deboned meat (Lee, 1999). The low process efficiency, high

freshwater consumption, and deleterious environmental impact of surimi plants

are creating in some regions political pressures for their shutdown. At present,

there is a pending court case filed by the National Environmental Law Center

(NELC) against owners and operators of a seafood plant in Oregon. NELC claims

that `the plant has been routinely violating the Clean Water Act, degrading local

waterways and threatening endangered salmon and steelhead' (NELC, 2003).

A chitosan-alginate treatment has been proposed recently at Oregon State

University as a new technology alternative to lower the biological oxygen

demand (BOD) of water discharged from surimi processing plants. Surimi wash

water (SWW) can be treated effectively with a chitosan-alginate complex

prepared at the optimum chitosan to alginate mixing ratio of 0.2 (w/w) and used

at a complex concentration of 0.1 kg/ton of SWW (Fig. 4.17) (Savant and

Torres, 2000, 2003; Wibowo, 2003; Wibowo et al., 2005a,b, 2006a,b). Chitosan,

the deacetylated derivative of chitin, is recovered from crustacean processing,

particularly shrimp. After the meat is extracted, the shells are demineralized

using hydrochloric acid, washed and dewatered to obtain chitin which is then

deacetylated chemically or enzymatically. Alginate, on the other hand, is a

polysaccharide extracted from the cell walls of brown seaweeds and used in the

food industry as a thickener, stabilizer or gelling agent.

The crude protein content of the insoluble solids recovered by the chitosan-

alginate complex technology is over 70% (Wibowo et al., 2005b, 2006a), while

the amounts of recovered proteins vary with the concentration present in SWW,

which ranges from 0.5±2.3% (Lin and Park, 1996; Morrissey et al., 2000). After

Fig. 4.16 Supernatant appearance after 10-min incubation of the pH precipitated fishproteins with an anionic flocculant of high molecular weight (b) as compared to an

untreated control (a).


treatment with the polymeric complex, the proteins are recovered by centri-

fugation and can be sent to a disposal site, incorporated into surimi, or sold as a

feed ingredient for feeds. Recovering protein from SWW not only produces

protein for food and feed production, but also generates treated water for

potential reuse in the plant.

Proteins recovered from SWW have high concentrations of essential amino

acids. Animal studies have demonstrated the superior nutritional value and the

safety of SWW solids recovered by Chi-Alg when tested at the levels recom-

mended by commercial producers of animal feeds, i.e., under 15% (Wibowo et

al., 2005a). These studies showed no difference in feed consumption and growth

rate, while post-mortem examination of internal organs showed no visible signs

of damage caused by feeding the experimental diet. Blood analysis using 20

indicators confirmed the superior nutritional value and safety of SWW solids

recovered by Chi-Alg. Subsequent studies demonstrated that 100% substitution

of dietary protein by these SWW proteins was also safe and nutritionally

equivalent or superior to other protein sources showing higher protein efficiency

ratio (PER) and net protein ratio (NPR) than the casein control (Wibowo et al.,

2006b). This outcome has economic implications for the region where a surimi

plant is located. For example, in Oregon, not a major poultry producer, an

estimated 100 thousand tonnes of feed are needed to sustain broiler production

representing an excellent market opportunity for recovered SWW proteins.

Many protein sources have been employed to improve the mechanical

properties of surimi gels. The most frequently used are egg white and whey

protein concentrates; other sources such as leguminous extracts and porcine

plasma protein have been proposed, too. These proteins are added to inhibit the

Modori phenomenon, i.e., the proteolytic degradation of fish myosin when gels

are incubated at about 60ëC, or to improve the setting phenomenon associated

with improved mechanical properties by the action of endogenous and added

transglutaminase enzymes (An et al., 1996; GarcõÂa-CarrenÄo, 1996; SaÂnchez et

al., 1998; Benjakul et al., 2001). It appears that when added to surimi, low

concentrations of SWW proteins can improve their mechanical properties with

minimum impact on colour (RamõÂrez et al., 2006).

Fig. 4.17 Surimi wash water (SWW) treated with a chitosan-alginate polymericcomplex prepared at a 0.2 weight ratio and used at rates lower and higher than the

recommended value (0.1 kg/ton SWW). (a) Untreated SWW, (b) SWW adjusted to pH 6,(c±f) SWW adjusted to pH 6 and after addition of 0.05, 0.1, 0.2 and 0.3 kg/ton SWW.


4.6 Conclusions

The amounts of by-products generated from processing aquatic foods and the

volumes of underutilized by-catch, discards, and low-value fish are staggering.

At the same time, overfishing of several species is a common problem.

Aquaculture has experienced great growth during the past 25 years, filling the

supply gap that cannot be provided using natural resources. However, the

environmental impact of this industry is beginning to slow its growth. It appears

that the only great opportunity is to improve fish processing so as to better

utilize existing resources. The new technology of isoelectric precipitation and

solubilization of fish muscle proteins with concurrent separation of fish oil for

the processing of by-product provides a great opportunity on a large scale. In the

particular case of the surimi industry, the new technology based on using natural

polymeric complexes for the treatment of process water, offers an opportunity to

alleviate its environmental impact, freshwater use and low yield limitations. In

addition to basic research on recovering valuable fractions, it is necessary to

develop applications for the recovered products. Government agencies, industry

and academia must collaborate in this effort.

4.7 References

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5.1 Introduction

5.1.1 Waste from primary processing

Filleting is a primary method of processing fish in many countries around the

world. In the preparation of fish for today's consumer up to 50% of the whole

fish is commonly discarded as waste (Windsor and Barlow, 1981). This com-

prises skeletal structure, intestinal organs, and also a large amount of edible fish

muscle that cannot easily be removed from the bone structure by conventional

fish filleting processes.

There is also additional wastage arising as a consequence of further processing

of fish fillets, e.g. skinning, incorrect weights, and visual appearance problems

such as blood spots. Notable exceptions to this are trout and herring, which are

usually sold or processed whole. Archer et al. (2001) estimated the quantity of

on-shore processing waste in the UK from demersal fish processing as 154 143

tonnes per year. The principle use for the waste has been for fishmeal production

(for which the processor will receive about £20/tonne waste). Fishmeal is

primarily used as animal feed. Hence for the primary fish processors the result is

a large difference in income between the fillets and the waste (Fig. 5.1).

Some specific types of waste, however, are utilised for human consumption,

e.g. cod livers for production of cod liver oil. Conversion of constituents of these

wastes into food for human consumption, useful products, or extraction of

valuable components prior to use for fishmeal would be both an improved

utilisation of the resource and could be financially beneficial to the primary

process. This chapter details the increased processing yield by flesh recovery

from by-products.

5

Increased processed flesh yield byrecovery from marine by-productsK. D. A. Taylor and A. Himonides, University of Lincoln, UK andC. Alasalvar, TUBITAK, Turkey

5.1.2 Physical processing methods and by-product recovery from filleting

waste

The remaining flesh attached to the filleting waste of fish, such as cod and

haddock, that cannot be removed by the filleting process, can be up to 60% of

the waste's weight (Ravichander and Keay, 1976). The recovery of this flesh is a

much studied field of waste utilisation. A variety of methods have been

developed for the separation and recovery of fish flesh. Generally, these can be

divided into mechanical and non-mechanical techniques (Grantham, 1981).

Non-mechanical separation techniques, consist of chemical and biochemical

methods, which normally involve the enzymatic, or acid proteolysis of waste

material and the recovery of the hydrolysed (solubilised) flesh, by means of

centrifugation, or filtration. The application of heat and the separation of the

denatured protein (flesh) from the skeletal bones, using pressurised water, has also

been reported (Grantham, 1981). Although the yields of recovery are generally

high with these methods, protein functionality is often irreversibly changed.

Mechanical separation techniques normally result in products that retain most of

the nutritional value and organoleptic characteristics of fish flesh. Simple physical

removal of tongues and cheeks can provide a nutritional and valued by-product.

The application and further development of the mechanical recovery of fish

flesh is desirable because it provides maximum yields at a reasonable processing

cost and produces a product (minced fish) that offers scope for further improve-

ment. As long as there is a demand for such products, the mechanical separation

of fish flesh from filleting waste offers a profitable alternative to the production

of fishmeal or fish silage.

5.2 Recovery of flesh from filleting waste

5.2.1 Fish cheeks and tongues

The cheeks and tongues of large fish, such as cod and haddock, are often

removed manually. Equipment to remove the cheek is commercially available,

Fig. 5.1 Price for fish and fish waste.


but has not been widely adopted (Regenstein, 2004). The meat from tongues and

cheeks can be used in pies, fishcakes or retextured products. However, fried cod

cheeks and tongues are considered as delicacies in different parts of the world

(e.g., Italy, Canada, and Spain). Hence they can command similar or higher

prices to fillets.

5.2.2 Flesh-bone separators

The development of flesh-bone separators started in Europe and North America

and evolved rapidly from the first machines available, taken from other

industries (fruit and meat industry), to the later specialised equipment of the fish

industry (Drews, 1976).

Most of the mechanical separation techniques are based on the physical

screening of flesh from non-flesh components through a perforated filter. Such

devices should have the ability to separate and recover as much of the remaining

flesh from the waste material as possible, without destroying the fibroid

structure of the flesh. This is normally achieved by pressing the raw material

through perforations small enough to retain the bones, but big enough to allow

the flesh to pass through. The smaller the perforations, the smaller the chances

are for a bone to pass through. However, very small perforations also means

unwanted reduction of the flesh particles, with serious results on the products

texture (Drews, 1976). Thus, there has to be a compromise between quality (in

terms of texture, number of bones, etc.) and also quantity (yields) of recovered

fish flesh. Flesh-bone separators can be divided into three main groups

depending on the operation principles used (Grantham, 1981).

· A belt and a drum is used by Baader, Pibun, Prince, Seffelaar and Looyen,

Yanigiya, and Yiedmaster. Many modifications of that system have been

developed.

· A screw feed and perforated cylinder system is used by Beehive.

· Two concentric cylinders (the inner perforated and rotating) which break up

the bones and separate flesh by a micro-groove principle are used by Paoli.

All systems have their own advantages and disadvantages. The belt and drum

systems benefit from readily adjustable pressure although belt wear can be high

when using raw materials with large, or hard bone particles. Most separation

equipment was designed to be quick to dismantle, so that cleaning can be easy

and thorough. It is absolutely essential that all parts of the machinery and

equipment that may come in contact with the product are hygienically clean in

order to minimise bacterial contamination. The screw cylinder systems do not

have the same wear problems, but generate higher shear rates and consequent

textural damage. In general, these systems are more expensive, but all give

similar yields ranging from 60±80% for whole fish to 30±70% for filleting

waste. Despite the apparently simple operating principles, the relationships

between pressure, perforation size, and perforation area with yield, contaminant

levels, and shear damage, are complex (Grantham, 1981).

Increased processed flesh yield by recovery from marine by-products 93

The Baader flesh-bone separator

The Baader separation technique is based on the physical screening of flesh from

non-flesh components through a perforated filter (Drews, 1976; Grantham,

1981). This is performed by a thick elastic conveyor belt and a metallic

perforated drum (Figs 5.2 and 5.3). Both belt and drum are driven by a gear

motor at the same speed.

The raw material goes through an opening in a feed hopper onto the belt,

which partly encompasses the drum and is pressed tightly against it by an

adjustable roller. The material is squeezed through the perforation, to the interior

of the drum and then it is discharged from the end of the drum by a stationary

screw. Thicker bones get embedded in the elastic bed which squeezes off all the

flesh around the bone. Bones and pieces of skin remain on the outside of the

drum and are removed by a scraper blade.

The rotation of the belt and drum are designed to minimise grinding or

tearing of the material which could destroy the fibroid structure of the flesh. A

small perforation size could also damage the flaky texture of the flesh, so even

though there is a range of drum hole sizes (from 1 to 10mm), perforations of 3

or 5mm give the best results in terms of compromising an acceptable texture

with a low percentage of bones escaping in to the mince and a high yield

(Drews, 1976; Grantham, 1981).

Fig. 5.2 Baader flesh-bone separator.


5.2.3 Minced fish

The term minced fish is very often used to describe the mechanically recovered

fish flesh. This term is one of convenience rather than accuracy since mincing

occurs naturally during separation rather than deliberately as if it were passed

through a mincer. It is indeed a product that retains most of its textural

characteristics compared with fish which has been minced (Ravichander and

Keay, 1976).

Mincing accelerates the deconformation, aggregation and cross linking of the

myofibrillar proteins. This results in a loss of the extractability and contractility

of the actomyosins, with a consequent increase in the objective toughness,

granularity and drip loss and a decrease in water binding capacity, emulsification

capacity, gel-forming ability, and rheological properties. These reactions are

exacerbated through the cross linking of the proteins with formaldehyde, which is

derived from the breakdown of trimethylamine oxide (TMAO). This is an

enzyme related reaction which is accelerated by mincing and the mixing of blood

and other organs with the flesh and predominantly occurs during frozen storage.

The gadoid fish species and particularly hake and cod are very susceptible to this

reaction (Babbitt et al., 1974). Denaturation in frozen mince products is also

accelerated by the rapid pH drop resulting from accelerated glycolysis during

mincing and by temperature cycling during frozen storage (Grantham, 1981).

Fat degradation can be accelerated by mincing, due to dispersion of fat

degrading enzymes and oxidation catalysts and the increase of the surface area.

Fig. 5.3 Baader flesh-bone separator showing the perforated drum and minceproduction.


Pelagic species are more susceptible to fat degradation, especially when

processed whole, due to the high lipid content and particularly the high levels of

unstable polyunsaturated lipids located on the skin, tissues, viscera and brain

that may contaminate the flesh during separation. Processing of filleting waste

(frames and flaps) from lean demersal species such as cod and haddock is thus

less susceptible to fat oxidation, due to negligible lipid content and absence of

contaminating materials such as skins, viscera, and brains.

One of the primary reasons for low acceptance of minced fish products is due

to the aesthetic deterioration that occurs during mechanical separation of the

flesh. Mincing normally results in a product that is darker in colour and more

heterogeneous than the actual flesh in the raw material. This is due to the natural

mixing of components such as blood, pigments, skin, and membrane particles,

bones, and other material that can pass through the perforations of the drum.

Consequently, this aesthetic deterioration is not so marked in mince produced

fish with coloured flesh such as salmon and salmon mince which can have a

value as high as £2000/tonne. Salmon mince can be used in fishcakes, minced

salmon nuggets, pastes, pates, and low cost ready meals.

Although there is an enormous number of minced fish products that have

been studied, reported, and developed (Bligh and Regier, 1976; King, 1976;

Regenstein, 1980), only a few of these are successfully established in the world

market. According to the same authors, poor recognition of the true value and

potential of fish mince is often associated with the trend to develop products that

mimic existing fish products, rather than creating something new that exploits

the natural properties of fish mince.

The world market of fish mince is dominated by products derived from

frozen fish mince blocks and surimi-derived products (Grantham, 1981; Suzuki,

1981). Frozen mince blocks are usually produced from fish mince, derived from

white fish such as cod and haddock. Fish mince is also incorporated into fillet

blocks. These blocks are intermediate material for the production of a range of

products such as coated fingers, steaks, cakes, sausages, patties, loaves, and

burgers. Some of these are simply formed and coated products whilst other are

totally transformed, reformed, and textured products (Grantham, 1981).

The manufacture of surimi initiated in Japan where it is still believed to be

the primary source of the population's protein intake. Surimi-based products

have also been introduced to North America and Europe. This market is now

facing a rapid growth. Surimi is basically frozen water-washed fish mince with

cryoprotecting substances (Suzuki, 1981). It is primarily manufactured from

Alaska pollack and other gadoid fish minces, although recent studies have

shown that even pelagic species can give acceptable products (Grantham, 1981;

Suzuki, 1981). Surimi is an intermediate processed seafood product, used in the

formulation/fabrication of a variety of products (Hall and Ahmad, 1997), such as

kamaboko and chikuwa (Suzuki, 1981). Fish mince is also used for the produc-

tion of canned products (Regenstein, 1980; Grantham, 1981), dried and inter-

mediate moisture products (IMP), fermented products, and for the production of

animal feed (Taylor and Alasalvar, 2002).


5.2.4 Problems of fish mince

Mechanical separation of flesh from demersal species such as cod and haddock

can accelerate the degradation of lipids, proteins and bacteriological quality and

also results in a product that is often aesthetically unacceptable. However, one of

the great advantages that minced fish offers is the possibility of great control

over product flavouring, texture, appearance, and storage properties. Minced

fish can be mixed with a variety of foodstuffs, including other fish. In sub-

sequent processing, a range of textural variations can be induced by mechanical

mixing, often in the presence of salt and other additives, and furthermore the

addition of flavour and other desirable additives is more effectively

accomplished (Ravichander and Keay, 1976).

With respect to lipid oxidation, the use of antioxidants is the most widely

studied and applied method of stabilisation. However, other techniques such as

glazing and oxygen impermeable packaging were found to inhibit the oxidative

deterioration of mince blocks. Protein stability and functionality enhancement of

de-boned flesh is a widely studied subject, mainly due to the particular suscepti-

bility to protein degradation and also to the high inherent functionality of mince.

With respect to protein stabilisation during frozen storage, a wide range of

stabilisers and cryoprotecting agents, such as polyphosphates, sugars, poly-

saccharides, and peptides, have been studied (Grantham, 1981).

The study of protein functionality involves both the preservation of the

inherent functionality of fish mince and/or the enhancement of certain functional

properties depending on the uses of the material. For example, many cryo-

protecting agents, such as phosphates and salts, which are used for the stabilisa-

tion of mince during frozen storage and thus maintain the inherent functionality

of mince, are also used as functionality enhancers for their solubilisation,

complex forming, and gel-forming effects. Hydrocolloids are used extensively

to increase viscosity, water binding, and gel-forming capacities. A wide range of

proteins are used to improve binding properties. Sugars are used in dried mince

products to reduce denaturation, whilst starches are used as thickeners and

gelling agents. Furthermore, fats and oils are used to improve succulence in

combination with added proteins as emulsifiers (Grantham, 1981).

Whiteness and colour homogeneity of fish mince derived from white flesh fish

are prerequisites that determine retail price and also consumer acceptance, with

the whiter mince attracting a much higher price (Meacock et al., 1997). Methods

of improving mince appearance involve treatment of the fish material prior to

mechanical separation, whitening of the recovered flesh, or masking of the mince

colour with coloured additives. The addition of sawdust waste from band sawing

of frozen blocks of white fish to fish mince can also improve the whiteness.

When processing whole fish, the removal of heads and guts is essential prior

to separation. This minimises the contamination of the recovered mince from

coloured, non-flesh components such as blood, skin, and brain fragments, and

improves the appearance and also storage life of mince (Ravichander and Keay,

1976; Connell and Hardy, 1982). Contamination of mince also occurs during the

mechanical separation of cod and haddock frames, due to the crushing of the


backbone section and the subsequent mixing of the blood with the separated

flesh. Thus, the removal of this section prior to separation improves the

appearance of the recovered mince (Ravichander and Keay, 1976; Connell and

Hardy, 1982).

5.3 Recovery of flesh from demersal species

The mechanical recovery of the remaining flesh attached to the frame and flap,

which cannot be removed by the filleting process is a much studied field of

waste utilisation and is a potential alternative to fishmeal production. With the

advent of drum flesh bone separators, fish mince production from the filleted

fish frames and trimmings began, with up to 10% of the whole fish weight or

25% of the gutted weight of an averaged sized cod being recovered (Connell and

Hardy, 1982). Table 5.1 shows the recovery of minced flesh from various parts

of cod waste. This recovered flesh is nutritionally similar to the fish fillet.

However, the visual appearance of mince which is derived from the filleting

waste (flaps and frames) of white fish such as cod or haddock, is poor,

principally due to the presence of pigments such as haemoglobin from blood and

other haem proteins, mixed with the flesh during separation. This discolouration

does not present a major problem in the case of non-white fish such as salmon.

There are also associated quality problems, i.e. the lack of texture and the

poor microbial profile that can result from poor handling and storage of the

waste, and the additional processing required for the production of fish mince.

Consequently, the amounts paid for the mince are generally mediocre, in the

region of £800/tonne for white fish mince. Fish mince colour partly determines

retail prices and consumer acceptance, with the whiter minces attracting a higher

price than those contaminated with traces of blood. In the UK, fish mince is

classified as Class A (little discolouration), Class B (discoloured with blood and

haem proteins), and Class C (discoloured), and the value ranges from £400 to

£1000/tonne. For these reasons, development of a method of producing a white

or whiter fish mince is commercially desirable.

The use of recovered flesh is primarily for human consumption in products

such as fish cakes or fish fingers. As a major factor in consumer unacceptability

for mince from white fish is the fish mince colour, several approaches have been

investigated to overcome this.

Table 5.1 Recovery of minced cod fish using Baader 694 flesh-bone separator

Recovery (%) Weight of fish (%)

Whole frame 72±82 8±10Frame with back bone removed 55±65 6±8

(of whole frame)Cod flaps 60±67 3±4


5.4 Quality and improvement of fish mince

5.4.1 Washing

Washing or soaking of waste with water, prior to separation, removes part of the

superficial contamination of the waste (blood, clots, and impurities) and has a

small effect in improving the colouration (i.e. in reducing the red pigmentation)

of the recovered mince (Ravichander and Keay, 1976; Grantham, 1981).

Direct water washing of fish mince can elute part of the blood and improve

the colour of mince to an off-white colouration (Ravichander and Keay, 1976;

Grantham, 1981; Connell and Hardy, 1982). Alkali or acid washing is believed

to improve the separation of blood (Steinberg et al., 1977; Grantham 1981).

Common problems generated from the washing of the mince include the

removal of the excess water and the significant loss of soluble proteins, unless a

method of recovering the proteins is applied (reverse osmosis, ultrafiltration, or

coagulation).

5.4.2 Use of high pressure

Possible decolouration of haemoglobin in fish mince via the use of high pressure

has been postulated by Hoover et al. (1989), but textural changes are a possible

side-effect of such a process.

5.4.3 Removal of back bone prior to flesh bone separation

Most of the blood and discoloration is in/along the spinal cord. Thus, during

mechanical separation of mince from cod and haddock frames, the crushing of

the backbone section results in mixing of this blood with the separated flesh. The

removal of this section prior to separation improves the appearance of the

recovered mince (Ravichander and Keay, 1976; Connell and Hardy, 1982).

Himonides (2001) investigated the effect of removal of the backbone by a V

cut prior to flesh bone separation. This process provided a much better quality

mince than that of the whole frames with only about 25% reduction in recovery

of flesh from the frame (Table 5.1). The recovered minced flesh accounted for

6±8% of the weight of the original cod. Whilst this was shown to work well in

the laboratory, it is uneconomic on a commercial scale (until an effective

machine is available for the removal of the backbone) owing to the labour

required.

5.4.4 Whitening the mince with hydrogen peroxide (H2O2)

Hydrogen peroxide has been used to bleach white fish mince such as that

derived from cod (James and McCrudden, 1976; Young et al., 1980; Connell

and Hardy, 1982), but problems have been reported concerning textural changes,

and loss of proteins and amino acids through dissolution and oxidation.

The study of Young et al. (1980) revealed that the application of 0.75% H2O2

at alkaline pH has a remarkable bleaching effect on mince derived from cod

waste. James and McCrudden (1976) found that 1% sodium polyphosphate


added into the whitening solution has a synergetic effect on both whitening and

texture. They also believed that the nutritional value of treated fish was retained.

The effect of the treatment on the oxidation state of the lipids, measured by

peroxide and epoxide values appeared to be negligible. The same authors,

however, found a significant loss of protein due to dissolution, increase in

insolubilisation of protein and loss of sulphur-containing amino acids, even

under relatively mild treatment conditions. According to the same authors, other

workers found no change in the amino acid content of other proteins treated with

H2O2. The effect of H2O2 on the amino acid content and protein digestibility of

saithe was investigated by Raksakulthai et al. (1983), who found that treatment

of samples with 3.2 g/L H2O2 for a period of 18 h oxidised methionine. Oxida-

tion mainly occurred during the first five hours of treatment. Methionine was not

entirely oxidised even after the addition of a further 3.2 g/L H2O2. The authors

also stated that other amino-acids seemed to be essentially unaffected by the

oxidant, while digestibility was slightly increased by the treatment. Also, many

workers have reported the disinfecting action of H2O2 on various foods

including fish (Stout and Carter, 1983; Wheaton and Lawson, 1985; Shibamoto

et al., 1993).

In spite of the reported difficulties using H2O2, Himonides et al. (1999)

considered that this had a commercial and technical potential for whitening the

mince. They observed that the discolouration of flaps is mainly a superficial

phenomenon and consequently treated whole flaps with H2O2 prior to separation

for the production of fish mince. This differs from previous works in which the

whitening treatment occurred after the flesh was separated and minced. The

application of the bleaching agent prior to mincing should minimise protein

insolubilisation and also the loss of water-soluble proteins, with minimal effect

on the nutritional value or texture compared to the direct application of H2O2 on

fish mince. The effect of concentrations of peroxide and time of soaking is

shown in Table 5.2.

Samples were removed from the H2O2 solution after the time shown, soaked

in water for 10 min and then assessed visually for colour improvement, and

textural damage. Immediately after treatment, flaps (particularly those of small

size) may appear to have a loose flesh texture, with areas of skin that appear to

be swollen owing to the oxygen trapped under the surface of the skin. This

textural effect, however, is minimised after soaking flaps in water, or even after

leaving the flaps out of solution, for a short period of time (30 min), during

which the oxygen bubbles dissipate. For industrial applications, concentrations

of 5±8 g H2O2/kg were recommended, with a soaking time of 90 min.

Visual sensory evaluation of the mince derived from standard and treated

flaps (soaking for 1.5 h in 8 g/L H2O2) showed noticeable improvement in the

elimination of the red colouration. The degree of improvement depends on the

initial condition of flaps and presumably on the depth of blood discolouration in

the flesh. Colourimetric measurements confirmed a highly significant reduction

of the red pigment (a values) and also a reduction of the yellow pigment towards

the grey area (b values). No significant difference was detected between the taste


and odour of treated and standard cooked mince, by the sensory evaluation

carried out before and after 8 months cold storage (±28ëC) of the samples.

Microbiological analysis showed that treatment with H2O2 significantly

reduces the total viable count of cod flaps. No residual H2O2 was detected in

flaps immediately after treatment, using oxygen electrode technique (the limit of

detection was 3�g/g) (Himonides et al., 1999). Immediately after treatment and

washing of flaps, it is possible that there may be residual H2O2 trapped in the

flesh. It has been demonstrated, however, that hyperoxidase (catalase), which

exists in the flesh cells, converts residual H2O2 into O2 and H2O, particularly

during mincing, owing to the release of the enzyme from the cells. The existence

of the enzyme was confirmed in cod and haddock mince and was found to be

present in the fish mince even after the eight months of frozen storage period.

Since H2O2 cannot be detected after treatment and the products (O2 and H2O)

are non-toxic, H2O2 can be characterised as a processing aid rather than a food

additive. As a result, and according to the food legislation, the declaration of the

use of H2O2 by the processor is not necessary. The procedure developed for and

used by industry is shown in Fig. 5.4.

5.4.5 Whitening the mince with titanium dioxide (TiO2)

An alternative to removing the discolouration, or bleaching the mince, is to

mask the colour using whitening chemicals, such as TiO2 (Grantham, 1981;

Connell and Hardy, 1982), vegetable fat based agents (Ravichander and Keay,

1976), hydrophilic colloids such as milk, gum hydrocolloids, mixtures of sugars,

surfactants, and fats. Polyphosphates can have a similar whitening effect via the

dispersion of myosin sol.

Common problems with colour masking include the generation of a non-

uniform colour, (given the highly variable colour of fish mince), the possible

artificial appearance of the resultant colour, and the necessity to produce an

emulsion, or suspension, which is capable of surviving the cooking/freezing

process, in order to maintain the uniform colour. Consequently, Meacock et al.

Table 5.2 Whitening effect of a range of H2O2 concentrations on flaps, for differenttime intervals

H2O2 (g/L) 5 min 10 min 30 min 40 min 60 min 120 min

0.5 0 0 0 0 0 01 0 0 0 s.i. s.i. s.i.2.5 s.i. s.i. s.i. s.i. s.i. s.i.5 n.i. n.i. n.i. 3 3 38 n.i. n.i. n.i. 3 3 310 3 3 3 3 3 3

p.t.c. p.t.c. p.t.c. t.c. t.c. t.c.

0: no improvement, s.i.: small improvement, n.i.: noticeable improvement, 3: good whitening effect,p.t.c.: possible textural change, t.c.: textural change.


(1997) investigated the use of TiO2 coupled with a variety of dispersing agents

with the aim of identifying a system to overcome these problems and which also

would not affect the taste, odour, or texture of the material.

To determine which suspension agents might be most suitable, a series of

dispersed suspensions using TiO2 were prepared, using mixtures which were

chosen on their existing use as suspension agents in a variety of products. Stable

dispersions were achieved with several agents but only xanthan gum gave a

stable and non-odorous dispersion. However, a `musty' taste and odour

developed after a few weeks storage at room temperature of the dispersed

TiO2 suspension dispersed with xanthan gum. Thus, such dispersed suspensions

Fig. 5.4 Improved whitening of cod and haddock flaps using H2O2.

Fig. 5.5 Titanium dioxide based whitener.


should be used within 3 days of production. Fish mince produced using TiO2 in

water produce an uneven dispersion, with a toothpaste-type appearance to fish

mince products after cooking.

The TiO2/xanthan gum dispersion is easily incorporated into the mince, with

excellent distribution. It gives an even increase in whiteness which is stable after

frying the product or microwaving. Although at concentrations of up to 250 ppm

of TiO2, the whitening effect is negligible, the whiteness increases with further

increasing concentrations of TiO2. A level of 1 g of TiO2/kg mince gives a most

acceptable level of whiteness compared with the colour of cooked cod fillet. At

this level, no differences were detected by expert panels, triangle tests, or the

texture analyser regarding the resulting texture of whitened fish mince. Figure

5.5 summarises the whitening procedure.

5.4.6 Masking colour

As a major problem with full acceptance of minced flesh from white fish is the

colour, a possible alternative approach is to develop products which the

consumer would accept as being of a definite acceptable colour, other than

white. Products such as curried fish cakes and tomato flavoured fish cakes have

been developed (Meacock, 1994), but not yet produced on a commercial scale.

Initial consumer trials indicated that masking of the colour of the mince is an

acceptable method of upgrading, but suitable retail outlets have to be identified

for products such as curried cod fish burgers.

5.5 Future trends

Fish mince production is a viable process and is currently carried out using both

white fish and salmon filleting wastes. However, in the case of white fish waste

the use and hence the market for the mince is limited due to the colour of the

resultant mince. The value of white fish mince is approximately £800/tonne, but

does depend on quality, which is measured primarily by whiteness. The

recovery of 10% of the weight of the fish as fish mince is potentially financially

viable, and is practised by several firms. The additional costs include purchase

of a flesh-bone separator (which could be from £14,000 for a second-hand one

to £40,000), labour, electricity, and packaging/freezing. There are possible

techniques for improvement of the colour and the alternative approach to

develop products which the consumer would accept as being of a definite

colour.

Technological developments are likely to assist in the improved and

economic recovery of edible flesh from the by-products of filleting and other

primary processes. For example, an EU project funded to develop a process for

automated tuna head meat recovery reported that optimisation of the prototype

machine will make the production of automatically recovered meat

economically feasible (Stefansson, 2001). The incentive for improved utilisation


is threefold as it 1) reduces waste, and the problems of disposal are likely to

increase, 2) increases the utilisation of harvested fish for food, and 3) could

increase the profitability of processing firms.


In addition to the standard scientific literature, the following organisations are a

source of advice concerning the recovery of flesh from fishery by-products.

Sea Fish Industry Authority

St Andrews Dock

Hull, HU3 4QE

United Kingdom

Tel: +44 (0)1482 327 837

Fax: +44 (0)1482 223 310

Web-site: www.seafish.org

Baader UK Ltd

Nautilus House, Prospect Point

35 Waterloo Quay

Aberdeen, AB11 5BS

United Kingdom

Tel: +44 (0) 1224 597320

Fax: +44 (0) 1224 597321


Web-site: www.baader.com

5.7 References

ARCHER M, WATSON R, DENTON J W (2001), `Fish waste production in the United Kingdom

± The quantities produced and opportunities for better utilisation', Hull, UK,

Seafish Report No. SR537, The Seafish Industry Authority.

BABBITT J K, CRAWFORD D L, LAW D K (1974), `Quality and utilisation of minced fish

muscle', 2nd Technical Seminar on Mechanical Recovery and Utilisation of Fish

Flesh, Boston, MA.

BLIGH E G, REGIER LW (1976), `The potential and limitations of minced fish', in Keay, J N,

Proceedings of the Conference on the Production and Utilisation of Mechanically

Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 73±

77.

CONNELL J J, HARDY R (1982), Trends in fish utilisation, Surrey, UK, Fishing News Books

Ltd.

DREWS J (1976), `Development of fish boning machines', in Keay, J N, Proceedings of the

Conference on the Production and Utilisation of Mechanically Recovered Fish

Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 25±27.


GRANTHAM G J (1981), Minced fish technology: a review, Rome, Italy, FAO Fisheries

Technical Paper No. 216.

HALL G M, AHMAD N H (1997), `Surimi and fish-mince products', in Hall, G M, Fish

Processing Technology, 2nd edn, London, Blackie Academic & Professional, 74±92.

HIMONIDES A T (2001), `The improved utilisation of fish waste with particular reference to

the enzymic hydrolysis of fish frames for the production of fish protein

hydrolysates', PhD Thesis, University of Lincoln, Lincoln, UK.

HIMONIDES A T, TAYLOR K D A, KNOWLES M J (1999), `The improved whitening of cod and

haddock flaps using hydrogen peroxide', J Sci Food Agric, 79, 845±850.

HOOVER D G, METRICK C, PAPINNEAU A M, FARKAS D F, KNORR D (1989), `Biological effects

of high hydrostatic pressure on food micro-organisms', Food Technol, 43, 99±107.

JAMES A L, MCCRUDDEN J E (1976), `Whitening of fish with hydrogen peroxide', in Keay, J

N, Proceedings of the Conference on the Production and Utilisation of Mechanic-

ally Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station,

54±55.

KING F J (1976), `Past, present and future uses of minced fish in the USA', in Keay, J N,

Proceedings of the Conference on the Production and Utilisation of Mechanically

Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 78±

81.

MEACOCK G (1994), Ùnpublished data', University of Lincoln, Lincoln, UK.

MEACOCK G, TAYLOR K D A, KNOWLES M J, HIMONIDES A T (1997), `The improved whitening

of minced cod flesh using dispersed titanium dioxide', J Sci Food Agric, 73, 221±

225.

RAKSAKULTHAI N, AKSNES A, NJAA L R (1983), Èffects of hydrogen peroxide and of

sulphite and humidity on the amino acid composition and digestibility of fish

protein', J Sci Food Agric, 34, 619±626.

RAVICHANDER N, KEAY J N (1976), `The production and properties of minced fish from

several commercially important species', in Keay J N, Proceedings of the

Conference on the Production and Utilisation of Mechanically Recovered Fish

Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 18±24.

REGENSTEIN J M (1980), `The Cornell experience with minced fish', in Connell J J,

Advances in Fish Science and Technology, Surrey, UK, Fishing New Books Ltd.,

192±199.

REGENSTEIN J M (2004), `Total utilization of fish', Food Technol, 58, 28±30.

SHIBAMOTO T, LEONARD B, TAYLOR S (1993), Introduction to food toxicology, San Diego,

CA, Academic Press.

STEFANSSON G (2001), `Developing a process for automated tuna head meat recovery',

Final project report to the EU 35-01. FAIR CT-98-9079.

STEINBERG M A, SPINELLI J, MIYAUCHI D (1977), `Minced fish as an ingredient in food

combinations', in Proceedings of the Conference on the Handling, Processing and

Marketing of Tropical Fish, London, Tropical Products Institute and Ministry of

Overseas Development, 245±248.

STOUT V, CARTER G (1983), Àmes test for mutagenicity on Pacific whiting treated with

hydrogen peroxide', J Food Sci, 48, 492±495.

SUZUKI T (1981), Fish and krill protein: processing technology, Essex, Applied Science

Publishers Ltd.

TAYLOR T, ALASALVAR C (2002), Ìmproved utilisation of fish and shellfish waste', in

Alasalvar, C, Taylor T, Seafoods ± Quality, Technology and Nutraceutical

Applications, Berlin, Germany, Springer, 123±136.


WHEATON F W, LAWSON T B (1985), Processing aquatic food products, New York, John

Wiley & Sons Inc.

WINDSOR M, BARLOW S (1981), Introduction to fishery by-products, Surrey, UK, Fishing

News Books Ltd.

YOUNG K W, NEUMANN S L, MCGILL A S, HARDY R (1980), `The use of dilute solutions of

hydrogen peroxide to whiten fish flesh', in Connell J J, Advances in Fish Science

and Technology, Surrey, UK, Fishing New Books Ltd., 242±250.


6.1 Introduction

About 30% of total fishery landings is composed of by-catch (unconventional,

unexploited), but fish processing wastes can also be considered as underutilized

(Venugopal and Shahidi, 1995). In the past, part of this wasted material has often

been dumped or used without treatment for animal feed or as fertilizer. However,

due to the worldwide decline of fish stocks, a better use of by-catch and by-

products is deemed necessary. Today, particularly with the introduction of

refined enzyme technologies, the potential for taking advantage of value added

molecules already present in fish and invertebrate by-products is very high.

Employing enzymatic methods for protein or lipid recovery in fish or shellfish

processing as an alternative to mechanical or chemical treatments, which often

damage the products and reduce product recovery, renders it possible to produce

a large and diversified range of products for different applications. Among

products of interest are enzymes, lipids, chitin and chitosan, calcium, nucleic

acid, pigments, and biologically active peptides from fish protein hydrolysates.

Enzyme technology has evolved to become an integral part of the food

industry. This is due to the highly specific nature of enzymes. Enzymes are

active at very low concentrations and under mild conditions of pH and tem-

perature, which results in fewer unwanted side-effects in the production process

(Shahidi and Kamil, 2001). The enzymatic processes vary according to the

nature of the treated by-products. Usually, the type of enzyme which is

employed for extracting or solubilizing by-products may be directly associated

with the nature of the molecules to be extracted. For example, proteases are

utilized for the solubilization of the protein part of by-products but in recent

works, proteases were also used to enhance lipid extraction from fish tissues.

6

Enzymatic methods for marineby-products recoveryF. Guerard, University of Western Brittany, France

This chapter surveys the recent literature on the possibilities of processes

involving enzymatic biotransformation of fisheries and aquaculture by-products.

The interest of the extracted molecules from a nutritional or a medical point of

view will also be described.

6.2 Overview of by-products extracted by enzymatic methods

6.2.1 Definition of wastes and by-products

According to Rustad (2003), there is not any single definition of what is a `by-

product', but usually, by-product indicates something that is not regarded as an

ordinary saleable product but can be used after treatment. Thus `waste' refers to

products that cannot be used for feed or food but have to be composted, or

destroyed (e.g., by burning).

Recovery of edible portions in seafood processing is traditionally low,

ranging only from 20 to 50% and the resulting non-edible part consists of head,

viscera (entirely or partly: heart, spleen, stomach, intestines, piloric caeca, liver,

gall bladder, gonads, milt and roe), shell, skin and flesh remaining on the bone

(An and Visessanguan, 2000; Aspmo et al., 2005a; GueÂrard et al., 2004; Taylor

and Alasalvar, 2002). In addition, when harvesting fish and crustaceans, many

species that are not used for human food are also caught. These `trash' fish can

consequently be processed into useful products. The other by-product sources

are fresh fish, crustaceans, and molluscs, of high or good edible quality but not

having found any buyer. These could then either be frozen for subsequent

selling, or destroyed according to the regulations governing the fish markets.

Therefore, a large part of such material could alternatively be used as high

quality raw material. All these examples indicate clearly that there is no

established and permanent distinction between by-products and wastes.

6.2.2 Estimate of the available quantities of by-products

In the literature, there are only a few papers dealing with the yield of by-product

collected starting from the entire animal in terms of weight or percentage of

head, frame, skin, viscera (Fig. 6.1). For example, in cod fillet production, as

much as 60% of the whole fish is by-products, the backbone yielding about 15%

of the fish weight (Gildberg et al., 2002). In a typically automated salmon

filleting line, the fillets account for approximately 59±63% of the total weight

for a salmon with body weight of 5±6 kg. Other products from the filleting line

are salmon frame (9±15%), head (10±12%) and trimmings (1±2%) (Liaset et al.,

2003). Shell waste from shrimp Crangon crangon processing is a good source of

chitin and proteins with 17.8% and 40.6% in amount, respectively (Synowiecki

and Al-Khateeb, 2000). The lobster heads correspond to two-thirds of the total

weight with about 20% meat (Viera et al., 1995).

In 2002, more than 133 million tonnes (MT) of fish and shellfish were landed

(capture fisheries) or produced (aquaculture) (FAO report, 2004), of which about


100 MT were used for direct human consumption. The remainder (32 MT) was

used for the manufacture of fishmeal including feed for aquaculture, fish oil and

other non-food uses. The amount of processed fish (frozen, cured and canned)

for human consumption was estimated at 39 MT. This could be translated into

about 20 MT of fish by-products potentially available as a source of raw

material.

These data reveal that a large potential source of raw material exists globally

from the fisheries industry. The basic recoverable components of fish frames and

guts are mainly proteins and oils, while crustacean shell wastes provide calcium

carbonates and chitin.

6.3 Enzymatic extraction methods

Enzymatic methods have become an important and indispensable part of the

process used by the modern food and feed industry in order to produce a large

and diversified range of products for human and animal consumption (Shahidi

and Kamil, 2001).

6.3.1 Extraction of lipids

Health effects and PUFA sources

The beneficial effects of marine oils on human health were scientifically

recognized three decades ago and have been well supported by many scientific

Fig. 6.1 Fish by-products and their possible use (compiled from Andrieux, 2004;Gildberg et al., 2002; GueÂrard et al., 2004; Liaset et al., 2003).

Enzymatic methods for marine by-products recovery 109

studies and reviews over the past decade (BergeÂ and Barnathan, 2005; Hunter et

al., 2000; Kroes et al., 2003; Shahidi and Wanasundara, 1998; Vanschoonbeek

et al., 2003 ; Ward and Singh, 2005). They have been attributed to the long chain

n-3 polyunsaturated fatty acids (PUFAs) characteristic of marine oils, notably

cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-

docosahexaenoic acid (DHA). The n-3 PUFAs inhibit tissue eicosanoid bio-

synthesis, reduce inflammation, and lower serum triacylglycerol and cholesterol

levels (Osborn and Akok, 2002). EPA has a beneficial effect on the cardio-

vascular system and inhibits very low-density lipoprotein (VLDL) formation,

cell proliferation, and the inflammatory and allergic response (Uauy and

Valenzuela, 2000). DHA is a major structural component of the grey matter of

the brain and the eye retina and an important component of heart tissue,

consequently DHA plays an important role in brain development and retinal

function of the foetus and infants (Ward and Singh, 2005).

The long chain n-3 PUFA may be obtained from marine mammals' oils (e.g.,

seal and whale blubber), and fish oils (e.g., menhaden, salmon, mackerel, cod,

herring, sardine, capelin and tuna), as well as marine algae or derived from �-linoleic acid by a series of chain elongations and desaturations (Ward and Singh,

2005). In 2003±2004, the global production of fats and oils was expected at 12

805 million tonnes from which 82% were of vegetable origin (BergeÂ and

Barnathan, 2005). Fish oils are still the least expensive natural source of

preformed long-chain PUFA and the n-3 PUFA content and EPA/DHA ratios in

marine oils tend to vary with season, both in quality and quantity (Wanasundara

et al., 2002). Aidos et al. (2002a) studied the oil composition changes of herring

fillets and by-products over the year. They demonstrated that even if the herring

by-products were throughout the year an adequate raw material for fish oil

production, herring by-products were richer in PUFAs during the summer. On

the contrary, farmed salmon (Salmo salar) slaughterhouses generate high-

quality offal at a relatively constant rate. In Norway, by-products of farmed

salmon species were estimated at 133 000 tonnes in 2002, of which 8000 tonnes

were used directly for salmon oil production (Skara et al., 2004). Efficient

transportation or close proximity to a processing plant facilitates transformation

of very fresh raw materials, in which initial post-mortem oxidation processes are

limited.

Today, the main sources of fish oils are pelagic species caught in large

quantities and often not used for other purposes than fishmeal and oil

production, but fish oil can also be produced from offal from the processing

industry (Aidos et al., 2001). In fatty fish species, lipids are localized mainly

under the skin, around the intestines or in the white muscle and the oil content

varies but it can reach up to 21% in herring and 18% in sardine (BergeÂ and

Barnathan, 2005). The type of fat differs in various parts of the fish and organs

(Aidos et al., 2002b). Pre-cooked tuna heads are known to be rich in DHA

(Chantachum et al., 2000). In the cod viscera, the lipid content varies within the

range of 1±10%, while the liver contains 42±78% lipids (Dauksas et al., 2005).

The lipid concentrations of the farmed Atlantic salmon (Salmo salar L.) viscera


and fillet were 24.1 and 5.5%, respectively. Except for C18:0, C18:2, C20:1(n-

9), C20:4(n-6), C22:5(n-3), there were no significant differences in fatty acid

concentration between the viscera and the fillet total lipid extracts (Sun et al.,

2002). On the contrary, herring head by-products and their oil presented the

highest amount of saturated fatty acids and the lowest �-tocopherol and PUFA

contents compared to mixed and headless herring by-products (Aidos et al.,

2002b).

Concentration of n-3 PUFA

Typical processes for extraction/concentration of marine oils commonly involve

cooking the raw material by steam under pressure with or without the presence

of water. Partial cooking sterilizes the oil, denatures protein and facilitates oil

release. Following this stage, the cooked material is pressed and centrifuged and/

or filtered to recover the oil from the micelle. Then PUFA concentrates may be

obtained by several methods including chromatographic methods, distillation

method, low temperature crystallization, supercritical fluid extraction, urea

complexation and enzymatic methods including lipase-catalyzed hydrolysis and

lipase-catalyzed esterification (Aidos et al., 2003; Shahidi and Wanasundara,

1998; Shimada et al., 2001).

Experimental designs have been used to optimize the process parameters

from the fish oil pilot plan in terms of oil quality (oxidative status) and yield

(Aidos et al., 2003; Linder et al., 2005b) and to investigate the storage stability

(lipid oxidation) of different processed oils (Skara et al., 2004). Each technique

has its own advantages and drawbacks, as reviewed by Wanasundara et al.

(2002).

Enzyme-catalyzed process for lipid extraction

The main enzymatic processes applied to fish oil recovery from by-products and

whole fish can fall into two categories. The first one is the protease-catalyzed

hydrolysis in order to extract the polyunsaturated fatty acids from the starting

material (whole fish or selected by-products). This is a recent approach for lipid

extraction by using a pre-hydrolysis step with a large spectrum of proteases in

order to disrupt tissues and cell membranes. The second category concerns the

lipase-catalyzed hydrolysis and esterification in order to enrich PUFAs in the

oils and/or to produce different forms and compositions of PUFAs and/or to

produce structured lipids. These enzymatic transformations occur once the oil is

extracted from the starting material by traditional processes or by the use of

proteases. Lipases may offer a degree of selectivity for particular fatty acids, not

observed with acid or base catalyst (see Chapter 17). The examples presented

below describe some applied enzymatic procedures using proteases for their

ability to release oil content from the marine by-products.

Use of proteases on lipid extraction from fish by-products

Several commercial enzymes from plant origin such as papain and animal origin

such as chymotrypsin or microbial sources such as ProtamexTM, FlavourzymeÕ,


NeutraseÕ 0.5L, AlcalaseÕ 2.4L can be used for this purpose (Dumay et al.,

2004; Linder et al., 2005a; Dauksas et al., 2005; Slizyte et al., 2005a). Linder et

al. (2005a) carried out solvent-free extraction of oil from by-products of Atlantic

salmon at moderate temperature for a short duration (50±60ëC; 30±120 min) by

selective enzymatic hydrolysis using three commercial proteases (NeutraseÕ

0.5L, AlcalaseÕ 2.4L and FlavourzymeÕ). Reaction kinetics were monitored

by measuring the degree of hydrolysis using the pH-stat method, in order to

preserve the functional and nutritional values of hydrolysates. The amount of oil

(17%) obtained after 2 hours was close to that obtained using the classical

method with solvent. In a second step, the lipolysis of the oil was carried out

with Novozym SP398 and followed by a filtration using a hydrophobic

membrane (Fig. 6.2). DHA increased from 9.9% to 11.6% and EPA changed

from 3.6% to 5.6%. A re-esterification of the free fatty acids in the permeate was

achieved using glycerol and Lypozyme IM to increase the amount of long chain

acylglycerols.

Dumay et al. (2004) also compared yield extraction for total lipids, phospho-

lipids, EPA and DHA, using four proteases (papain, chymotrypsin, ProtamexTM,

Fig. 6.2 Flowsheet for the production of hydrolysate and crude oil from salmon by-products (adapted from Linder et al., 2005a).


FlavourzymeÕ) to those obtained by organic extraction. The authors observed

that, despite using non-optimizing hydrolysis conditions, the use of proteolytic

enzymes for partially disrupting lean fish tissue had, most of the time, enhanced

the fat extraction. However, this increase was sometimes higher for total lipids

than for those complex lipids such as phospholipids, DHA and EPA which, in

fact, led to a reduction in the proportion of phospholipids, DHA and EPA among

the total lipid compounds extracted this way. Liaset et al. (2003) isolated

approximately 77% of total lipids present in salmon frames using ProtamexTM at

55ëC, pH 6.5 for 60 min. The resulting salmon oil was high in both EPA and

DHA. Slizyte et al. (2005a) and Dauksas et al. (2005) performed hydrolysis of

by-products from cod Gadus morhua (viscera, backbone, digestive track) using

respectively FlavourzymeÕ, NeutraseÕ or no enzyme. Four lipid containing

fractions were generated: oil, emulsion, FPH (fish protein hydrolysate) and

sludge. The authors demonstrated that the most important factor influencing the

yield of the different fractions was àdded water' rather than the type of enzyme

used, the highest yield of oil being obtained was when no water was added. The

fatty acids were distributed unequally among the four fractions. The sludge

contained up to 50% lipids with phospholipid content of up to 60% and the

highest amount of PUFA (EPA/DHA). In the oil fraction, the major lipid class

was triacylglycerols and the amount of triacylglycerols was similar in all

samples (95±98%), the second major compound in the oil fraction was

cholesterol (1.0 to 3.4% of total lipids) while the phospholipids were more

difficult to extract from the mixtures. The monounsaturated fatty acids (MUFA)

were the main compounds of the fatty acid methyl esters (FAMEs) in the oil

fraction and made up more than 50% of all FAMEs. The oil produced from

viscera tended to develop high levels of free fatty acids (FFA). FFAs are more

susceptible to oxidation than esterified fatty acids and a low level of FFA is an

important quality criterion in many food-related applications.

After the preliminary step of oil extraction by conventional methods or by

using proteases as described above, the enzymatic processes applied to fish oils

are lipase-catalyzed hydrolysis, esterification, or exchange of fatty acids in

esters. This provides an opportunity for the fat and oil industry to produce new

types of triacylglycerols, esters and fatty acids, and to improve the quality of the

existing products produced by employing conventional technologies (Shahidi

and Kamil, 2001; Osborn and Akok, 2002).

Lipase-catalyzed hydrolysis and esterification

Lipases (E.C.3.1.1.3) are enzymes that are primarily responsible for the

hydrolysis of acylglycerols in aqueous conditions. In a typical lipase-catalyzed

hydrolysis run, the first step consists of obtaining freshly prepared, refined,

bleached and deodorized marine oil. Then the lipase is dissolved in a selected

buffer and the oil is added to it and stirred at a predetermined temperature.

Samples are withdrawn at fixed time intervals until a desired hydrolysis per-

centage is reached. Alcohol is added quickly to deactivate the enzyme, and the

sample is titrated in order to determine its free fatty acid content and con-


sequently, the percentage of enzymatic hydrolysis. Then separation of the

enriched fraction is carried out (Shahidi and Kamil, 2001). However, since

lipase reactions are reversible, the enzymes can be used to promote synthesis

reactions such as esterification, interesterification, alcoholysis, acidolysis

reactions, the last three reactions often being grouped together into a single

term, i.e. transesterification (Table 6.1). These reactions are favoured when the

amount of water in the reaction mixture is restricted (Gandhi, 1997).

Thus, it is possible to attempt enzymatic purification of PUFA by selective

esterification or alcoholysis. In theory, by choosing the right lipase judiciously,

with respect to lipase triacylglycerol positional specificity, ester specificity and

fatty acid chain length specificity, and by varying substrate, water content and

use of hydrophobic solvents and other conditions, the reaction can be tailored to

produce different product forms (Shimada et al., 2001; Ward and Singh, 2005).

In addition, lipases have been frequently used to discriminate between EPA and

DHA in concentrates containing both of these fatty acids, thus providing the

possibility of producing n-3 PUFA concentrates with dominance of either EPA

or DHA (Wanasundara et al., 2002).There is an extensive literature concerning

different approaches to the preparation of n-3 fatty acid concentrates from

natural sources using lipases (Linder et al., 2002; Shahidi and Wanasundara,

1998; Shimada et al., 1998; Skara et al., 2004; Ward and Singh, 2005). Sun et al.

(2002) estimated the yield of EPA and DHA concentration in Atlantic salmon

viscera using lipase C. rugosa. One kilogram viscera produced 0.17 kg viscera

oil. One gram of viscera oil with 27.1% of EPA + DHA can be obtained from 4 g

of viscera oil with 11.7% EPA + DHA after incubation with lipase from C.

rugosa at 35ëC. The recovery of EPA + DHA from salmon viscera oil was about

58%. Therefore, from 1 kg salmon viscera, 11.4 g EPA + DHA can be produced.

In some cases, experimental design was used to optimize the production of

PUFA from marine oils (Wanasundara and Shahidi, 1998b; Linder et al.,

2005b). Some examples of modification of lipids from marine oil involving

lipase-catalyze process are given in Tables 6.2 and 6.3.

In conclusion of this section, applications of enzymes in bioprocessing are

especially advantageous because they act under mild conditions: mild tempera-

ture and pH conditions, ambient pressure, less solvent, give cleaner products-

attributes of `green chemistry', and can exert region- or stereospecific control

over reactions (Scrimgeour, 2005). Long chain PUFAs are highly labile and

reaction methods, which exploit extremes of pH and temperature can destroy the

Table 6.1 The two main categories in which lipase-catalyzed reactions may beclassified (Gandhi, 1997)

Hydrolysis RCOOR0 + H2O , RCOOH + R0OH

Synthesis Esterification RCOOH + R0OH , RCOOR0 + H2OInteresterification RCOOR0 + R00COOR* , RCOOR* + R00COOR0Alcoholysis RCOOR0 + R00OH , RCOOR00 + R0OHAcidolysis RCOOR0 + R00COOH , R00COOR' + RCOOH


all cis nature of n-3 fatty acids, such as EPA or DHA, by oxidation, cis-trans

isomerization or migration of double bond. A number of recent reviews and

books cover topics developed in this part (Ward and Singh, 2005; Osborn and

Akok, 2002; Shahidi and Wanasundara, 1998). Enzymatic oil extraction using

food-grade proteases could provide an interesting alternative as it presents two

major interests: the first one is the mild condition in which the enzymatic

process is performed, coupled or not with membrane technology. The valuable

components in the fish oil are not destroyed and oils with higher quality will

obtain higher prices. The second one is its versatility: optimal processing

conditions must be found to obtain tailor-made products according to the final

objective such as the purification of phospholipids, a high oil yield and/or a high

quality fish protein hydrolysate.

Subsequent modification of the extracted lipids by using lipases having high

stereoselectivity has found applicability in the site-specific modification of tri-

acylglycerol products enriched with EPA or DHA. Lipases offer many

advantages over traditional methods of concentration (chromatographic separa-

tion, molecular distillation, etc.) because such procedures involve extremes of

pH and high temperature, which may partially destroy the natural all cis n-3

PUFA by oxidation and by cis-trans isomerization or double bond migration.

Therefore, the lipase-catalyzed process provides a good alternative that could

also save energy and increase product selectivity. In addition, the enzymatic

hydrolysis method produces n-3 fatty acids in the acylglycerol form, which is

considered to be nutritionally favourable (Wanasundara and Shahidi, 1998a;

Wanasundara et al., 2002). The lipase catalytic efficiency is high, therefore a

relatively low amount of enzyme is required, especially when immobilized,

which enables their reuse and enhances their productivity.

The susceptibility to oxidation of fish oil during storage is a major problem

since the formation of undesirable odours and flavours caused by oxidation often

limit the shelf life (Skara et al., 2004). Most fish oils are produced under inert

gas or in closed containers to reduce oxidation by atmospheric oxygen (Ward

Table 6.2 Summary of general PUFA enrichment process using lipase (adapted fromWard and Singh, 2005)

Method Procedure

Enzyme splitting · Promote lipase selective esterification of more saturatedfatty acids

· Separate concentration of PUFAs in FFA fraction fromesterified saturated fatty acid

· Repeat esterification for further PUFA enrichmentPUFA transformations · Esterify PUFA free fatty acids to produce esters (ethyl-,

glyceryl-, sugar-, other)· Interesterification to enrich non-HUFA oils with PUFAs

PUFA: Polyunsaturated fatty acidHUFA: Highly unsaturated fatty acid


Table 6.3 Examples of lipase-transformations of PUFAs

Enzyme process PUFA Specific Reaction conditions Transformation References

substrate PUFA %concentration

1. Lipase- before Seal EPA 6.4% 4g Oil, 6mL phosphate EPA 9.75% Wanasundara and

assisted optimization blubber oil DPA 4.7% buffer, lipase CC* 200 DPA 8.6% Shahidi (1998a)hydrolysis (SBO) DHA 7.6% U/g oil, 40 h DHA 24%

after Seal EPA 6.4% lipase CC* 297 U/g oil, EPA 16.5% Wanasundara and

optimization blubber oil DHA 7.6% 26 h, 36ëC Shahidi (1998b)using CCRD** (SBO)

lipase CC* 342U/g oil, DHA 28.1%51 h, 39ëC

2. Lipase- Salmon EPA 67 mg/g 4 g Oil, 6mL phosphate EPA 106mg/g Sun et al. (2002)

assisted viscera oil DHA 74 mg/g buffer, lipase C. rugosa DHA 184mg/ghydrolysis (800U/g oil), 35ëC

3. Lipase- before Menhaden EPA 13.2% 4 g Oil, 6mL phosphate EPA 18.5% Wanasundara and

assisted optimization oil (MHO) DPA 2.4% buffer, lipase CC* 200 DPA 3.6% Shahidi (1998a)hydrolysis DHA 10.1% U/g oil, 40 h DHA 17.3%

after lipase CC* 370U/g oil, EPA 21.1% Wanasundara and

optimization 31 h, 37ëC Shahidi (1998b)using CCRD**

lipase CC* 314U/g oil, DHA 25.9%

34 h, 36ëC

4. Lipase- Sardine oil EPA 14.5% 12 g Oil, 18mL phosphate EPA 46.2% Gamez-Meza et al.

assisted DPA 1.3% buffer, lipase*** (0.25, 0.5, DPA 2.16% (2003)

hydrolysis DHA 12.5% 0.75% w/w oil basis) pH7 DHA 40.32%

5. Acidolysis Seal 5.4% 20:5n-3, Oil/fatty acid mole ratio SL containing Senanayake and(incorporation blubber oil 7.9% 22:6n-3 of 1:3, 45ëC, hexane, 24 h, 3.2% 20:5n-3, Shahidi (2002)

of capric acid) (SBO) 1% water, 10% 7.5% 22:6n-3,Lipozyme-IM 27.1% 10:0

*CC: Candida cylindracea from Amano Pharmaceutical; **CCRD: central composite rotatable design;***lipase: Pseudomonas cepacia lipase immobilized on chemically modified ceramic (CMC).

and Singh, 2005) but efforts to keep these products from oxidative rancidity

during processing, cooking, and storage are necessary (Uauy and Valenzuela,

2000). In the case of direct consumption of PUFA-containing oils, this is

overcome through use of capsules while microencapsulation techniques can

address this problem for incorporation of oils into dried foods (Ward and Singh,

2005). In addition to the oxidative instability of PUFA-containing oils is the

presence of co-extracted contaminants. Nevertheless, the crucial problem of fish

oils is their sustainability due to the worldwide decline of fish stocks. A better

use of raw material by-catch and by-products from fisheries as well, may be one

solution (BergeÂ and Barnathan, 2005).

6.3.2 Proteins

Enzymatic proteolysis as applied to proteinaceous fish by-products has been the

objective of numerous studies (reviewed by Kristinsson and Rasco, 2000;

Mackie, 1982). Fish protein hydrolysates (FPH) and other hydrolysates have a

range of potential applications, as ingredients in animal feed or food, as peptone

ingredient in microbial growth media, as fertilizer or as a new source of

bioactive peptides (Aspmo et al., 2005b; Byun and Kim, 2001; DufosseÂ et al.,

2001, 2003; GueÂrard et al., 2001a, 2003, 2005a; Je et al., 2005; Liaset et al.,

2000; Rousseau et al., 2001).

Proteases are among the best characterized enzymes and their use is well

established in the food industry. Proteases are classified in four major classes

according to the specificity of their peptide bond cleavage: serine proteinases

(E.C.3.4.21), cysteine proteinases (E.C.3.4.22), aspartic proteinases (E.C.3.4.23)

and metalloproteinases (E.C.3.4.24). Proteases are further characterized by their

hydrolyzing mechanism into endoproteinases or exoproteinases. The breakdown

of proteins into peptides is brought about primarily by endoproteinases which

cleave the peptide bond within protein molecules, usually at specific residues to

produce relatively large peptides (Kristinsson and Rasco, 2000). The

exopeptidases, including carboxypeptidases, aminopeptidases, and di- and tri-

peptidase, systematically reduce the peptides into amino acids. Endoproteinases

may be combined with exopeptidases in order to achieve a more complete

degradation.

Autolysis versus enzymatic hydrolysis

Biochemical processes for converting fish processing wastes and by-products

into fish protein hydrolysates may be carried out by employing an autolytic

process or by using added proteolytic enzymes. Two important examples of

utilization of autolytic digestion in product manufacturing are fish sauce and fish

silage production, in which hydrolytic enzymes from the fish itself play a key

role in the solubilization and degradation of the tissue proteins (Gildberg et al.,

2000). The biochemistry of the process is not well understood in detail (Martin

and Patel, 1991).


Enzymatic hydrolysis with added enzymes

On the other hand, the enzymatic hydrolysis using added proteases presents a lot

of advantages compared to autolytic process and chemical hydrolysis (Table

6.4). Addition of commercial exogenous enzymes to the fish tissue reduces the

time needed to obtain a similar degree of hydrolysis (DH) and allows a good

control of the hydrolysis, and subsequently of the size of the obtained peptide. In

addition, hydrolytic degradation products via racemization reaction observed

with both acid and alkaline hydrolysis, are not produced. The choice of the

hydrolysis process will depend on the targeted applications. For dietary use, or

in order to obtain a hydrolysate with a high nutritional and therapeutic value, the

protein hydrolysates should be rich in low molecular weight peptides, with as

few free amino acids as possible whereas large molecular weight peptides (more

than 20 amino acid residues) are presumed to be associated with the

improvement in the functionality of hydrolysates.

Research during the past 20 years has greatly enhanced understanding about

better processing of fish or shellfish by-products. A list of the most frequent

underutilized species or processing wastes investigated for the production of

hydrolysates, is presented in Table 6.5. Generally, underutilized fish, fish frames

or crustacean wastes are suspended in water and enzyme is added to the slurry. In

some cases, the meat is first heated in order to denature the endogenous proteases

(GueÂrard et al., 2001b). The reaction is allowed to proceed for less than one hour

to several hours, depending on the activity of the enzyme employed, process

temperature and other factors. After separation of solids, pH is adjusted and the

aqueous layer is clarified, and then dehydrated. Figure 6.3 outlines the main steps

of the process in the enzymatic solubilization of proteinaceous fish or

invertebrate by-products. The need to inactivate added enzymes by pH or heat

treatment at the end of the batch reaction adds to the processing cost and may be

improved by coupling the enzymatic hydrolysis with membrane technology in

order to perform continuous process. Continuous processes are used by many

companies for large-scale production of hydrolysates.

Most of the commercial proteases have been used successfully to solubilize

proteins from underutilized species or processing waste. Industrial proteinases

are mostly derived from GRAS microorganisms (AlcalaseÕ 2.4L, NeutraseÕ,

FlavourzymeÕ, UmamizymeÕ, ProtamexTM), and to a lesser extent from plant

(papain, bromelain, ficin) and animal sources (pepsin, trypsin). According to

Simpson (2000), there is only very limited use of marine proteinases by the

industry. The reasons for the rather limited use of marine digestive proteinases

include the relative paucity of basic information on these enzymes, the cyclical

nature of the source material (which precludes supply in a steady manner), and

the stereotypical attitude of the general public toward the source material: fish

offal. Some examples of proteolytic enzymes used to hydrolyze marine by-

products are presented in Table 6.5. By selecting both the enzyme and the

conditions of digestion, various degrees of hydrolysis or breakdown of the

proteins can be achieved in order to obtain products with a range of functional

and/or biological properties. The introduction of new proteases capable of


Table 6.4 Comparison between autolysis, chemical and enzymatic hydrolysis (adaptedfrom Diniz and Martin, 1997b; Kristinsson and Rasco, 2000; GueÂrard et al., 2005a)

Process Specificity Advantages Disadvantages

Autolysis process(fish sauce andsilage)

Action ofthe digestiveenzymes ofthe fishitself

Low costSimple operation

Mild reactionconditionsNo destruction ofamino acidsHigh nutritionalvalueNo enzyme addition

Improvement oforganolepticcharacteristics

Slow reactionMolecular weight out ofcontrolSubsequent deactivationof the enzymeLarge amount of salt (infish sauce)Enzymes with differentactivity requirementsHigh variations in thepresence of enzymesFinal product with badfunctionality

Acid/alkalinehydrolysis

Randomprocess

Fast reactionComplete hydrolysis

Low costHigh solubility

High temperaturesMolecular weight out ofcontrolLarge amount of saltUndesirable sidereactions (destruction oftryptophan, racemization,etc.)

Enzymatichydrolysis

Uniquespecificityof action oftheenzyme(s)

Control of themolecular weightMild reactionconditionsAttractive functionalproductcharacteristics(solubility,dispersibility,foaming, capacityand foam stability)Control of theproperties of theresulting productsFew side reactionsNo destruction ofamino acidsHigh nutritionalvalue

Cost of enzyme(s)

Subsequent deactivationof the enzyme(s)Complex process


Table 6.5 Some examples of proteolytic enzymes used to hydrolyze marine by-products including `trash fish' or underutilized species

Enzymes Suppliers Substrates Appli- Evaluation of hydrolysis Referencescations

Papain Solvay Herring (Clupea harengus) 1 TCA soluble N, TN, colour, Hoyle and Merrit(EC 3.4.22.2) Enzymes, Inc. sensory, MWDP (1994)

Sigma Lobster cephalothorax (Palinurus sp) 1 TL, FP, NSI Vieira et al. (1995)

Sigma Capelin (Mallotus villosus) 1,2 pH-stat, FP, PER, ED Shahidi et al. (1995)

Biochem Europe Atlantic cod (Gadus morhua L.) 3 DM, �-amino groups**, Aspmo et al.(7¨/kg)a viscera MWDP, TN, MS, SDS-PAGE (2005a,b)

Actinidin Biochem Europe Atlantic cod (Gadus morhua L.) 3 DM, �-amino groups**, Aspmo et al.(30¨/kg)a viscera MWDP, TN, MS, SDS-PAGE (2005a,b)

Bromelain Pineapple juice Yellowfin tuna (Thunnus albacares) np NR Raghunath (1993)(EC 3.4.22.4) canning waste

Biochem Europe Atlantic cod (Gadus morhua L.) 3 DM, �-amino groups**, Aspmo et al.(20¨/kg)a viscera MWDP, NT, MS, SDS-PAGE (2005a,b)

Trypsin Merck Sardine (Sardina pilchardus) viscera 2 MWDP Cancre et al. (1999)(EC 3.4.21.4) and heads

Pepsin Sigma Lobster cephalothorax (Palinurus sp) 1 TL, FP, NSI Vieira et al. (1995)(EC 3.4.23.1)

Atlantic cod (Gadus morhua) and 1 pH-stat, NP, MWDP Liaset et al. (2000)Atlantic salmon (Salmo salar) frames

Table 6.5 Continued


Crude proteinases Cod (Gadus macrocephalus) frame 1,2 10% TCA soluble nitrogen Kim et al. (1997);from tuna content, LPC, MWDP, FP Jeon et al. (1999)(Thunnus thynnus)pyloric caeca

Crude proteinase Hoki frames (Johnius belengerii) 2 Kim et al. (2003)from mackerelintestine

FlavourzymeÕ Novozymes Gold carp (Carassius auratus) % �-amino acids released*, Sumaya-Martinezfilleting by-products NR, ED et al. (2005)

Fish soluble concentrate (a by- DH%=AN/TN, Nilsang et al. (2005)product from canning industry) ED, AAC, SE

Atlantic cod (Gadus morhua L.) 1 �-amino groups/TN; PER, Slizyte et al.viscera free AA; MWDP; FP (2005a,b)

KojizymeTM Novozymes Fish soluble concentrate (by- DH%=AN/TN, Nilsang et al. (2005)product from canned fish industry) ED, SE

Fungal protease Sigma Lobster cephalothorax (Palinurus sp) 1 TL, FP, NSI Vieira et al. (1995)type II fromA. oryzae

Newlase A from Amano Atlantic Cod (Gadus morhua) frames 4 Viscosity, MWPD Ferreira and HultinRhizopus niveus (1994)

AlcalaseÕ 2.4 L Novozymes Capelin (Mallotus villosus) 1,2 pH-stat, FP, PER, ED Shahidi et al. (1995)(25¨/kg in2004)a

Shrimp (Crangon crangon) processing 1 pH-stat, PER, SE, EEA Synowiecky and Al-discards Khateeb (2000)

Atlantic cod (Gadus morhua L.) viscera 3 DM,�-amino groups**, Aspmo et al.MWDP, TN, MS, SDS-PAGE (2005a,b)

Tuna stomach (Tunus albacora) 1,2,3 pH-stat, MWDP GueÂrard et al.(2001a,b)

Herring (Clupea harengus) 1 TCA soluble N, TN, colour, Hoyle and MerritSE, MWDP (1994)

Herring (Clupea harengus) 1 DH-TNBS method; Liceaga-Gesualdo andSDS-PAGE, FP Li-Chan (1999)

Pacific whiting (Merluccius productus) 1 DH-TNBS method, NR, Benjakul andsolid wastes colour, SDS-PAGE Morrissey (1997)

Harp seal (Phoca groenlandica) 1 pH-stat, NSI, FP Shahidi et al. (1994);Shahidi andSynowiecki (1997)

Shrimp (Pandalus borealis) waste 2 MWDP Cancre et al. (1999)

Cod head (Gadus morhua) 2 MWDP Cancre et al. (1999)

Shrimp (Pandalus borealis) waste 1 TN Gildberg and Stenberg(2001)

Threadfin bream (Nemipterus japonicus) 1 pH-stat, SE, MWDP, Normah et al. (2004)SDS-PAGE

Shark muscle (Squalus acanthias) 1 pH-stat, NR, ED, PER Diniz and Martin(1996, 1997a, 1998)

Atlantic cod (Gadus morhua) and 1 pH-stat, NP, NSI, FP, MWDP Liaset et al. (2000)Atlantic salmon (Salmo salar) frames

Atlantic salmon (Salmo salar) heads 1 pH-stat, ED, MWDP Gbogouri et al.(2004); Linder et al.(2005a,b)

Table 6.5 Continued


AlcalaseÕ 0.6 L Novozymes Capelin (Mallotus villosus) 1,2 pH-stat, FP, PER, ED Shahidi et al. (1995)

NeutraseÕ 0.5 L Novozymes Pacific whiting (Merluccius productus) 1 DH-TNBS method, NR, color, Benjakul andsolid wastes SDS-PAGE Morrissey (1997)

Harp seal (Phoca groenlandica) 1 pH-stat, NSI, FP Shahidi et al. (1994)

Atlantic cod (Gadus morhua) and 1 pH-stat, NP, MWDP Liaset et al. (2000)Atlantic salmon (Salmo salar) frames

NeutraseÕ 0.8 L Novozymes Atlantic cod (Gadus morhua L.) viscera 1 �-amino groups/TN; PER; Slizyte et al.(15¨/kg)a free AA; MWDP; FP (2005a,b)

Atlantic cod (Gadus morhua L.) viscera 3 DM, �-amino groups**, Aspmo et al.MWDP, TN, MS, SDS-PAGE (2005a,b)

UmamizymeÕ Novozymes A/S Tuna stomach (Tunus albacora) 1 pH-stat, MWDP, NR GueÂrard et al. (2003)

ProtamexTM Novozymes Atlantic cod (Gadus morhua L.) viscera 3 DM,�-amino groups**, Aspmo et al.(42¨/kg)a MWDP, TN, MS, SDS-PAGE (2005a,b)

Atlantic Salmon (Salmo salar L) frames 1 NR, Lipid recovery Liaset et al. (2003)

a: approximative price as specified by suppliers. Prices may vary depending on purchase order quantity (Aspmo et al., 2005a).1: Dietary protein source; 2: biological activities; 3: peptone; 4: fertilizer; np: not precise.AAC: amino acid composition; �-amino groups; **: concentration of �-amino groups evaluated using OPA; DM: dry matter, EEA: essential amino acids; ED:experimental design; FD: factorial design; FP: functional properties; FVFA: free volatile fatty acids; LPC: length of the peptide chain in hydrolysate using the TNBSmethod; MS: mass spectrometry; MWDP: molecular weight distribution of peptides; N: nitrogen content; NP: nutritional properties; NR: nitrogen released; NSI:nitrogen solubility index; PER: protein efficiency ratio; pH-stat: pH-stat method; SC: soluble content; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; SE: sensory evaluation; TL: tyrosine level; TCA: TCA soluble nitrogen; TN: total nitrogen.

degrading bitter peptides (such as Flavourzyme from Novozymes) has

contributed to eliminating the problem of bitter hydrolysates.

The screening for a suitable enzyme in any process or experiment is very

important if the product is to have predetermined properties (Kristinsson and

Rasco, 2000). The screening process can be conducted in a variety of ways, and

there is no standard methodology for this selection. Recently, the relative

activities of six commercial enzymes (AlcalaseÕ 2.4L, NeutraseÕ 0.8L,

ProtamexTM, papain, bromelain, actinidin and a mix of plant proteases) were

measured for the hydrolysis of cod viscera at the natural pH of the substrate

without pH-control, in order to avoid adding more salt to the hydrolysate and to

limit the cost of the process (Aspmo et al., 2005a). However, in this experiment,

viscera endogenous enzymes worked together with the added proteases and

maximized the process efficiency, thus making it difficult for a comparative

study of the proteases. The results showed that there is a great variation among

the performances of commercial enzymes. Highest yields in solubilized dry

matter were obtained with AlcalaseÕ 2.4L and papain, AlcalaseÕ 2.4L clearly

performing the best and leading to solubilization of close to 95% of the dry

matter. Endogenous enzymes made an important contribution to viscera

solubilization. The fish viscera hydrolysates gave good performances as

peptones for microbial growth (Aspmo et al., 2005b). Shahidi et al. (1995)

Fig. 6.3 Flowsheet for the enzymatic solubilization of proteinaceous fish or invertebrateby-products (adapted from Kristinsson and Rasco, 2000; GueÂrard et al., 2005a).


used AlcalaseÕ 2.4L to optimize the preparation of capelin protein hydrolysates.

Alcalase-treated hydrolysates exhibited superior protein recovery (70.6%)

compared with NeutraseÕ (51.6%) and papain (57.1%). AlcalaseÕ 2.4L and

NeutraseÕ were studied by Benjakul and Morrissey (1997) on the solubilization

of Pacific whiting solid wastes (solid wastes resulting from surimi production).

AlcalaseÕ 2.4L had a higher proteolytic activity than NeutraseÕ. The final

hydrolysate had high protein content (about 80%) and an amino acid composi-

tion comparable to fish muscle. Other enzyme preparations from microbial

origin have shown good potential for hydrolyzing proteinaceous by-products to

make highly functional hydrolysates, including FlavourzymeÕ, ProtamexTM, and

UmamizymeÕ (Table 6.5). UmamizymeÕ performed as effectively as AlcalaseÕ

2.4L for the tuna waste solubilization, however, UmamizymeÕ stability during

the hydrolysis process was lower than that of AlcalaseÕ 2.4L (GueÂrard et al.,

2002).

Although proteases operating at alkaline pH have often been used for

hydrolysis of fishery by-products, Ferreira and Hultin (1994) reported the use of

a fungal enzyme (the Newlase A from Rhizopus niveus, Amano) in order to

liquefy cod fish frames under acidic conditions. The main advantage of working

at low pH is the stabilizing effect towards microbial spoilage. The authors

concluded that Newlase A was a useful catalyst in any situation where a pH

between 3 and 4 was to be used and where it was desired to reduce the viscosity

of fish frames without excessive hydrolysis.

In some cases, experimental designs were employed to optimize hydrolysis

conditions. Shahidi et al. (1995) used response surface regression (RSREG)

procedure of the statistical analysis system to fit a quadratic polynomial

equation to the experimental data. The three-dimensional response surface

indicated that both the AlcalaseÕ 2.4L concentration (13.9±83.9AU/kg crude

protein) and the treatment temperature (45±65ëC) affected the degree of

hydrolysis and thus the protein recovery. Response surface methodology was

also used by several authors in order to study the effects of pH, temperature,

enzyme/substrate ratio and substrate concentration on the degree of hydrolysis

of crayfish by-products (Baek and Cadwaller, 1995), canned tuna processing

(Nilsang et al., 2005), dogfish muscle (Diniz and Martin, 1996, 1997a), and gold

carp by-products (Sumaya-Martinez et al., 2005). The resulting equations were

adequate for predicting the DH under any combination of values of the

variables. For example, Sumaya-Martinez et al. (2005) used a Box-Behnken

factorial design with four independent variables (Temperature T, pH, substrate/

buffer ratio S:B, and enzyme concentration E). All regression coefficients were

significant and the model obtained showed a good fit with the experimental data.

A maximum DH% was obtained at the following critical values for

FlavourzymeÕ: pH � 5.9, T � 53ëC, S:B � 14.7%, and E � 80 LAPU (leucine

aminopeptidase units) gÿ1.Thus, among numerous proteases tested, AlcalaseÕ 2.4L, an alkaline enzyme

produced from Bacillus licheniformis and developed by Novozymes for the

detergent industry, has been repeatedly proven by many researchers to be one of


the most effective enzymes to solubilize proteins (Diniz and Martin, 1997b;

Aspmo et al., 2005a). Unfortunately, these comparative studies were not

undertaken using standardized relative enzyme activity and most researchers

compare enzyme activity on (i) a weight basis of enzymes used in the reaction

mixture, or (ii) when using a system based on Anson Units, did not use the

enzymes at the same AU, or (iii) did not inactivate the endogenous proteases

before adding exogenous enzymes. Kristinsson and Rasco (2000) suggested

assaying enzymes using a synthetic substrate such as azocoll or casein in order

to obtain a uniform level of proteolytic activity for all the enzymes used.

Quantification of the proteolysis extent

The hydrolysis reaction must be carefully controlled in order to maintain a

uniform quality of the end products. The degree of hydrolysis (DH), which is

defined as the percentage of cleaved peptide bonds, is most commonly used to

describe hydrolysis of food protein and serves as the controlling parameter for

the hydrolysis reaction. The pH-stat technique consists of adding base for

titration of the released �-amino groups, thus maintaining the pH constant (at pH

values above 6.5). Equation (6.1), which relates the DH to alkali consumption, is

as follows (Adler-Nissen, 1982):

%DH � B� Nb� 1

�� 1

MP� 1

htot� 100 �6:1�

where B is the volume (mL) of base added, Nb is the normality of the base, � is

the average degree of dissociation of the �-NH groups, MP is the gram of

protein in the reaction mixture, htot is the total number of peptide bonds. The

principle of the pH-stat method has been used by many workers for kinetic

studies and the DH gives a measure of the enzyme hydrolytic efficiency (Table

6.5).

In addition to the pH-stat method, the extent of proteolysis may also be

quantified by the depression of the freezing point, which is indicative of the

increasing osmolarity (osmometry), or by the increase in solubility in trichlor-

acetic acid (Kristinsson and Rasco, 2000). DH values determined by different

methods are often not directly comparable. Base consumption and osmometry

methods are easy to perform, allowing continuous monitoring of the hydrolysis

process, whereas the estimation of soluble nitrogen content using the Kjeldahl

method is time-consuming and cannot be used as an on-line process control tool

(Panyam and Kilara, 1996). The trinitrobenzenesulfonic acid (TNBS) method

developed in 1979 by Adler-Nissen is always used to determine the degree of

hydrolysis of food protein hydrolysates. However, the o-phtaldehyde (OPA)

method used for analysing the protein hydrolysate DH, has been found to be more

accurate, easier and faster to carry out than the TNBS method (Nielsen et al.,

2001). This method has a broader application range and is environmentally safer.

Silvestre (1997) provided a review of various methods used for the analysis of

protein hydrolysates and discussed the potential and limitations of the different

techniques. Finally, hydrolysates can be characterized according to the peptide


size in order to check that hydrolysates can be produced in a reproducible manner.

This is a very important point when the objective is the production of hydrolysates

with biological activities. In this case, size exclusion chromatography is a simple

and quick method for the evaluation of peptide molecular weight. New size

exclusion chromatography supports in FPLC mode such as the SuperdexÕ Peptide

HR 10-30 (Pharmacia Biotech, Sweden), with fractionation range from 100 to

7,000 Daltons allowed accurate separations of enzymatic hydrolysates. However,

the fractionation range values can only serve as guidelines, especially because the

elution behaviour of peptides in non-dissociating media is influenced by

adsorption and aggregation and because of the underestimation of small peptides

and free amino acids (GueÂrard et al., 2001b).

Some properties of enzymatically hydrolyzed by-product proteins

Functional properties

The functional properties of proteins can be defined as those properties that

affect the processing, storage stability and organoleptic quality of the formulated

food in which they are present (Vojdani and Whitaker, 1994). It has been

reported that FPH possess desirable functional properties such as high solubility

over a wide range of pH and excellent wettability (Shahidi et al., 1995; Vieira et

al., 1995). These properties are the consequence of enzymatic degradation of the

original protein to smaller peptides (Diniz and Martin, 1997b). As observed by

Kristinsson and Rasco (2000), the emulsifying capacity (EC) of hydrolysates

decreased with increasing DH. Hydrolysates with low DH, have high EC while

extensive hydrolysis resulted in a drastic loss of emulsifying properties. Shahidi

and Synowiecki (1997) reported the use of a seal protein hydrolysate (bland

taste) as phosphate alternative for enhancing the water-binding capacity and

improving the functional properties of thermally processed meat products.

Functional properties of fish protein hydrolysates are extensively discussed by

Kristinsson and Rasco (2000).

Biological activities

In an extensive paper, FPH were recently described as a new source of bio-

logically active substances (GueÂrard et al., 2005a). A wide range of biological

activities including antioxidant activities (GueÂrard et al., 2005b; Jun et al., 2004;

Je et al., 2005), neuroactive peptides (Bernet et al., 2000; Bordenave et al.,

2002), hypotensive (Byun and Kim, 2001) and immunoactive peptides (Bogwald

et al., 1996; Gildberg et al., 1996), hormonal and hormonal regulating peptides

such as calcitonin gene related peptide (CGRP), cholecystokinins and gastrins

(Fouchereau-PeÂron et al., 1999; Rousseau et al., 2001; Ravallec-PleÂ et al., 2001)

have been associated with FPH or with some purified sequences derived from

FPH. In addition, the results of several studies have shown that fish peptones

may be an excellent nitrogen source for microorganisms including bacteria

requiring complex growth media (Aspmo et al., 2005b; DufosseÂ et al., 2001,

2003; Gildberg et al., 1989; Ghorbel et al., 2005; Martone et al., 2005; Vasquez

et al., 2004).


In conclusion to this section, enzymatic extraction and/or solubilization of

proteinaceous by-products (e.g., heads, frames) have been developed for many

years. The resulting hydrolysates have great potential to be produced and sold as

functional food or feed ingredients. The occurrence of many biologically active

peptides in marine by-products is now well established. In all cases, the control

of the enzymatic reaction is very important. Uncontrolled or prolonged

hydrolysis of proteins may result in the formation of highly soluble peptides,

completely devoid of the functional properties of native proteins and may

promote the formation of bitter peptides.

6.3.3 Skin, bones, fin, scales, cartilage

By-products from the fish processing industry create large amounts of skin,

bones and scales in which structural proteins such as keratin, collagen and, to a

lesser extent, elastin have been found (Jongjareonrak et al., 2005a,b).

Extraction of collagen and collagen-derived products

According to Gomez-Guillen et al. (2002), about 30% of fish wastes consists of

skin and bone with a high collagen content. Heat denaturation of collagen

produces gelatine, a high digestible protein characterized by its rheological

properties varying according to the starting raw material. Until now, the main

sources of collagen were limited to those of land-based animals, such as bovine

and porcine skin and bone. However, the outbreak of bovine spongiform

encephalopathy (BSE) has resulted in anxiety among users of collagen and

collagen-derived products of land origin. Due to religious objections, the

collagen obtained from porcine sources cannot be used as a component of some

foods. As a consequence, increasing attention has been paid to alternative

collagen sources, especially fish skin and bone from seafood processing wastes

(Jongjareonrak et al., 2005a; Kittiphattanabawon et al., 2005). Collagen type I is

found in all connective tissue, including bones and skin. It is a heteropolymer of

two �1 chains and one �2 chain. It consists of one third glycine, contains no

tryptophan or cysteine and is very low in tyrosine and histidine (Muyonga et al.,

2004).

Isolation of acid- and pepsin-solubilized collagens

In general, the preparation of collagen from fish skin and bone is performed

according to the procedure described by Nagai and Suzuki (2000) with or

without slight modifications. Typically, the collagen extraction combine treat-

ments by NaOH to remove non-collagenous proteins and pigments then by butyl

alcohol to remove fat followed by an acid extraction using acetic acid. The

product obtained is referred to as acid-solubilized collagen (ASC) and, in some

cases, is subjected to limited hydrolysis with pepsin (E.C. 3.4.23.1). The product

is referred to as pepsin-solubilized collagen (PSC). With the limited pepsin

digestion, the cross-linkages at the telopeptide region are cleaved without

damaging the integrity of the triple helix (Hickman et al., 2000). As a


consequence, the triple helix structure is still predominant in both ASC and PSC,

resulting in similar thermal characteristics for both fractions.

Jongjareonrak et al. (2005a) used pepsin in order to solubilize collagens from

the skin of Browstripe red snapper (Lutjanus vitta). A yield of 4.7% on the basis

of wet weight was obtained. The pepsin-solubilized collagen (PSC) consisted of

two different � chains (�1 and �2), and was characterized to be type 1 with no

disulphide bond. The PSC peptide pattern was totally different from those of calf

skin collagen type I, suggesting differences in amino acid sequences and

collagen conformation. The thermal stability of PSC was much lower than that

of calf skin collagen, due to its lower hydroxyproline content (86 residues versus

94 residues per 1000). Morimura et al. (2002) developed a procedure for the

extraction of protein and production of peptides by enzymatic hydrolysis from

the spine of yellowtail fish wastes containing collagen by evaluating the

effectiveness of 16 commercial enzymes for degradation of pretreated fish bone.

One protease from Bacillus species appeared to be superior to those originating

from fungi for the degradation of collagen with an optimal 83% degradation

efficiency using 5 g.lÿ1 of substrate and 0.25 g.lÿ1 enzyme. The resulting

hydrolysate showed a mean degree of polymerization of about three, and

appeared to be a composite of oligopeptides. The hydrolysates had a high anti-

radical activity (IPOX50, 0.18 and 0.45mg/mL) and a high potential for

decreasing blood pressure (IC50, 0.16 and 0.41mg/mL), suggesting the

hydrolysate had suitable properties for use as additive in food materials.

A three-step membrane reactor was designed to prepare the enzymatic

hydrolysis of gelatine extracted from Alaska pollack skin. Successive digestions

with AlcalaseÕ 2.4L, PronaseÕE, and collagenase were performed in order to

produce angiotensin converting enzyme (ACE) inhibitory effect peptides and

antioxidant peptides (Byun and Kim, 2001; Kim et al., 2001). The isolated anti-

ACE peptides were composed of Gly-Pro-Leu and Gly-Pro-Met and showed

IC50 values of 2.6 and 17.13�M, respectively. Two antioxidant peptides com-

posed of 13 and 16 amino acid residues were also purified.

Ogawa et al. (2003) isolated acid-soluble collagen (ASC) and pepsin

solubilized collagen (PSC) from the skins of black drum (Pogonias cromis) and

sheepshead seabream (Archosargus probatocephalus). The yields of ASCs on

dry basis from black drum and sheepshead seabream, were estimated at 2.3 and

2.6%, and the yields of PSC were 15.8 and 29.3%, respectively. Analyses of

molecular weight profile, amino acid composition, and secondary structure

showed that the skin collagens from both species were typical type-I collagen.

The amino acid composition of ASC and PSC for both species was closer to calf

skin ASC than to cod skin ASC.

Jung et al. (2005) reported the enzymatic solubilization of the skeletons

discarded from industrial processing of hoki (Johnius belengerii). The efficiency

of an enzymatic preparation extracted from the intestine of bluefin tuna

(Thunnus thynnus) containing tryptic and collagenic enzymes was compared to

other commercial enzymes such as AlcalaseÕ 2.4L, pepsin, collagenase and

papain. The tuna intestine crude enzyme (TICE) efficiently degraded the hoki


bone matrices composed of collagen, non-collagenous proteins, carbohydrates

and minerals, in comparison with other enzymes tested. Total bone hydrolysates

liberated by TICE were 32.1% of total bone, from which an oligophospho-

peptide with 23.6% phosphorus, a molecular mass of 3.5 kDa and calcium

binding activity was purified. The fish bone oligophosphopeptide prepared by

enzymatic digestion of the bone could be utilized as a nutraceutical with

potential calcium-binding activity. Gildberg et al. (2002) used a combination of

gentle enzymatic hydrolysis and chemical extraction to recover both gelatine

and a calcium-rich residual bone fraction with favourable nutritional properties.

The functional properties of gelatine extracted from shark cartilage were lower

than that of porcine skin gelatine but their fat-binding capacity was higher (Cho

et al., 2004).

Some characteristics of fish collagens are presented in Table 6.6. The quality

and, consequently, the specific applications of both collagen and gelatine are

highly related to their purity and functional properties. The denaturation

temperatures (Td) of collagen from cold-water fish are lower than those of

warm-water fish and seemed to be correlated with a lower content of imino acids

(proline and hydroxyproline) compared to those of temperate and warm-water

fish (Jongjareonrak et al., 2005a; Kittiphattanabawon et al., 2005). The presence

of pigments and fish odours may restrict the potential use of fish collagen and

gelatine. However, although fish gelatine does not form particularly strong gels,

it is well-suited for certain industrial applications, e.g. microencapsulation, light-

sensitive coating and low-set-time glues (Rustad, 2003).

Chondroitin sulphate from shark cartilage

Shark cartilage and purified components of cartilage have traditionally been

credited with a number of medical benefits, including anticancer effects (Hassan

et al., 2005), fibrinolytic activities (Ratel et al., 2005), and beneficial effects on

osteoarthritis, progressive systemic sclerosis and neurovascular glaucoma, as

well as other diseases. The immunomodulating activity of shark cartilage was

associated with large molecular masses and the active compounds are extracted

from shark cartilage pulverized in a complex medium without any enzymatic

process (Kravolec et al., 2003). Chondroitin sulphate, a bioactive compound of

shark cartilage, is known to have the same therapeutic functions as described

above. Chondroitin sulphate is a typical mucopolysaccharide sulphate. There are

three isomers differing in the position of the sulphuric acid groups. The pro-

motion of crude shark cartilage extracts as a cure for cancer is highly criticized

by Ostrander et al. (2004) for at least two significant negative outcomes: a

dramatic decline in shark populations and a diversion of patients from effective

cancer treatments.

Lignot et al. (2003) described a low cost process producing chondroitin

sulphate (CS) in non-denaturing conditions. The first step consisted of an

enzymatic extraction using papain at 65ëC, pH 6.5 for 3 hours, followed by a

tangential filtration to concentrate and purify CS up to a volume concentrate

ratio (VCR) of 4. The performances of UF and MF membranes were compared


Table 6.6 Imino acid content and thermal transition temperature of acid soluble collagen (ASC) and pepsin soluble collagen (PSC) (type I)

Source of collagen Imino acids Tm (ëC) Td (ëC) Yield (%) References(Pro+Hyp)content per

1000 residues

Black drum (Pogonias cromis) skin ASC 199.8 34.8 34.2 2.3dwb Ogawa et al. (2003)Sheepshead skin ASC 205.1 33.5 34.0 2.6dwb Ogawa et al. (2003)Black drum (Pogonias cromis) skin PSC 197.1 35.1 35.8 15.8dwb Ogawa et al. (2003)Sheepshead skin PSC 198.1 33.6 34.3 29.3dwb Ogawa et al. (2003)Cod (Gadus morhua) skin ASC 130.3 13.0 ND npBrownstripe red snapper (Lutjanus vitta) ASC 212.0 31.5 np 9 Jongjareonrak et al. (2005a)Brownstripe red snapper (Lutjanus vitta) PSC 221.0 31.02 np 4.7wwb Jongjareonrak et al. (2005a)Hake (Merluccius merluccius) skin collagen 191.0 np np np Montero et al. (1990)Trout (Salmo irideus) skin collagen 180.0 np np np Montero et al. (1990)Fish (Pagrus major) scale collagen PSC 180.0 np 28.85 np Ikoma et al. (2003)Bigeye snapper (Priacanthus macracanthus) skin ASC 211.0 30.37* np 6.4wwb Jongjareonrak et al. (2005b)

28.85**Bigeye snapper (Priacanthus macracanthus) skin PSC 187.0 30.87* 1.1wwb Jongjareonrak et al. (2005b)

29.38**Ocellate puffer fish (Takifugu rubripes) skin PSC 170.0 np 28.0 44.7dwb Nagai et al. (2002)Adult Nile perch (Lates niloticus) ASC 193.0±200.0 np 36.0 58.7±63.1 Muyonga et al. (2004)Chub mackerel skin (Scomber japonicus) PSC np 25.6 np 49.8dwb Nagai and Suzuki (2000)Bullhead shark skin (Heterodontus japonicus) PSC np 25.0 np 50.1dwb Nagai and Suzuki (2000)Porcine dermis PSC 220.0 np 40.69 np Ikoma et al. (2003)Porcine skin ASC 205.0 np 37.0 np Morimura et al. (2002)

dwb: dry weight basis; np: not precise; Tm: thermal transition; wwb: wet weight basis*in deionized water; ** in 0.05mol.kgÿ1 acetic acid.

in terms of flux and selectivity. The 0.1�m-pore size membrane appeared to be

most efficient to separate CS from other compounds.

6.3.4 By-products from the shellfish industry

By-products from shellfish processing (crabs, shrimps, lobsters, etc.) are a good

source of chitin, chitosan and proteins, which consist of approximately 33%

protein and 33% chitin (Martin and Patel, 1991). Chitin is the second most

abundant natural biopolymer polymer after cellulose. Chitosan is the

deacetylated (to varying degrees) form of chitin, which, unlike chitin, is soluble

in acidic solutions. Chitin and chitosan are interesting polysaccharides with

unique properties that offer a wide range of industrial applications. They may be

employed to solve numerous problems in environmental and biomedical

engineering (Ravi Kumar, 2000). The food applications of chitin and chitosan

are numerous: these include preservation of foods from microbial deterioration

due to their antimicrobial activity, formation of biodegradable films, clarifica-

tion and deacidification of fruit juices, etc. (Shahidi et al., 1999). Chitosan may

find applications as a preservative coating for herring and Atlantic cod in

reducing or preventing moisture loss, lipid oxidation, and microbial growth

(Jeon et al., 2002).

The processing of crustacean shells mainly involves the removal of proteins

and the dissolution of calcium carbonate, which is present in crab shells in high

concentration (Ravi Kumar, 2000). Usually, a simple base extraction is

employed. The alkali removes the protein and deacetylates chitin simultaneously.

However, this process produces waste liquid containing base, proteins and

protein degradation products, and results in commercial products of inconsistent

physico-chemical characteristics. Chitinolytic enzymes would be a prime tool for

converting chitin into oligomeric units without the use of the chemical depoly-

merization such as concentrated hydrochloric acid. There are many reports of

purification and identification of chitinolytic activities in marine species, the

origin of the gastrointestinal chitinases (endogenous origin or from microflora)

being disputed (see the review of Shahidi and Kamil, 2001). However, the main

enzymatic processes use proteolytic enzymes for the shell digestion in order to

recover chitin and nutritionally valuable protein hydrolysates. For example,

Synowiecky and Al-Khateeb (2000) reported the digestion of Crangon crangon

processing discards preliminary demineralized using AlcalaseÕ 2.4L at 55ëC and

pH 8.5. Recovered protein hydrolysate contained, on a dry basis, 64.3% of

protein (N � 6.25), 6.24% lipids and 23.4% of sodium chloride. The authors

concluded that the enzymatic deproteinization of the shrimp shells using

AlcalaseÕ 2.4L was suitable for isolation of the chitin containing only about 4%

of protein impurities and also for production of protein hydrolysate with good

essential amino acid index (125.4) and protein efficiency ratio (2.99).

Simpson and Haard (1985) performed extraction of carotenoproteins from

shrimp processing discards using trypsin and a chelating agent. The product

recovered contained about 80% of the protein and carotenoid pigments present


in shrimp offal. A new process for advanced utilization of shrimp wastes

including enzymatic hydrolysis was recently described by Gildberg and

Stenberg (2001). The authors demonstrated the recovery of amino acids,

nitrogen and astaxanthin by AlcalaseÕ 2.4L pre-treatment of shrimp (Pandalus

borealis) processing wastes before further processing in chitosan. The AlcalaseÕ

2.4L treatment did not influence negatively either the recovery or the quality of

chitosan produced from the shrimp heads and scales. The nitrogen recovery was

about 70% as compared to only 15% by conventional method. The yield and

quality of chitosan was not affected by the enzymatic treatment. In addition, a

concentrate of astaxanthin was recovered and could constitute a valuable

supplement in salmon feed, improving both the growth and the disease

resistance of the fish.

According to Shahidi et al. (1999), most physiological activities and func-

tional properties of chitin and chitosan oligomers clearly depend upon their

molecular weights and a chain length of at least five residues is required. These

oligomers may be more advantageous than chitin and chitosan as polymers in

the field of food additives and nutraceuticals in human health, because chitin and

chitosan could not be degraded in the human intestine due to the absence of

enzymes such as chitinase and chitosanase. In this context, chitin and chitosan

may behave as dietary fibres which are excreted without any degradation in the

intestine.

6.4 Traceability of by-products

All commodities recovered from by-products may be prepared from a large

number of species and from starting materials that exhibit much wider

compositional variations. The following section will examine the question of

accurate determination of fish species at the various steps of processing, when

the by-products are presented in the form of fragments, the origin of which it is

impossible to determine. Another aspect of the traceability of the products, in

terms of contaminant and toxin contents will not be discussed in this section.

A method of genetic identification applicable to fresh fish samples as well as

to derived products, has recently been described (GueÂrard et al., 2005a). The

first step of the protocol consisted of extraction, purification and amplification

of a DNA fragment from muscle, skin, liver, bone or cartilaginous material

using PCR technology. The DNA can also be directly sequenced using either

one or the other external primer that was used for the amplification process or

with an internal primer. This protocol was illustrated with data obtained for two

processed (boiled, washed and dried) samples of `dry shark fin' collected from a

fish market in Asia, for which it was impossible to obtain any indication of

origin. After checking for possible misinterpretations of the results of electro-

phoresis, the sequences obtained in an easily exploitable forms from the two

`dry fin' samples were aligned and compared using the program (Blast) and a

sequence library established for phylogenetic purposes. From a comparison of


shark sequences, using only one (partial) marker sequence, the authors

demonstrated the processed sample was composed of two different species of

shark from the genus Sphyrna.

Thus, the use of genetic markers for studying genetic diversity and population

structure of marine resources, for stock identification and fishery management,

is a powerful, reliable and easy technique on fresh, ethanol-preserved, dry,

boiled or even processed samples. However, there is a need to complete this

approach, assessing methodologies for acquiring data on a large panel of

commercial fish and shellfish species and related by-products. It is also

necessary to draw up consensual protocols and to adapt the choice of the

markers to the level of the need for identification. In addition, it is of primary

importance that the sequences used as references were given with all the desired

precision on specimens identified without any ambiguity.

6.5 Conclusions and future trends

Bioconversion of fishery processing by-products is receiving increasing

attention with the realization that the by-products contain valuable components

which can be utilized for conversion into useful and high-value products.

Physical and chemical processes applied to fishery by-products have

demonstrated their limits in terms of functionality and quality of the final

products, while enzymatic processes appear to be prominent among those which

will need to be developed for the upgrading of fishery by-products, because they

offer the possibility of obtaining tailored products. With regard to future trends,

emphasis should be laid on the following aspects:

· The production of fish oils is a mature technology, with more than one

decade of experience and refinement. Due to (i) environmental pressures

demanding cleaner processes, (ii) existence of a market for new products

based on fish oils, and (iii) highly labile nature of PUFAs, the application of

enzymes in bioprocessing is especially advantageous due to environmentally

friendly processes. Increased efforts should be focused on research for more

specific lipases and proteases in order to control production of tailor-made

products.

· Fish protein hydrolysates prepared using enzymatic processes under

controlled conditions will probably find new uses and markets. Additional

research is needed for optimization of the enzymatic processes (e.g., choice

of more specific enzymes, development of models for prediction of

hydrolysis degree and biological activities) in order to develop hydrolysates

enriched in tailored peptides suitable for the production of specific food with

active compounds (antihypertensive, antioxidant, etc.).

· Further research work is needed on scaling up of laboratory-tested processes

to commercial applications based on improved understanding of the

mechanisms of enzymatic hydrolysis in heterogeneous media and on the

reproducibility of the enzymatic processes.


· The potential for genetic traceability of the raw material needs to be

developed with the use of genetic markers for accurate determination of fish

species and by-products at various steps of processing.

· Much greater efforts are required to recover by-products of higher quality (for

example, low microbial load, low content in heavy metals and toxins) and for

the training of people working on the recovery of fishery by-products. In

addition, efforts should be focused on the recovery and primary stabilization

of by-products such as storage at low temperature or freezing.

· The potential for upgrading of by-products from fish processing industries

needs to be improved. Efficient transportation or close proximity to a

processing plant will facilitate processing of very fresh raw material, in which

initial post-mortem oxidation processes are limited.

· Last but not least, so that the by-products have a durable future and may be

transformed in new products with high added value, they will have to be

treated with the same care as the products intended for human consumption.


This work was performed within the Integrated Research Project

SEAFOODPlus, Contract N FOOD-CT-2004-506359. The partial financing of

this work by the European Union is gratefully acknowledged. In addition, we

thank Mr. Jean-Jacques Le Yeuc'h for reviewing the English language of this

document.

6.7 References and further reading

ADLER-NISSEN J (1979), `Determination of the degree of hydrolysis of food protein

hydrolysates by trinitrobenzenesulfonic acid', J Agric Food Chem, 27(2), 1256±

1262.

ADLER-NISSEN J (1982), `Limited enzymic degradation of proteins : a new approach in the

industrial application of hydrolases', Chem Tech Biotechnol, 32, 138±156.

AIDOS I, VAN DER PADT A, BOOM R M, LUTEN J B (2001), Ùpgrading of maatjes herring by-

products: production of crude fish oil', J Agric Food Chem, 49, 3697±3704.

AIDOS I, VAN DER PADT A, LUTEN J B, BOOM R M (2002a), `Seasonal changes in crude and

lipid composition of herring fillets, byproducts, and respective produced oils `, J

Agric Food Chem, 50, 4589±4599.

AIDOS I, MASBERNAT-MARTINEZ S, LUTEN J B, BOOM R M, VAN DER PADT A (2002b),

`Composition and stability of herring oil recovered from sorted by-products as

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7.1 Introduction

Fish has an amino acid composition which makes it an excellent source of

nutritive and easily digestible protein. Fish proteins also possess properties that

make them good agents of water holding, gelation, fat binding, emulsification

and foaming (Xiong, 1997; Kristinsson and Rasco, 2000). For these reasons they

are an attractive food source and ingredient for various food applications. The

great demand for quality fish protein in the world is growing at a faster pace than

can be met with traditional resources, and has in many places led to significant

over fishing, often requiring governmental intervention (Kristinsson and Rasco,

2002). Despite the current bleak situation and the economic disruption this has

caused, there are still abundant sources of fish that are underutilized in the sense

that they are not utilized as human food, most notably the fatty pelagic fish

species and processing by-products. The underutilized species are normally

small dark muscle pelagic fish species, and make up to 40±50% of the world fish

catch (FAO, 2000). Only about 40% of the small pelagic species caught are

utilized for human consumption (Unido, 1990). The potential exists to develop

high-value functional protein products from these species. Furthermore, many

fisheries operations lead to substantial amounts of by-catch consisting of a

complex array of different fish species. For example, it has been reported that

over four pounds of by-catch is caught by shrimp boats in the Gulf of Mexico for

every pound of shrimp using conventional fishing gear (Cushman, 1998). This

material is typically discarded back to the sea with little attempt at recovery, and

represents an enormous amount of high quality protein which can be utilized for

7

Chemical processing methods for proteinrecovery from marine by-products andunderutilized fish speciesH. G. Kristinsson, A. E. Theodore and B. Ingadottir, University ofFlorida, USA

human consumption and furthermore could create a substantial dividend for the

local fishing industry. Even with the best preventative gear available to reduce

by-catch it will inevitably always be a sizable portion of the catch or about 2±3

pounds fish per pound of shrimp (Gulf and South Atlantic Fisheries Foundation,

Inc, personal communications). Discarding the product makes little economic

and environmental sense. The same holds true for fish processing by-products

which are typically composed of fish frames generated from filleting, including

visceral material, and are usually discarded or utilized in animal feed or

fertilizer.

Using conventional technologies to process fish and creating value added fish

products generally leads to limited utilization of the animal and large amounts of

protein-rich by-product materials are lost and not recovered. For example, even

the most efficient filleting operations will always yield great amounts of protein-

rich by-products, up to 60±70% of the fish depending on species (Mackie, 1982;

Kristinsson and Rasco, 2000). This material is high in quality protein and lipids,

and other valuable compounds which could be utilized for human consumption.

The global aquaculture industry is also growing at a rapid rate and should not be

overlooked as it will lead to more processing by-products in the coming years,

which could provide a sizable source of quality food protein and lipids.

The advantages of a process aimed at isolating high-quality food protein from

underutilized fish species and by-products are obvious. To upgrade products

made from pelagic species and by-products would not only add economic value

and assist the seafood industry, it would also be a more responsible use of these

sources. Major efforts have been undertaken in both academia and industry in

the past century to reach the goal of economic recovery and utilization of

proteins from underutilized species of fish and byproducts (Kristinsson and

Rasco, 2000, 2002). Most of these efforts have been met with limited success.

Some of the key hurdles in the successful and economic recovery of fish proteins

include: (a) the processes have to be able to process the material with as little

pre-processing as possible (ideally whole fish), (b) the processes have to be able

to utilize low-value sources of fish such as fatty pelagic fish species, trimmings

and frames, and (c) the processes have to be able to yield a consistent, func-

tional, palatable and stable product (Kristinsson and Rasco, 2000, 2002). Pelagic

species, by-catch and by-products present the fish processor with numerous

difficulties with respect to their utilization. These raw materials are very

complex, including bones, skins, connective tissue, abundance of oxidatively

unstable lipids, large amounts of pro-oxidants (blood and heme proteins),

unstable muscle proteins of low functionality and in some cases high levels of

active proteases (Okada, 1980; Hultin, 1994; Hultin and Kelleher, 2000). The

above factors hamper their direct consumption and greatly limit the possibilities

to economically recover functional proteins from them using conventional

techniques. Various attempts have been taken towards this goal in the past but

with limited success. Both chemical and enzymatic processes have been

developed with the goal to recover functional ingredients from these materials,

most notably proteins and lipids. One of the oldest methods to recover proteins

Chemical processing methods for protein recovery 145

from fish muscle is surimi processing which includes washing ground fish

muscle with water to leech out undesirable water soluble proteins and lipids.

This approach, however, has limited utility on very complex raw materials such

as whole fish or by-products. Early attempts on complex raw materials included

harsh chemical extraction processes where both proteins and lipids could be

effectively extracted, but in many cases functionality was lost and palatability

was low. This led to a growing interest in the production of fish protein

hydrolysates, which are extracted with enzymes under milder conditions, and are

discussed in detail in Chapter 10. Using proteases to extract fish proteins does,

however, lead to modifications in their functionality, and often a reduction in

certain key functional properties. Therefore, there was still a need to develop a

mild chemical extraction process, which could be used on challenging raw

materials but not compromise protein functionality. This has recently been

achieved with a novel acid and alkaline solubilization/precipitation process

developed by Hultin and coworkers (Hultin and Kelleher, 1999; Hultin et al.,

2004). This chapter discusses some of the principal chemical processes

developed primarily with the goal of extracting functional fish proteins.

7.2 Chemical extraction: fish protein concentrate

One of the earliest attempts to recover protein from by-products and under-

utilized species for use as a human food was the production of fish protein

concentrates (FPC). Fish protein concentrates are produced by using chemical

solvents and sometimes high temperatures to extract and separate proteins from

other components of the raw material (e.g., fat). The National Marine Fisheries

Service (NMFS) in the US (then Bureau of Commercial Fisheries) initiated a

large research program in this area in the early 1960s with the goal of finding

ways to produce FPC on a large scale to stimulate the US seafood industry and

also fight the global protein malnutrition problem (Snyder, 1967). The process

of making FPC is relatively straightforward. Solvent extracted FPC (type-A

FPC) is produced by extraction with isopropanol or azeotropic extraction with

ethylene dichloride. Ethanol has been successfully used as well. Figure 7.1

shows one example of FPC processing (Sikorski and Naczk, 1981). The raw

material is ground and then extracted with isopropanol at 20±30ëC for 50

minutes. The supernatant is then collected and extracted two times, first at 75ëC

for 90 minutes with isopropanol and then at 75ëC for 70 minutes with azeotropic

isopropanol. This gives a final supernatant fraction which is then dried, milled

and screened to separate out bone pieces. The final product should be largely

colorless and odorless and primarily consist of protein (<1% lipids) with high

biological value. This relatively harsh process does, however, take its toll on the

functionality of the proteins. Type-A FPC is poorly soluble or dispersible in

foods, which greatly limits its applicability (Cheftel et al., 1971; Mackie, 1974;

Venugopal et al., 1996). Relatively poor emulsification properties have also

been reported (Cheftel et al., 1971; Mackie, 1974; Venugopal et al., 1996).


Temperature during extraction has an impact on the functionality of the protein.

For example, it has been reported that FPC produced at 50ëC had significantly

lower emulsifying properties compared to FPC produced at 20ëC (Dubrow et al.,

1973). Both had very low solubility. Some studies have, however, reported that

FPC has good foaming properties over a wide pH range (pH 2±11), although this

functional property may be of limited interest for a fish protein ingredient

(Sheustone, 1953; Hermansson et al., 1971; Kinsella, 1976). Despite major

problems with protein functionality, solvent extraction has been the method of

choice for fatty pelagic fish species (e.g., sardine, herring and capelin) since the

protein is effectively separated from the oil, thereby improving oxidative

stability. It has been reported that isopropanol is a slightly more efficient solvent

than ethanol for fatty fish species since it removes more oil (Moorjani et al.,

1968). However absolute ethanol was able to produce FPC of lighter color and a

more neutral flavor (Moorjani et al., 1968).

Very few recent studies have been reported on the production and use of FPC,

since more successful protein extraction techniques are now available (e.g., fish

protein hydrolysates (Chapter 10) and fish protein isolates made with pH-shift

processing, see below). There are, however, a handful of papers from the last

10±15 years which demonstrate that solvent extracted FPC may find good uses if

Fig. 7.1 An example of fish protein concentrate production using isopropanol as theextracting solvent (adapted from Sikorski and Naczk, 1981).


it is produced properly. For example Vareltzis and coworkers (1990) used

ethanol extraction to make FPC from sardines and added it to hamburger patties.

These authors reported that that the overall functional properties of the

hamburger, i.e. water binding and cooking yield, increased with addition of FPC.

Penetration depth and shear force value of the FPC added hamburger also

indicated a better hamburger patty. However, on the downside, the hamburgers

were found to have a slightly unfavorable fishy flavor.

Although FPC on its own may possess poor properties in many cases, several

studies have shown that FPC may be a good substrate for enzymatic hydrolysis

to make fish protein hydrolysates (FPH) (Cheftel et al., 1971; Hale, 1972;

Spinelli et al., 1972; Quaglia and Orban, 1987a,b; Hoyle and Merritt, 1994).

This is because it provides a largely oil-free substrate and has partially denatured

proteins which are highly susceptible to enzymatic hydrolysis (Kristinsson and

Rasco, 2002). Enzymatically hydrolyzed FPC have generally greatly improved

solubility and dispersibility compared to the parent FPC, while some other

functional properties such as foaming would be reduced (Hermansson et al.,

1971; Kristinsson and Rasco, 2002). Taste and odor problems are generally

minimized for FPH when FPC is the starting material (Hale, 1972). For example,

Hoyle and Merritt (1994) found that FPC made from herring with ethanol

extraction and then hydrolyzed with either Alcalase or papain produced a FPH

with a reduced bitterness and less fishy odor compared to FPH made directly

from the herring. However, general poor functionality, off-flavors and colors,

high cost of production and possible traces of solvent in the final product have

made solvent extracted FPC commercially unsuccessful regardless of intensive

efforts (Mackie, 1982).

7.3 Chemical hydrolysis

Proteins can be chemically hydrolyzed with either acid or base with the help of

high temperatures. Chemical hydrolysis is a relatively inexpensive and simple

method to extract fish proteins from by-products and several processes have

been proposed for the acid or alkaline hydrolysis of fish (Hale, 1972). Chemical

hydrolysis is, however, a difficult process to control and because of that leads to

end products with variable composition and functionality (Blenford, 1994;

Skanderby, 1994). Furthermore, hydrolyzing proteins at very low or high pH,

sometimes in the presence of chemical solvents at very high extreme tem-

peratures, generally yields products with reduced nutritional qualities, poor

functionality and restricts their use to products such as seafood flavorings

(Webster et al., 1982; Loffler, 1986).

Acid hydrolysis is a more commonly used method to hydrolyze fish proteins

than alkaline hydrolysis. Acid hydrolysis of fish protein normally involves

adding strong hydrochloric acid or sulfuric acid to the fish raw material and then

extensively hydrolyzing the proteins at a high temperature, sometimes under

high pressure. Total hydrolysis can be achieved in 18 hours at 118ëC in 6N


hydrochloric acid (Thomas and Loffler, 1994), although those conditions would

rarely be used. The resulting fraction containing the hydrolyzed proteins is then

neutralized to pH 6.0±7.0 and dried or concentrated (Thakar et al., 1991). The

extensive hydrolysis leads to a product of very high solubility and dispersibility,

while other functional properties are largely destroyed (Kristinsson and Rasco,

2002). Due to the pH neutralization, the hydrolysate can contain a large amount

of salt (NaCl) which can reduce the palatability of the product. Another

downside of the acid hydrolysis process is the destruction of tryptophan, which

is an essential amino acid. This limits its use as a protein ingredient in food or

animal feed.

A handful of publications have shown that acid hydrolysis may find a use as a

protein recovery process. Orlova and coworkers (1979) reported a relatively

promising process where they used acid hydrolysis on whole fish, followed by

steam distillation to remove aromatic substances, then filtering and concen-

trating the extracted and hydrolyzed protein. The concentrate was successfully

used in dehydrated soup cubes and as a microbial media (Orlova et al., 1979).

Acid hydrolysis (sometimes with the aid of acidic proteases) is also commonly

used to convert underutilized species and processing by-products into fertilizer,

due to the simple operation, low production cost and extensive hydrolysis which

makes the peptides/amino acids easily utilized by plants (Kristinsson and Rasco,

2002).

The use of alkali (mainly sodium hydroxide) to hydrolyze protein can result

in poor functionality and adversely affect the nutritive value of the final product.

Despite these drawbacks, limited alkali treatment is used in the food industry to

recover and solubilize a broad range of proteins (Kristinsson and Rasco, 2002).

Very few studies have been published on the alkaline hydrolysis of fish proteins.

One of the reported key benefits of alkaline hydrolysis is to help modify and

improve functional properties of otherwise highly insoluble FPC (Sikorski and

Naczk, 1981). For example, Tannenbaum and coworkers (1970a,b) developed a

small-scale batch process that utilizes very high pH (12.5) and 95ëC for 20 min.

The final product consisted of large peptides, some which were relatively

insoluble at the isoelectric point of myofibrillar proteins, but demonstrated an

overall improvement in functionality compared to the original FPC. These

authors reported that the alkaline-treated FPC could be used as a milk substitute,

giving a product of far superior properties to that obtained with the original FPC,

which had very low solubility and dispersibility.

7.4 Surimi processing

Surimi originated in Japan where it has been a traditional food source for cen-

turies. Currently, Japan consumes about 70% of the surimi produced worldwide.

In Japan, the popular surimi-based products include: satsuma-age, chikuwa,

kamaboko, flavored kamaboko, hanpen/naruto, and crab sticks. Imitation crab

chunks, flakes, and sticks are the most popular form of surimi consumed in the


United States and Europe where it's gaining much popularity, and consumption

is growing at a rapid rate every year and the product receives a premium price

(Anonymous, 2001). China, Russia, and South America have also recently

discovered surimi-based crab sticks and are becoming major users (Park, 2000).

Surimi once referred to the ground fish paste formed during the manufac-

turing of the surimi-based product kamaboko. Surimi now describes mechanic-

ally deboned then washed fish muscle which is used as an ingredient for a range

of imitation seafood products, primarily crustacean and shellfish substitutes. It is

important not to confuse fish mince with surimi. Fish mince is a starting material

of surimi, not surimi itself. Briefly, in conventional surimi processing the raw

material is minced, mechanically deboned, washed with water, strained,

dewatered, cryoprotectants added and the product packaged and finally frozen

in blocks until used. The remaining myofibrillar protein concentrate demon-

strates enhanced functional properties, such as gel-forming ability, water

holding capacity, and fat-binding (Okada, 1992). Surimi is often modified for

long-term storage or further processed into other seafood products, such as

imitation crab meat, by incorporating additional components such as flavoring

agents, sugars, and salts. The primary fish species used to make surimi in Japan

and in the United States is Alaskan pollock. However, other species such as

menhaden (Brevooritia tyrannus), red hake (Urophycis chuss), Pacific whiting

(Merluccius productus), and spiny dogfish (Squalus acanthias) are being used in

the surimi industry (Gwenn, 1992). For many years the industry was dependent

on supply and availability of fresh fish. However, the discovery of adding

cryoprotectants to surimi in order to prevent protein denaturation during freezing

revolutionized the industry (Park and Lanier, 2000) which was no longer

dependent on fluctuations in supply of fresh fish.

Surimi manufacture is a multi-step process, as shown in Fig. 7.2. Fish heads

are removed, guts are cleaned, and bones are removed with large amounts of

water to separate the waste material from the muscle tissue. The muscle is then

minced by passing the material through a perforated screen and collecting the

mince. During the mincing process, tough cartilage, skin, and bones do not pass

through the mesh screen, thus removing further undesirable material from the

muscle. By removing blood, skin, membranes, and other materials, the muscle

becomes more stable and yields a higher quality product (Park and Morrissey,

2000). The next phase in surimi processing is the washing step. The number of

washing cycles and water volume depend on many factors, such as fish species,

facility type and capacity, initial fish quality, and desired final surimi quality.

Generally, the fish to water ratio is between 1:5 and 1:10, although more modern

operations are able to use ratios as low as 1:2. Washing with water removes

components that can have negative effects on gelation (e.g., sarcoplasmic

proteins, although this is debatable) and compounds that can cause flavor, odor,

stability and color problems.

The product after washing primarily consists of myofibrillar protein, with a

significant decrease in amount of blood, soluble proteins, connective tissue and

fat (which are mostly removed during washing) as compared with the starting


material. By removing or at least decreasing the amount of undesirable

compounds in the fish, the surimi texture, color, flavor, and storage quality is

increased. During the entire process the temperature should be maintained low

enough to prevent protein denaturation which varies according to species

(Ohshima et al., 1993; Park, 2000). The washed muscle is then refined, which

removes any remaining bone pieces, skin, scales and connective tissue. The

material is then dewatered two or three times by centrifugation, screening or

pressing. Dewatering is necessary because during the process water is absorbed

(approximately 100% increase) due to repulsion of negatively charged proteins

in the washed mince (which is at pH 6.4 to 7.0). The water reduces the repulsion

by separating the proteins. Addition of salt (0.1 to 0.3% NaCl or a combination

of NaCl and CaCl2) to the wash water further reduces the repulsive forces by

shielding negative charges which allows the proteins to be in closer contact with

each other, thus expelling water and reducing the tendency of the tissue to

absorb water.

Since surimi is generally frozen after dewatering, it is important to protect the

functional properties of the product during storage. By adding cryoprotectants to

the washed refined material prior to freezing, protein denaturation and aggrega-

tion are reduced, which would otherwise result in reduced gelation ability of the

proteins (Park and Lanier, 2000). The most common cryoprotectants used in the

surimi industry are sorbitol and sucrose at ~8±9%, along with a 1:1 mixture of

sodium tripolyphosphate, and tetrasodium pyrophosphate at ~0.2±0.3%. These

Fig. 7.2 Processing steps in conventional surimi processing.


compounds are uniformly distributed throughout the surimi by using a silent

cutter (Park and Morrissey, 2000). Prior to freezing, proteolytic enzyme

inhibitors are sometimes added along with cryoprotectants to prevent proteolytic

degradation of proteins during heating. For example in Pacific whiting surimi

manufacturing enzyme inhibitors have to be added as well as the application of a

very rapid heating rate to minimize proteolytic degradation of muscle proteins

(Klesk et al., 2000; Park and Lanier, 2000). Surimi is then frozen in blocks or in

chips or chunks (Park and Morrissey, 2000).

High quality surimi has generally only been produced from lean white fleshed

fish such as Alaska pollock. However, much effort has been put into how to make

good surimi from dark fleshed underutilized species as well as by-products. Most

of these attempts have led to products with poor gelation properties, in part due to

the low pH of the muscle of these species and different protein isomers in dark vs.

white muscle. Considerable color and lipid stability problems are also

encountered with surimi from dark muscle species and by-products due to the

high amount of lipids, pro-oxidants and pigments (Okada, 1980; Hultin and

Kelleher, 2000). Studies have shown that oxidative problems can be reduced with

proper processing techniques. For example, having the wash-water alkaline or

mincing fish tissue underwater (preferably in an alkaline solution) may reduce

rancidity and improve gel strength (Hultin and Kelleher, 2000). Also applying

antioxidants early on during processing may significantly increase gel strength

and oxidative stability (Kelleher et al., 1994). Many researchers have also

proposed ways to remove the dark muscle of these species and process only the

light muscle (e.g., Langmyhr et al., 1988; Shimizu et al., 1992; Spencer and

Tung, 1994). In some cases this has led to high quality surimi, comparable to that

from Alaska pollock, which is the industry standard (Ishikawa et al., 1977;

Suzuki and Watabe, 1987). However, this practice becomes very expensive as it

requires filleting and then deep skinning the fillets to remove the dark muscle,

which is highly unfeasible for small pelagic fish species due to prohibitive cost of

labor. Also, the more dark muscle is removed so is some light muscle and thus

yield drops. In addition, utilizing only the light muscle of fish will lead to

substantial loss of protein. Even just obtaining the fillets from these species can

be too cost prohibitive. Also, even if fish can be headed and gutted, some tissues

such as the black skin layer on their belly flaps and the kidney tissue along the

backbone make processing whole fish far more difficult than just the muscle

(Hultin and Kelleher, 2000). The requirement to use very fresh, preferably lean

white fleshed fish fillets and the high labor needed for conventional surimi

processing, therefore makes it a relatively expensive process. The large quantities

of water used also increase the cost of this process.

7.5 Fish protein isolates: pH-shift processing

Two recent processes, developed by Dr Herbert O. Hultin and coworkers (Hultin

and Kelleher, 1999; Hultin et al., 2004, 2005), involving acid and alkaline


solubilization and isoelectric precipitation of muscle protein are specifically

designed to recover highly functional, stable proteins from low value

underutilized fish and by-products. These new processes have shown great

promise for both cold and warm water fish species and are currently being

commercialized for several species. Fish proteins can be solubilized without the

addition of salt at very low and high pH values. The high and low pH values give

the muscle proteins a large net charge, causing them to solubilize. At the same

time as the proteins solubilize at extremes of pH, the cellular lipid membrane

encasing the myofibrillar proteins is disrupted causing a dramatic drop in

solution viscosity (Kristinsson, 2002; Kelleher et al., 2004). This allows new

approaches to be taken to economically recover fish muscle proteins to produce

functional protein isolates from fish sources of low value (Hultin and Kelleher,

1999; Hultin et al., 2004). The process (Fig. 7.3) involves subjecting a diluted

slurry (5±10 fold dilution) of finely homogenized muscle tissue to either a very

low pH (~2.5±3) or very high pH (~10.8±11.2) at low temperatures. The

solubilization of the muscle proteins, cellular membrane disruption and dramatic

Fig. 7.3 Schematic representation of the acid and alkaline processes used in theproduction of functional fish protein isolates. The process involves solubilizing muscleproteins at low or high pH, separating them from undesirable muscle components viacentrifugation and recovery of the proteins of interest by isoelectric precipitation. Thefinal protein isolate can then be used directly, or stabilized with cryoprotectants and

frozen until used. (Adapted from Kelleher and Hultin, 2000.)


drop in viscosity enable the cellular lipid membranes to be separated from the

soluble proteins by centrifugation (Kelleher and Hultin, 2000), at the same time

removing solids such as bones, scales and neutral fat, which are not desired in

the final product (Fig. 7.4). The soluble proteins are then collected and

recovered by adjusting the pH to ca. 5.2±5.5, the isoelectric pH of most muscle

proteins (primarily myofibrillar proteins), causing them to aggregate and

precipitate to give a protein pellet, i.e. the protein isolate.

There are several significant benefits of these processes compared to harsh

chemical extraction and hydrolysis processes and surimi processing. Whole fish

with skin and bones, and fatty fish can potentially be utilized in the acid- and

alkali-aided processes because proteins are selectively separated and recovered

from undesirable muscle components. This is not feasible using typical surimi

processing without greatly negatively affecting the recoveries and quality

(Hultin, 2002). In the acid- and alkali-aided processes protein recoveries are

normally significantly higher compared to many other recovery processes.

Significant amounts of proteins are often lost during the production of FPC as

well as hydrolysis. Conventional surimi processing leads to the loss of almost all

sarcoplasmic protein during the washing steps, upwards of 35% of the total

protein. Multiple washings furthermore lead to myofibrillar protein solubiliza-

tion and consequently some loss of these proteins as well (Lin and Park, 1996).

The loss of these proteins during conventional surimi processing is responsible

for the significant decrease in yield. The sarcoplasmic proteins are, however,

largely recovered in the acid- and alkali-aided processes, thus substantially

increasing yield. As a testament to this, using Pacific whiting fillets as the

starting material, a conventional three-washing cycle surimi processing yielded

only 40% recovery, compared with 60% recovery using acid-aided processing

(Choi and Park, 2002). Kristinsson and coworkers (2005) published data on

catfish muscle, and found that the acid- and alkali-aided processes had

Fig. 7.4 The three phases that develop after the first centrifugation step at low or highpH. The soluble protein is collected and sedimented by isoelectric precipitation aided by

centrifugation to prepare the protein isolate.


significantly higher protein recovery than a lab-scale surimi process (Table 7.1).

Similar recoveries were recorded for herring light muscle; 74% for acid-aided

process and 68% for alkali-aided process (Undeland et al., 2002). The protein

recovery can be further increased when the first centrifugation step is omitted

(Kristinsson et al., 2005). This is possible for certain raw materials which are

stable towards lipid oxidation, and can bring the protein recovery close to 90%.

The bottom sediment from the first centrifugation can also be reprocessed to

increase the level of protein extracted (Kristinsson and Demir, 2003). The

isoelectric precipitation step in the acid- and alkali-aided process also aids in the

higher protein yields as compared to conventional surimi processing. This is due

to the protein having a zero net charge at their pI, thus leading to aggregation

and precipitation of the proteins. Surimi processing, on the other hand, does not

involve reducing the native pH, resulting in a moderate negative charge on the

protein molecules which gives them more solubility and thus more proteins are

leached out during washing.

Generally, the acid-aided process has resulted in slightly higher protein yields

compared to the alkali-aided process with a few exceptions (e.g., tilapia).

Studies by Kristinsson and coworkers (2005) have demonstrated that more of the

sarcoplasmic proteins are recovered for the acid-aided process compared to the

alkali-aided process, due to more aggregation at pH 5.5 as a consequence of a

more improperly refolded protein structure. Many of the sarcoplasmic proteins

are not greatly affected by the high pH in the alkaline process, and are thus

partly or fully native when readjusted to pH 5.5. That pH is away from the

isoelectric point of many sarcoplasmic proteins, and thus they don't ready

aggregate and co-precipitate with the aggregated myofibrillar proteins. The

higher recovery of sarcoplasmic proteins for the acid process, however, means

that more heme proteins are recovered with the isolate, which can have negative

consequences on color, odor and eventually flavor. More retention on sarco-

plasmic proteins in the isolate does, on the other hand, translate to fewer

pollution problems since the processing water has a low biological oxygen

demand due to its relatively low protein content. This is not the case for surimi

processing.

A major advantage of the acid and alkali isolation processes is that undesir-

able compounds, like skin, bones, microorganisms, cholesterol, membrane

lipids, and other contaminating materials are removed during the first centrifug-

Table 7.1 Comparison of protein recovery and lipid reduction for the acid-aided, alkali-aided and surimi processes (adapted from Kristinsson et al., 2005)

Protein recovery Lipid reduction (neutral and polar)

Alkali-aided process 70.3 � 2.9%b 88.6 � 2.8%c

Acid-aided process 71.5 � 4.5%b 85.4 � 2.0%b

Surimi processing 62.3 � 3.1%a 58.3 � 7.8%a

Means within one column having different superscript letters are significantly different (p < 0:05).


ation step (Hultin and Kelleher, 2000). Work on catfish has demonstrated that

both processes lead to a significant reduction in aerobic bacteria and also a

longer bacterial shelf life compared to surimi from the same starting raw

material (Kristinsson, 2004). This work demonstrated that bacteria are both

killed/injured by the high and low pH and are also removed during the first

centrifugation step in the bottom sediment. The processes also have the potential

to remove lipid soluble toxins such as mercury and PCBs, and along with the

reduction in bacteria give a safer fish protein product. Removal of most lipid

components in the acid and isolation procedures can lead to greater oxidation

stability and decreased off-odor development as compared with conventional

surimi where membrane lipids mostly remain (Hultin and Kelleher, 2000). The

great reduction in lipids is a key step in this process, since many materials of

interest are rich in triacylglycerols (neutral storage lipids) and in particular

membrane phospholipids due to high amounts of mitochondria in dark muscle

(Hultin, 1994). The substantial absence of membranes and neutral lipids in the

protein isolate clearly distinguishes the acid and alkali processes from presently

available processes. Cholesterol is also reduced in the process due to the

removal of membranes (Mireles Dewitt et al., 2002). Due to the higher

unsaturation of phospholipids vs. neutral lipids and their greater surface area

exposed to the cell aqueous phase, they are known to be the main substrate for

oxidative reactions in muscle foods (Shewfelt, 1981; Gandemer, 1999). Their

removal is therefore expected to greatly enhance the oxidative stability of the

final protein isolate. This is not achieved in conventional processes unless

organic solvents are used, which destroys protein functional properties.

Kristinsson and Demir (2003) studied four different species and reported total

lipid reduction of 58.3, 72.1, 10.4 and 16.7% for surimi processing (catfish,

Spanish mackerel, mullet and croaker, respectively), 85.4, 76.9, 58, 38.1% for

the acid-aided process, and 88.6, 79.1, 81.4 and 68.4% for the alkali-aided

process. Undeland and coworkers (2002) reported ~70% reduction in neutral

lipids and ~50% reduction in membrane phospholipids for herring white muscle

using both acid and alkali-aided processes. Undeland and coworkers (2005) later

reported that including centrifugation in the solubilization step to remove

membranes led to about 50% less development of secondary oxidation products

(as measured by TBARS analysis). Kristinsson (2004) also demonstrated that

catfish protein isolates have lower TBARS values on storage when centrifuga-

tion is included in the solubilization step, compared to skipping the centrifuga-

tion. Kristinsson and Demir (2003) showed that the alkali-aided process gives a

more oxidatively stable protein isolate at pH 5.5 than the acid-aided process, and

in many cases is more stable than surimi (Fig. 7.5). Substantial lipid oxidation

was seen during the acid-aided process. Petty and Kristinsson (2004) investi-

gated oxidation at low and high pH in detail for Spanish mackerel muscle

homogenates and showed extensive oxidation development at low pH but almost

no development at high pH. When the homogenate was adjusted from low and

high pH to pH 5.5 or 7, the isolates from the acid-aided process oxidized

significantly more than those form the alkali-aided process. The same was seen


in a model system with trout hemoglobin and washed cod muscle, and it appears

that the hemoglobin (one of the main catalysts/mediators of oxidation in fish

muscle) was effectively stabilized at high pH but became highly pro-oxidative at

low pH (Kristinsson and Hultin, 2004). Undeland and coworkers (2005) also

reported extensive oxidation for herring isolates made with the acid-aided

process. This oxidation could be effectively reduced and delayed by employing

proper antioxidant treatments, such as erythorbate, EDTA and sodium

tripolyphoshpate (Undeland et al., 2005). Undeland and coworkers (2005)

demonstrated that if antioxidative treatments (metal reducing agents and metal

chelators) are incorporated early on in the extraction process, e.g. during

homogenization, oxidative stability of the isolate can be improved. Speed of

processing at the extreme pH appears to be important. Lipid oxidation can be

somewhat reduced if the system is at low pH for a very brief time before being

adjusted to pH 5.5 (Hultin, 2004; Petty and Kristinsson, 2004).

Heme proteins are more effectively removed with the alkali-aided process

compared to surimi processing, resulting in a product that is whiter and more

stable to lipid oxidation. Heme proteins are also protected from denaturation and

autoxidation during high pH treatment at low temperature (Kristinsson, 2002).

The acid aided process, however, leads to the denaturation of heme proteins and

thus co-precipitation with muscle proteins when they are adjusted to pH 5.5

(Kristinsson and Hultin, 2004; Kristinsson et al., 2005). This leads to an isolate

with darker color and more oxidative problems (Kristinsson and Demir, 2003).

Choi and Park (2002) reported that whiteness was lower in acid-treated Pacific

whiting isolates as compared with conventional surimi. This lower whiteness of

Fig. 7.5 Levels of oxidation for surimi and acid and alkali isolates at day 3 ofrefrigerated storage (4ëC) as assessed by TBARS (secondary oxidation products). Acidisolates were made using pH 2.5 as the solubilization pH, while alkali isolates were madeusing pH 11. Isolates were recovered at pH 5.5 and stored at 4ëC. Surimi was made by bywashing ground muscle in 3 volumes of water 3 times, with the last wash containing 0.3%NaCl to aid in dewatering. The pH of the surimi was not adjusted and was from pH 6.5±

6.6 for all species. Surimi was also stored at 4ëC.


the acid isolates was attributed to higher b* values, indicating a more yellow

appearance. Kristinsson and Demir (2003) demonstrated that color was

generally good for isolates made from several warm water species using the

acid- and alkali-aided processes as compared to surimi from the same species.

Table 7.2 shows the color for catfish and croaker isolates and surimi. Isolates

had higher L* values and whiteness than surimi, however the acid isolates had

higher yellowness, possibly since more denatured heme proteins are found in

these isolates (Kristinsson and Demir, 2003).

It has been found that functional properties are retained, decreased (in few

cases for the acid process) or often significantly improved (most notably for the

alkali process) using the pH-shift processes to recover fish proteins. The main

functional property of extracted fish proteins is their ability to form strong and

elastic gels with high water-holding capacity. Research shows that the ability of

isolates to form gels varies, depending on species and conditions used to make

the isolate. Hultin and Kelleher (2000) reported that acid-aided isolates made

from Atlantic cod and mackerel produces good gels. Later it was found that

Pacific whiting surimi from a 3-cycle washing method made stronger gels than

gels from the acid-aided process (Choi and Park, 2002). Work by Kristinsson

(2004) has demonstrated that the acid-aided process in some cases does form

better gels than surimi but in some cases worse. All studies by this group,

however, clearly show that the alkali-aided process produces superior gels over

both the acid-aided process and the surimi process. For example, Ingadottir and

Kristinsson (2004, 2005) reported significantly higher gel strength and elasticity

for tilapia isolates made with the alkali-aided process compared to the acid-

aided process and surimi (Fig. 7.6). Davenport and Kristinsson (2004) did also

report using oscillatory rheology and torsion testing that catfish protein isolates

from the acid-aided process have significantly lower gel-forming ability than

isolates from the alkali-aided process, and hypothesize that this could be due to

some very different effects on protein structure at acid vs. alkali pH. Another

study by Yongsawatdigul and Park (2001), demonstrated that rockfish protein

isolates produced from the alkali-aided process had better gel-forming ability as

compared to the acid-aided and conventional surimi processes. The lower

performance of the isolates from the acid-aided process could be due to

Table 7.2 Color of protein isolates and surimi according to Hunter L*, a* and b* values(adapted from Kristinsson and Demir, 2003)

Sample L* a* b*

Catfish alkali PI 75.0 � 0.7 ÿ3.0 � 0.2 0.2 � 0.4Catfish acid PI 73.8 � 0.4 ÿ3.6 � 0.2 5.7 � 0.3Catfish surimi 70.4 � 1.1 ÿ0.9 � 0.2 0.7 � 0.4

Croaker alkali PI 67.6 � 0.4 ÿ2.1 � 0.2 4.2 � 0.4Croaker acid PI 69.8 � 0.7 ÿ2.0 � 0.3 7.8 � 0.4Croaker surimi 64.7 � 2.4 0.2 � 0.1 6.4 � 0.9


Fig. 7.6 Shear stress (kPa) and strain values of gels produced from tilapia acid protein isolate, alkali protein isolate and surimi. The acid isolateswere made by using pH 2.9 as the solubilization pH, while the alkali isolates were made using pH 11. Isolates were recovered at pH 5.5. The surimiwas made by washing ground muscle in 3 volumes of water 3 times, with the last wash containing 0.3% NaCl to aid in dewatering. A protein pastewas made with or without the addition of 2% NaCl, and adjusted to pH 7.2. No cryoprotectants were added. Pastes were cooked in steel tubes at 80ëCfor 30 min to form a gel. The gels were stored in a cold room at 4ëC for 48 hours prior to testing with a Torsion Gelometer. (Adapted from Ingadottir

and Kristinsson, 2005.)

proteolysis which has been seen during the low pH solubilization step (Choi and

Park, 2002; Undeland et al., 2002; Ingadottir and Kristinsson, 2004).

Proteolysis is a major problem for any muscle protein extraction process as

it will lead to adverse effects on protein functionality, particularly gelation and

water-binding. Both Ingadottir and Kristinsson (2005) and Undeland et al.

(2002) found proteolysis of myosin at low pH. Kristinsson and Demir (2003)

reported the same findings for Spanish mackerel. For some species, proteolysis

can also occur at the recovery pH, i.e. pH 5.5. Choi and Park (2002) showed

that cathepsin B and L activity was higher in an acid-treated Pacific whiting as

compared with a 3-cycle washed surimi, leading to poorer gel-forming ability

for the isolate. The differences between the cathepsin activity levels were due

to cathepsin B and H removal during repeat washing and pH 5.5 being the

optimum pH for cathepsin L activity (An et al. 1994; Choi and Park, 2002).

However, cathepsin H was removed from the surimi and inactivated in the acid

isolate. Therefore, this enzyme did not contribute to decreased gel-forming

ability in either sample. Some species have, however, not demonstrated any

proteolysis at low pH, e.g. cod (Hultin and Kelleher, 2000) and catfish

(Kristinsson et al., 2005). Cod isolate made with the acid-aided process makes

a good gel, while the catfish isolate made with the acid-aided process makes a

poorer gel than isolates from the alkali-aided process. The difference has been

linked to how the muscle proteins respond differently to changes in pH.

Davenport and Kristinsson (2003) reported that catfish myosin adjusted to a

low pH (2.5) and then readjusted to pH 7 had significantly less gel-forming

ability compared to myosin adjusted to high pH (11) and then readjusted to pH

7. The difference was not attributed to proteolysis, but rather some changes

within the protein, which are yet to be fully understood. Previous work with

cod myosin demonstrated that acid and alkali treated myosin performed about

the same as untreated myosin (Kristinsson and Hultin, 2003). This

demonstrates that proteins from different species do respond differently to

these processes.

The theory has long been that the sarcoplasmic proteins interfere with gel

formation, possibly by binding to the myofibrillar proteins on heating (Okada,

1980; Shimizu et al., 1992; Park et al., 1997). This is one of the arguments why

these proteins are removed in surimi. However, some recent studies have

challenged this belief and have shown that gel strength is either equal or, in fact,

enhanced by the presence of the sarcoplasmic proteins (Morioka and Shimizu,

1990; Ko and Hwang, 1995). The presence of the sarcoplasmic proteins in the

isolates from the acid and alkali process does not appear to negatively impact the

gel strength of the final product (Hultin and Kelleher, 1999) but gel mechanism

may be different as these proteins have been acid or alkali denatured. This may,

however, be species dependent. The acid-aided process does recover more of the

sarcoplasmic proteins, and as has been discussed above, some species produce

acid isolates of poor functionality, which could be linked to the higher levels of

denatured sarcoplasmic proteins. Kristinsson and Crynen (2003) studied the gel-

forming ability of myofibrillar and sarcoplasmic proteins from catfish, indivi-


dually and in combination. The results indicated that sarcoplasmic proteins

subjected to a low and high pH and added to myofibrillar proteins subjected to

the same treatment improve the gel-forming ability of the overall system

compared to myofibrillar proteins alone. The results suggested that adverse

changes in the myofibrillar proteins at low pH (pH 2.5) and positive change in

the myofibrillar proteins at high pH (pH 11) may explain the difference between

the performance of the acid and alkali isolates, rather than changes in the

sarcoplasmic proteins. This is being investigated in more detail.

It is a commonly held view that denaturing fish muscle proteins has a

detrimental impact on their functional properties (Konno et al., 1997;

Visessanguan and An, 2000). As the muscle proteins experience very low or

high pH in the acid and alkaline processes, one might assume that they lose their

functionality since they are partly denatured. Preliminary work by Kristinsson

(unpublished data) on the cellular organization of the muscle cell using phase

contrast microscopy showed that as pH is either lowered or increased, the

contractile element within the cell is being distorted as its protein constituents

are being progressively more charged, repelling each other and eventually they

become solubilized and any remnants of the muscle cell are lost. Upon pH

readjustment to pH 5.5 the cell structure is clearly not recovered and an

aggregate of partially denatured muscle proteins is observed. These findings are

supported by recent electron microscope data by Wright and Lanier (2005).

Studies on the molecular level with myosin have demonstrated that the protein

subunit assembly and tertiary structure are greatly affected by the low and high

pH, and are not reversibly fully refolded or reassembled on pH readjustment to

neutrality (Kristinsson and Hultin, 2003). Furthermore, the ATPase activity of

myosin is almost completely lost on acid or alkali treatment (Kristinsson and

Hultin, 2003). Other workers have also shown that isolates made with the

process have little or no ATPase activity, yet they have good gel-forming ability.

Studies frequently report on the positive relationship between functional ATPase

activity and functionality of muscle proteins (Katoh et al., 1979; Ooizumi et al.,

1981; Konno et al., 1997). Interestingly, even when all ATPase activity is lost in

the isolate, some of them have improved gelling capability compared to proteins

still with high ATPase activity. These findings suggest that a native structure is

not required for good gel-forming ability, and a partly pH denatured protein

may, in fact, be better suited to form quality gels, perhaps through different

mechanisms than native proteins.

7.6 Other processes using low or high pH

Only a handful of other workers have described processes for fish proteins that

utilize high or low pH. These processes, however, differ considerably in nature

to the one described above and the end uses are quite different. Cuq and

coworkers (1995) reported on the acid solubilization of fish muscle proteins at

pH 3 using aqueous acetic acid for the purpose of producing edible packaging


films. A process was reported by Shahidi and Venugopal (1993) where minced

Atlantic mackerel, herring or capelin is homogenized in aqueous liquids,

including acetic acid at pH 3.5. Venugopal and Shahidi (1994) later reported a

process where Atlantic mackerel is suspended in water and acetic acid at a pH of

3.5. The use of acetic acid in these processes, however, in many cases increases

viscosity of these fish protein suspensions and in some cases reduces it

insufficiently so that cellular lipid membranes cannot be separated from the fish

proteins. The volatile acetic acid also potentially leads to a strong odor to the

final material which may limit their use as food products. Some of the above

processes also involved washing steps which remove the water soluble sarco-

plasmic muscle proteins, which are retained in the acid and alkaline processes

described previously.

Alkaline pH has also been used in the processing of fish muscle and muscle

from other sources. One common use of high pH is to recover protein from

deboned meat (McCurdy et al., 1986; Opiacha et al., 1994), not, however,

involving separation of membrane lipids from the alkali solubilized protein.

Alkaline conditions have been used previously in the manufacture of surimi

from fish, where alkali or compounds with buffering capacity at high pH are

added to the wash water to increase pH. The wash water pH in these processes

is, however, considerably lower than the pH used in the alkaline process

employed in this proposal. The increase of wash water pH reportedly yields a

product with improved gelling abilities, brighter color and lower lipid content

(Shimizu et al., 1992; Jiang et al., 1998; Hultin and Kelleher, 1999). On the

other hand, yield drops considerably in these processes, since presumably

increased protein solubility as pH is increased would increase the amount of

proteins removed in the washing steps. For example, it has been reported that

processing surimi from mackerel light muscle using alkaline wash water led to

only 40% protein recovery (Hultin and Kelleher, 2000).

7.7 Future trends

To meet the increasing demand for quality fish proteins and products containing

fish proteins, it is of great importance to utilize our raw materials more

responsibly as well as to find new resources of fish to utilize. Processing

methods which can employ inexpensive raw material and whole fish instead of

fillets would create a significant economic advantage to the fish protein

ingredient industry, as new sources of fish unsuitable for conventional processes

could enter the market, and production could be increased to meet the world

demand. This chapter has reviewed some of the chemical processes which can

be employed to recover fish proteins, as well as lipids. From looking at research

and industrial applications of these processes, the pH-shift processing appears to

have the most promise on by-products and underutilized species. To increase the

success of these processes and their products it is essential to put more research

efforts into the commercial applications of these proteins as food ingredients or


as food products. More research should be focused on the utilization of extracted

fish proteins for human consumption rather than animal consumption, although

the latter cannot be overlooked. One very promising food application being

studied is the use of isolated fish proteins as water-binders in seafood products.

It has been found that these proteins can effectively compete with and

outperform phosphates as water-binders. This could have a significant meaning

for the seafood processing industry. Future research efforts should also be

directed towards ways to effectively stabilize the proteins against functional

changes as well as finding efficient and economic ways to stabilize the isolated

proteins against oxidative changes (e.g., lipid oxidation during processing and in

the final isolate). The future for functional fish protein ingredients is promising

and with the right mindset from industry, government and academia great

progress can be made in the near future.


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Part II

Food uses of marine by-products

8.1 Introduction: by-catch, discards and by-products

The evolution of the estimated world fish production from 1950 until around

1990 was basically characterized by an almost continuous growth, which was

followed by a levelling off until the end of the 1990s. The increased global

production further recorded was due to aquaculture catches, which in that period

had an average annual growth rate of 5.3%, excluding China's production (FAO,

2002). In 2000, reported global capture fisheries attained 77 to 78 million tonnes

(China not included) with relative percentage distribution shown in Fig. 8.1. In

this FAO report it is also pointed out that 70.8% (63 million tonnes) of the

estimated production was used for direct human consumption and the remaining

was destined for non-food products, mostly for the manufacturing of fish meal

and oil. The highest share of the total catch used for fish meal production was

38% in 1970.

The global per capita food fish supply has increased from about 7 kg in 1950

to 15 kg in 2000 (Valdimarsson and James, 2001). However, some decrease

occurred from 1987 to 2000 because the world's population increased more

quickly than the total production of food fish (FAO, 2002). Nevertheless, these

mean values mask important regional differences such as those between

developed and developing countries where the per capita consumption is 29 and

12.5 kg, respectively (Valdimarsson and James, 2001).

The previously mentioned production levels for the total world fisheries do

not include the discards, which are potentially useful for food or feed. According

to Alverson et al. (1994) such levels of catch include the portion of the catch

8

By-catch, underutilized species andunderutilized fish parts as foodingredientsI. Batista, Fish and Sea Research Institute (IPIMAR), Portugal

returned to the sea as a result of economic, legal, or personal considerations.

Another term frequently used is by-catch, which refers to the total catch of non-

targeted animals (Kelleher, 2005). Alverson et al. (1994) estimated 27.0 million

metric tonnes (MMT) of global discards annually, based on a target catch of 77

MMT. Nevertheless, these figures do not include data from freshwater,

molluscan and recreational fisheries. Discarding of fish or its components

constitutes a loss of valuable food with negative consequences for the

environment and biodiversity. Figure 8.2 shows discard weight in the oceans

and Mediterranean and Black Sea regions. The discarded catch from Pacific

Ocean represents the majority of total world discards and about 55% of them are

originated within the Northwest Pacific fisheries.

Shrimp fishery accounts for 35% of the global marine discards and the ratio

of discarded weight to landed weight is 5.2. However, there are important

differences from a gear-specific perspective. Shrimp trawls present the highest

by-catch to landed ratio (in weight basis), which was around 11.8; the lowest

ratio was recorded for pelagic trawls (less than 0.01).

In 2000, more than 60% of total world fishery production suffered some type

of processing problems as reported by FAO (2002). The demand for fresh fish

has generally increased in the last four decades, but in the 1990s the estimated

demand rose from 28 MMT in 1990 to 52 MMT at the end of this decade. Since

1960, the share of frozen fish also increased but canned fish remained constant

at around 10% and production of cured fish gradually declined from 20 to 10%

of the total amount.

The processing industry generates a significant amount of by-products, which

depends on the species and type of product. These by-products are mainly used

for fish meal production or other low price ingredients, but sometimes are just

dumped. This means that by-products together with the by-catch constitute an

important percentage of total catch. In the United Kingdom it was estimated that

only 43% of the total catches end up as product for human consumption and the

Fig. 8.1 Distribution of the world fisheries production in 2000 (FAO, 2002).


remainder is classified as waste (Archer, 2001). The waste material comes

mainly from the on-shore processing sector (35% of the resource) whereas the

contribution of discards and processing waste at sea represents 17 and 5%

respectively. In Norway, it is estimated that 80±90% of the by-products

generated at sea are dumped and only 10% of the by-products from the on-shore

fish processing is used in products for human consumption (Sandbakk, 2002).

8.2 Key drivers

The declining of traditional fish stocks coupled with strict management measures

have led to the exploitation of other fish species usually considered underutilized.

It has been also recognized that discards into the sea are responsible for a variety

of biological, ecological, environmental, economic and social costs (Alverson et

al., 1994). Thus, the importance of determining the amount and variability of

discards has been underlined (Connolly and Kelly, 1996; Blasdale and Newton,

1998; Allain et al., 2003). The development of a commercial market for discarded

species may contribute to reducing wastage. The Individual Quota system

established in Iceland induced the fleet to utilize unconventional species, which

were not covered by this quota system (Valdimarsson, 1998). The utilization of

these species was encouraged by guaranteeing a minimum price, which was

supported by a so-called By-Catch Bank. This Bank acted as a matchmaker

between the fish owners and marketing firms for selling the fish. The development

of this project permitted commercial exploitation of some unconventional species

including grenadiers, starry rays and deep-sea rosefish among others.

The disposal of fish waste at sea from vessels or other marine vessels is also a

main concern. For instance, most demersal fish are processed to some extent at

Fig. 8.2 Estimation of discard weight (%) by oceans (Alverson et al., 1994).

By-catch, underutilized species and underutilized fish parts 173

sea before landing and it is estimated that gutting waste represents 16% of the

total fish weight (Archer, 2001). In the United Kingdom the processing waste at

sea accounted for about 5% (ca. 42 600 tonnes) of the total catches (Archer,

2001). In Norway most of the by-products from fish processing are used as raw

material for different purposes but about 150 000 tonnes (ca. 29% of the total

by-products) are still dumped into the sea (RUBIN, 2005). The high volume of

these by-products, their nutritional value and potential industrial applications, as

well as the environmental problems resulting from sea dumping, have led to the

introduction of several regulations to control the disposal of such wastes at sea.

This is the case of the Food and Environment Protection Act 1985 in UK to

control the disposal of waste at sea, and the regulations in Iceland where it is

compulsory for vessels to return the fish livers to shore or in Norway where the

total fish catch must be landed.

Environmental protection is nowadays a major concern for governments,

particularly in developed countries. In the European Union, Regulation 1774/

2002 was adopted laying down health rules concerning animal by-products not

intended for human consumption. This new legislation on the disposal of animal

waste by-products has major implications for the seafood industry. Important

aspects of this legislation are banning the landfill of all untreated animal by-

products and the inclusion of the shell from shellfish in animal by-products.

8.3 Using the by-catch and underutilized species

The by-catch is usually a combination of many species, particularly that from

tropical shrimp fisheries, which could attain 200 different species. In the tropical

and subtropical regions, for instance, the main discards from the shrimp fisheries

include species from the Carangidae, Mullidae, Synodontidae, Gerreidae, and

Nemipteridae families, among others (Alverson et al., 1994). The fish size is

also quite variable and may include large and medium size (15±25 cm) market-

able species as well as small fish of less than 14 cm in length. The small fish size

constitutes as much as 50% of the catch (Allsopp, 1982). Generally, it is

estimated that between 24 and 69% of the fish from by-catch have a market,

depending on the fishing ground. However, fish that are saleable vary con-

siderably from country to country.

The large species do not usually represent a problem because they can be sold

in the traditional forms. Thus, the main difficulties arise from the smaller species

due to the large amounts of fish caught, the species variability, the range of sizes

and the low value. This last factor normally determines the final utilization of

the by-catch, which is constituted by a large proportion of underutilized species.

However, the designation of underutilized species is quite dependent on the

consumers' food habits in a given population. Thus, one fish species could be

underutilized in a certain region but the main fish supply in another one. For

instance, black scabbard fish (Aphanopus carbo) is considered an underutilized

species in Ireland, but it is the dominant fish species consumed in the Portuguese


island of Madeira. In the Pacific artisanal and subsistence fisheries, the fish

discards are very few because almost all the catch is used for consumption. In

Africa the consumption of sun-dried tiny species of less than 4 cm long is quite

frequent. Another example is red fish (Sebastes spp.), which was a by-catch of

cod (Gadus morhua) fisheries in the Northwest Atlantic in the 1950s. However,

after the introduction of improved freezing facilities on board, this species is

nowadays a very much-appreciated species in the European market. There are

also examples of very-low priced species that became very expensive such as

monkfish (Lophius spp.).

The underutilized species are often discarded and only the last catch of the

trip is kept on board where the holding facilities are primarily used for the

targeted species. This also means that they are not well preserved, thus reducing

the quality and the price as a consequence. These species pose several problems

related to recovery, handling and preservation on board and later on their

processing. Regarding the fish recovery, several approaches have been tried to

minimize the by-catch. The reduction of fishing effort and time, the area

closures of fishing grounds; and enforced prohibitions on discharges are some

management measures introduced. Another type of measure involves the

utilization of more selective gears. A few examples are: (i) the introduction of

gear modifications (raised footrope, cutaway trawl); (ii) changes of the cod end

characteristics (mesh size, twine thickness, number of mesh in the

circumference); (iii) the increase of mesh size or the inclusion of grids, square

mesh panels, and separator mesh panels; and (iv) the installation of sound

emissions devices or use of electric pulse fields. All these devices permit the fish

to escape due to its size, mobility or response to electric pulses. Nowadays, the

grids are used in fish or crustacean bottom trawl in several European, American,

and Australian fisheries (Fonseca et al., 2005a,b).

The fish sorting and grading after catching are critical steps, which may

represent a strong workload for the crew if it is done manually. However, a wide

variety of equipment has been developed allowing mechanization of some of

those operations. Olsen (1992) reported that Danish industrial fish trawlers are

equipped with a rotating sorter for separating white fish by-catch from the

industrial fish. There are also several types of graders based on the fish thickness

available in the market and well adapted to handle pelagic species (Sùrensen and

Mjelde, 1992; Olsen, 1992).

The recent development of equipment based on computer vision represents a

powerful tool to address the problems of fish processing industry resulting from

the diversity of species, sizes, shapes and colours as well as processing methods.

This equipment is intended for cutting portions and fillets, for shape, length and

size grading as well as for quality grading. Computer vision for sorting fish was

applied by Zion et al. (1999) to three freshwater species. According to these

authors the fish species detection was successfully performed regardless of size

and orientation. Storbeck and Daan (2001) also applied computer vision and a

neural network to fish species recognition. The vision system measured a

number of features of fish using a camera perpendicular to a conveyer belt. The


results of classification by the neural network showed that more than 95% of the

fish could be classified correctly. For Gunnlaugsson (1997), this equipment was

successfully introduced in fish processing plants increasing their efficiency and

productivity.

The use of computer vision equipment in portioning machines has presented

opportunities for accurate portion preparation of a predefined weight or size,

better utilization of the raw material and labour savings. The grading equipment

using computer vision can recognize automatically different shapes and forms.

Shape graders can be used to grade fresh or frozen products and to separate

different sizes of fillets or portions as well as individual shrimp or prawns head-

on or headless. In the case of quality grading, the vision equipment has been

used in the shrimp industry to separate unpeeled or not fully peeled shrimp from

properly peeled shrimp. It has also been used to grade salted fish according to

colour shades, blood spots, cuts and size. Another type of product is herring and

salmon fillets, which are graded according to the colour of flesh and silver

mirror on skin side for herring or the colour uniformity of smoked salmon slices.

Sensor technology was also developed for quality inspection and grading of

seasonal herring roe sacks to be implemented in a prototype-grading machine

(Croft et al., 1996). In a recent paper, Brosnan and Sun (2004) reviewed the use

of computer vision to a large range of food products inspection.

Another important aspect is fish preservation on board, which presents

special problems when dealing with small-sized species caught in very large

quantities and high temperatures as is the case in the tropical countries. Fish

deteriorates more rapidly than other animal products, but the spoilage of small

fish tends to be faster than large fish (Burt and Hardy, 1992). Thus, there is a

need to efficiently cool the fish as quickly as possible just above the freezing

point. Icing fish in boxes is a widespread practice for storing and transport.

There are many types and shapes of boxes and the materials of construction

include wood, aluminium, plastic (HD-polyethylene), fibreboard, and expanded

polystyrene (styrophor). Their utilization depends on tradition and specific needs

and requirements. For instance, fibreboard and styrophor boxes are non-

returnable and frequently used for dispatching fish by air. Boxing has several

advantages over other processes of fish storage (bulk, shelves). The use of boxes

reduces static pressure on fish and also facilitates the unloading of fish. Despite

the advantages of fish boxing there are some key points to consider for their full

utilization on board as referred to by Olsen (1992): (i) adequate handling rate of

filling in to prevent quality loss due to delays in icing; (ii) sufficient icing to chill

the fish to 0ëC and to keep this temperature until unloading; (iii) appropriate

hold construction to guarantee safe and easy stacking of the boxes; and (iv) good

hold insulation to prevent heat transfer. The installation of a small mechanical

refrigeration plant is also recommended.

The introduction of bulk chilling systems on board has been indicated as an

alternative to the traditional method of icing in boxes. The fixed RSW (refrig-

erated sea water) and CSW (chilled sea water) tanks have been successfully used

for different fish species (Olsen, 1992; Hassan, 2002). These systems are very


fast and effective chilling methods, but their installation on board has to be

carefully considered in every fishery and adapted to each vessel. For RSW

systems, some recommended practices needed to be followed to render the

maximum shelf life of the catch. According to Kraus (1992), the temperature

homogeneity between ÿ1.5ëC and 0ëC during storage is very important. The

input of cooled seawater must be divided across the total bottom tank area and

the filling capacity of the tanks has to be taken into account depending on the

fish species (80% for mackerel and 70% for herring). It is also recommended

that at least one temperature sensor be installed on each tank at the warmest

place, which is in the suction box.

In the CSW system the seawater is chilled with ice and is a cheaper invest-

ment than the RSW system. The main practical difficulty is the stratification of

temperature due to differences in specific gravity between seawater and ice and

between water of different salinity and temperature. The most popular method

used to overcome the temperature stratification is by blowing compressed air

introduced at the bottom of the tank. This is the so-called `champagne' method

where a rapid heat transfer between fish and ice is achieved. The CSW systems

have been used in different types of fishing vessels including small coastal ones.

Graham et al. (1993) reviewed all aspects related to the utilization of ice to

chill and preserve fish. They also discussed the benefits and the disadvantages of

the use of different chilling technologies. At present, the use of RSW and CSW

systems on board is scarce, the main criticism being the need for major space in

the warehouse without significant improvement in the fish quality.

In recent years a new type of ice, known either as slurry ice or binary ice, has

been used as one cooling medium for chilling the catch or products, at sea or on

shore. Slurry ice is a mixture of ice microcrystals in brine or seawater, which

enables quick cooling and can keep a low temperature for a long time. Its faster

chilling rate derives from its higher heat-exchange capacity when compared with

the traditional flake-ice and RSW (PinÄeiro et al., 2004). The application of slurry

ice reduces the physical damage to fish due to the small size of the ice particles.

Slurry ice also has the advantage of being pumpable and permits the addition of

preservatives such as ozone or chemicals like inhibitors of melanosis (Huidobro

et al., 2002). The main disadvantages are the occurrence of cloudiness in the

eyes of gilthead seabream (Sparus aurata) and the development of dull colour in

shellfish. The initial relatively high investment costs are also another aspect to

take into account. The new chilling method using slurry ice has been applied to

many species as reviewed by PinÄeiro et al. (2004) and the incorporation of slurry

ice systems into fishing vessels has been expanded in recent years.

The use of underutilized fish species for consumption as fillets or portions

and in the preparation of a range of fish products has gained importance as a

result of the great pressure on the exploitation of traditional fish stocks. Thus,

several programmes have been developed envisaging the upgrading of many

species. The study of each species generally involves different aspects dealing

with the size, shape, type of scales, fat content and protein stability among

others. Venugopal and Shahidi (1995, 1998) provided a general view on the


upgrading of underutilized species and discussed the various possibilities for

their utilization for human consumption.

Freezing is also an important preservation technology on board. It is of major

importance in the case of long fishing trips, because the chilling techniques do

not allow the fish quality to be kept at high standards for long periods. Blast or

plate freezing are widely used on board the fishing boats and its choice depends

on different aspects such as the fish size or the type of processing.

In a study on the utilization of four underutilized species (sand eel, greater

sand eel, sprat and Norway pout) of the North Sea, different processing

alternatives were followed (Nielsen et al., 1992). Washed mince of sand eel

(Ammodytes tobianus) was prepared and used in kroepoek, an expanded snack

very popular in Malaysia, and in the case of greater sand eel (Hyperoplus

lanceolatus) hot smoked products were prepared. It was considered that the

production of good smoked greater sand eel was technologically possible, but it

has to compete with the well-established smoked herring production. Sprats

(Sprattus sprattus), traditionally processed into canned products in the Nordic

countries, were used in the preparation of canned sprat in two new marinade

sauces (lemon/oil and Escabeche), which were compared with the traditional

smoked sprat canned in oil. The lemon/oil product was considered more

adjusted to the market taste but the panellists preferred the traditional product.

Norway pout (Trisopterus esmarkii) was used to prepare minces and surimi. The

minces obtained under different conditions were found unacceptable as a

substitute for white fish mince or fish fingers due to colour and texture. The gel

strength of surimi was comparable with the gel of a good quality product but it

could not be graded as first quality because of a very light grey colour and tiny

spots of membranes.

Larsen (1992) reported the studies made for the upgrading of roundnose

grenadier (Coryphaenoids rupestris) and greater argentine (Argentina silus),

which are by-catch in the shrimp fisheries. The large scales in the trunk and tail

of roundnose grenadier have to be removed before skinning or filleting. The

heads represent about 35% of the gutted fish and the filleting yield by machine is

22±23% round fish. Many products can be prepared from this species, including

fresh or frozen skinned fillets, skin-on fillets as smoked product, as well as

skinned fillets cut into squares for use in pizzas, salads, and soups among others.

In the case of argentine the presence of two rows of parietal ribs represents a

problem because they are difficult to detect and remove. The yield of machine

filleting is on average 30% of the round weight after skinning. Argentine has

high gel-forming ability and is also very satisfactory for mince and surimi

production. Gormley et al. (1991, 1992, 1993, 1994) studied the effect of

freezing and thawing on the water-binding capacity of the argentine proteins and

considered it best to make products from fresh or frozen whole fish/fillets rather

than from frozen mince. Great argentine also afforded highly acceptable fish

cakes and enrobed prawn analogues. Maier et al. (1997) evaluated nine under-

utilized species for the manufacture of consumer fish products. Breaded nuggets

of different species were prepared and the results of the taste panel indicated that


all were favourable compared with cod, but Greenland halibut (Reinhardtius

hyppoglossoides) and greater argentine were the most outstanding. Cardinal fish

(Epigonus telescopus), Portuguese shark (Centroscymnus coelolepis) and

dogfish (Scyliorhinus stellaris) had the most favourable water-holding quality

and gel strength, indicating good potential for use in fish products.

In general, a larger number of underutilized species has been introduced in

products for human consumption. The increasing recognition of the nutritional

value of fish together with the development of new types of seafood products

have contributed to their utilization. The globalization of the fish market also

contributes to upgrading those species.

8.4 Using underutilized fish parts as food and foodingredients

The relevance of the actual concept of by-product as all the raw material, edible

or inedible, left over during the preparation of the main product (Gildberg, 2002)

has become very important. Many approaches have been tried for the upgrading

of fish by-products to produce food, feed or for extraction of valuable bio-

molecules. However, a great variety of fish parts has traditionally been

recovered worldwide for human consumption. A number of them have very

wide distribution while others are only characteristic of some regions. On the

other hand, the current globalization contributes to a generalized knowledge of

this type of products, which are considered delicacies in some places. Thus, this

section will focus on different products obtained from fish parts intended for

human consumption.

In Fig. 8.3 the different steps in fish handling and processing and the resulting

by-products intended for human consumption are shown.

8.4.1 Fish heads, cheeks and tongues

Fish heads are usually discarded or reduced to fish meal. However, there are

some markets where they are commercialized fresh or frozen for direct human

consumption and in some Asian countries the head is one of the favourite fish

parts. They are usually from large fish such as salmon (Salmo salar), meagre

(Argyrosomus regius), white grouper (Epinephelus aeneus), wreckfish (Poly-

prion americanus), conger (Conger conger), tuna (Thunnus spp.), etc. and are

sold whole and sometimes with the collar. They can be prepared in a variety of

ways including soups, fried and steamed dishes. Salmon heads, for instance, are

sold in several countries where the price per kilogram can be the same as the fish

itself, such as in the Taiwan market. The heads from tuna fish and from some big

size tropical fish are consumed grilled over an open fire or in an oven and are

know as kabutoyaki in Japan (Tùnsberg et al., 1996).

Other parts of the fish head can also be found in the market such as the jaw of

many local species in Taiwan, where they are used in soups, barbecues and fried.


The neck meat from salmon and trout is quite popular in the Japanese market

where it is sold fresh or frozen in small packs. Some neck meat is also smoked or

canned.

In Iceland, where fish is an important item in the diet, dried fish heads

softened in dairy whey are used to prepare various dishes. In Nigeria salted and

fermented cod heads are considered a delicacy. The processing of cod heads

intended for human consumption has a long tradition. The gill-covers, the

Fig. 8.3 General scheme for the utilization of fish by-products for human consumption.


tongues and the cheeks are manually recovered and salted onboard. Codfish

heads contain relatively little meat, but this fish part is a very much appreciated

delicacy due to the taste and particularly its texture. The tongues constitute

approximately 1±4% of the weight of the head, cheeks 5±10%, collar 15±20%,

and upper head meat 5±15% (Arason, 2002).

In order to reduce the manpower required for processing these by-products

specific equipment has been developed in Iceland. One machine splits fish heads

and tears the gills out. It is claimed that it can process all sizes of head without

special adjustments. Another processing machine separates cheeks and tongues

from the head.

Cod tongues are used to prepare a variety of dishes, for instance in Iceland,

Portugal and Spain. The processing of cod tongues involves washing thoroughly

in clean seawater and trimming of any loose skin or fragments. After draining,

the tongues are mixed with salt following a standard procedure. Salting requires

on average ten days and after curing, they are rinsed in light brine and repacked

in barrels with brine. The tongues gradually harden after nine months in salt,

becoming inedible.

8.4.2 Fish roe

Fish roe is the common name of fish eggs, particularly when they are in sacks. It

is a very common seafood commodity worldwide and it is collected from many

different fish species. Whole fresh or frozen fish roe is available in different

markets. In England, for instance, fresh cod roes are sold already boiled and they

can be eaten cold or sliced and then fried, grilled or used to prepare other dishes

(Bannerman, 1977). Another example is the consumption of roe from Southern

Atlantic hakes in Uruguay, where it is used to prepare traditional foods (Mendez

et al., 1992). As a rule, the roe from large fish is highly priced but those from

smaller fish such as menhaden (Brevoortia tyrannus) could also attain good

prices in Carteret County (US). Frozen or salted fish roe is also the raw material

for preparation of smoked, canned and several kinds of spreads. As reported by

Arason (2002), the increased fish roe prices in Iceland led to its utilization from

most of the ground fish. After gutting the fish at sea, roe is collected in insulated

plastic tubs and preserved with salt or frozen onboard the freezing trawlers.

There are many different products, prepared from fish roe, available in the

market but they can be grouped into three types: whole sacks, individual eggs

and pateÂ or spreads.

Caviar, the most famous roe product in Europe and Japan, is salt-cured and

preserved individual eggs. The most valued caviar is made from sturgeon caught

in the Caspian Sea. Sturgeon caviar may be produced from more than 20 species

of sturgeon and the most renowned are produced from the Russian and Iranian

beluga, osetra, and sevruga sturgeons (Bledsoe et al., 2003). There are also

caviars from the Kaluga sturgeons and Amur River sturgeons. The supplies of

sturgeon caviar have been decreasing as a result of the reduction in the avail-

ability of Caspian Sea sturgeon, which led to increasing attention to caviar


products from other fish species. However, the caviar produced from other fish

or aquatic species than sturgeon must be identified with the indication of the

common name of the species used. In the US, the most common source of black

caviar is the farmed white sturgeon (Acipenser transmontanus) because the pro-

duction from wild sturgeon is normally not permitted due to the near extinction of

certain sturgeon species and subspecies. Black caviar with very high quality and

reasonably good price is also produced from the roe of fresh water sturgeons

(Acipenser sp.), shovel-nose catfish (Hemisorubim platyrhynchos), and

paddlefish (Polyondon spathula).

Salmon roe is commonly processed into individual eggs (salmon caviar) or as

a whole sack. Salmon caviar is known as `red caviar' and is prepared from pink

(Oncorhynchus gorbuscha), silver (Oncorhynchus kisutch) and chum salmon

(Oncorhynchus keta). For the production of salmon caviar (ikura in Japanese) the

eggs must be absolutely fresh, free from blood and of clear colour and with good

consistency. After separation from sack membranes the eggs are cured in brine

following slightly different processes according to local practices. The brining

time varies with species, season, temperature, size, and degree of maturity. The

brined eggs are placed on wire-meshed screens and usually drained overnight.

After draining they are packed and stored at refrigerated temperatures.

The screening, i.e., the separation of eggs from each other and from the sack

membrane, is normally a manual and time-consuming process. As an alternative,

different enzyme preparations for splitting the linkages between eggs and the

connective tissue of fish roe sack have been tried. Collagenase from crab

hepatopancreas or proteolytic enzymes extracted from fish viscera were tried in

the production of salmon caviar. The yields of recovery achieved were around

80% using the former enzyme and ranged between 84 and 93% for fresh roe and

76 and 87% for frozen roe when the viscera enzymes were used (Bledsoe et al.,

2003). The product obtained with proteolytic enzyme from fish viscera had

better quality than that obtained with the crab collagenase, which partially

hydrolyzed the egg sheath resulting in caviar softer than that obtained by the

traditional mechanical screening process. The enzyme treated roe also presented

faster salt uptake than fresh fish roe.

The traditional production of cured whole salmon roe (sujiko) is made from

freshly caught Pacific salmon. The main species used for its production is

sockeye salmon (Oncorhynchus nerka), but chum and pink salmon are also used.

The intact roe sacks from salmon are separated from the other viscera and sorted

for quality. The salmon roe is then immersed in saturated brine containing

nitrites, polyphosphates and other additives and seasonings and gentle agitated

for about 20 min (Bledsoe et al., 2003). The product is then packed in layers into

poly-lined wood or plastic containers. Fine granular salt is applied between

layers and the product then cures at ambient temperature for several days after

packing. The finished product exhibits salt content ranging normally from 7 to

10% and is sold in frozen or refrigerated state. The consumer may use it without

any further processing or preparation. Sujiko is often prepared with a special soy

sauce based marinade. Barako is the designation in Japanese of a by-product of


sujiko production, which is constituted by singled-out eggs from broken or

rejected sacks of sujiko.

Lumpfish (Cyclopterus lumpus) roe is another source for production of

moderately priced caviar. The individual eggs are mechanically separated from

the roe sack and salted to 3±5% sodium chloride and immediately packed. In

another alternative process the eggs are salted to 10±14% and then delivered for

reprocessing, desalting, and repacking in retail packages (Sternin and Dore,

1993). Lumpfish caviar is generally black or red coloured and dyeing is part of

or after the curing process. The final product is vacuum packaged and kept

refrigerated. The salt concentration in brine of commercial lumpish caviar varies

between about 4.2 and 12.9% (Bledsoe et al., 2003).

Alaska pollock (Theragra chalcogramma) roe is commonly processed into

mentaiko (or mentiko) and tarako. In 2000, the total production of mentaiko and

tarako was around 30 470 and 25 970 tonnes, respectively. Mentaiko is whole, a

matched pair of sacks and its preparation involves brining and curing. It may be

dyed and/or seasoned with salt, sugar, monosodium glutamate, garlic and other

spices, sesame, chilli or other flavoured oils, soy sauce or sake.

Tarako is literally the name of cod roe in Japanese but now designates

smaller salted roes. It is actually produced mostly from pollock roe, which is a

by-product of the surimi production. The roe for the preparation of tarako is

removed, washed with seawater and soaked in about 15% sodium chloride

solution for 40±60 min and drained for about 1 hour (Chiou et al., 1989). The fat

content depends on the raw material but could be between 2 and 4%. The ash

content is relatively high (6.9%) due to salting. Pacific cod roe is also used to

prepare tarako in a similar manner.

Botargo or the Mediterranean caviar is a delicatessen product prepared from

dried salted roe from mullet or tuna. It is very much appreciated in the

Mediterranean countries (Southern Europe and North Africa) as well as in Japan

where it is called karassumi. There is also some production of dry-salted mullet

roe in the United States, where it is believed that the method of preparation was

introduced in colonial times by Englishmen who discovered it in Greece (Jarvis,

1950). The roe for the production of botargo must be fresh, of good colour and

the skin of the roe sack not broken. It must be neither over-ripe nor too under-

developed. The egg sack is taken out of the fish as soon as it is landed. The lobes

should be freed from blood, gall bags, bits of intestine, and black skin and then

washed thoroughly and allowed to drain for further salting. After size grading,

the lobes of roe are rolled in fine salt, kept with salt for 6 to 12 hours and rinsed

to remove the excess of salt. The sacks are then placed between two wooden

boards and compressed slightly for 2 to 3 days until most of the liquid drains

from the roe. A small piece of the muscle is left in the roe in order to avoid its

emptying and also to be used for hanging them during drying or smoking. The

drying process takes 6 to 10 days and is completed when the roe is reddish-

brown in colour and feels hard. The moisture content of the dried product is

typically between 28 and 32%. The colour is amber honey and the texture very

soft. During storage the colour becomes dark and more dried and hard.


The traditional product presented the original and transparent sack skin, but

nowadays it is covered with food grade paraffin to improve the shelf life and to

prevent lipid oxidation. This product is stored between 5 and 15ëC and its shelf

life is around eight months. The utilization of a vacuum preservation method

allowed the increase of shelf life of botargo, thus opening its commercialization

to new markets. Tuna roe, mainly from Thunnus thynnus, are also used to

produce botargo, which is normally dark brown in colour.

The fish roe of small pelagic fish such as sardine (Sardina pilchardus), which

is used to produce canned products, is usually not removed from fish and is

consumed like the fish flesh. Capelin (Mallotus villosus) is another pelagic

species whose eggs are used to produce faux tobiko, the roe from flying fish

(Cheilopogon furcatus). The European market for capelin eggs is estimated at

100 tonnes per year but the largest market is Japan. It is a high priced product,

normally sold under the term masago, used in sushi cuisine. In the case of

herring it can make up 12% of the body weight of the fish. For the European

market, herring roe is processed to give separate non-sticky eggs. The fish are

machine filleted, the roe sack removed and the small eggs separated from the gut

tissue. The eggs are then concentrated and the small pieces of gut tissue

discarded. After being immersed in 3±5% brine for up to two hours to reduce

stickiness, the eggs are dewatered and packaged.

For the Japanese market the whole herring roe is a delicacy and has a much

higher price. The highest value cured herring roe, kazunoko in Japanese, is

prepared from perfectly matched pairs of skeins. To obtain the highest quality

product the herring roe must have no defects, the fish should have reached the

exact, desired degree of maturity and the average roe content of 10% by weight.

Kazunoko is made from frozen herring; the roe is removed by hand or

mechanically and cured by brining process, which involves many steps. This

product attains a very high price in the Japanese market and is commonly

prepared as sushi or for other dishes.

Another high priced product made of herring roe is kazunoko kombu or

herring roe on kelp. It is a uniform, dense layer of herring eggs covering both

sides of a piece of kelp. The kelp covered with herring eggs can be harvested

either in the wild or from kelp suspended in pens where live herring are placed

just prior to spawning. The egg-coated kelp is washed, trimmed, cut to market

size and packed in brine. The finished product is usually consumed in soups,

salads, or as a side dish. Bledsoe et al. (2003) also reported the utilization of fish

roe from many other species to prepare caviar-type products. They are con-

sumed alone or mixed with different flavourings, or blended with butter, soft

cheese or other spreads.

The gonads of some invertebrates are also used to prepare roe-based foods.

Among them the roe from sea urchin, sea cucumber, crustaceans, and octopus

are important. Sea urchin roe (uni) are collected from several urchin species and

may be sold fresh, steamed, baked, sauteÂed, frozen, or canned. When used for

sushi, they are brined and treated with alum. Other types of products prepared

from sea urchin roe include neri uni, a paste obtained after roe fermentation,


mizu uni, obtained by a dry cure process, doro uni where the roe is washed with

dilute alcohol, drained and mixed with salt. Roe from sea cucumber has a good

market in China where it is sold at a price 50 times higher than that of sea

cucumber muscle. The roe from crustaceans (shrimp, lobster, and crab) is used

to prepare traditional dishes. The salted octopus (Octopus vulgaris) roe is

considered a delicacy in Greece, but it is not usually available in the market

since it is only produced at an artisanal level.

8.4.3 Fish milt

Milt, also known as soft or white roe, is the sperm-containing fluid of the male

fish. Milt is sold fresh or frozen, but canned milt, particularly from herring and

mackerel, is also commercialised. It can be used cooked or fried and eaten sliced

or chopped on canapeÂs or mixed in salads, soups and stews (Rustad, 2003). As

reported by Richardsen (1992), the chemical composition of herring milt is

82.5% water, 2% fat, 16.7% protein, and 2% ash for fish with 21% milt. Cod

milt has a similar composition (82% water, 1.1% fat, 14.5% protein, and 1.8%

ash) (www.fao.org) and is traditionally used in Japanese cuisine. In Japan, it is

mostly imported fresh (90%) and can be consumed raw, seasoned with vinegar

and few drops of soy sauce or cooked together with meat, cod fillets and

vegetables (Tùnsberg et al., 1996).

8.4.4 Fish stomachs

Fish stomachs are traditionally consumed in Iceland as well as in Japan, Korea

and other Eastern countries (Archer, 2001). In Iceland, the stomach of some

species (cod, tusk, Brosme brosme, ling, Molva molva) are removed just after

catch and immediately frozen or kept in ice. A popular Icelandic dish is prepared

with cod stomach filled with a piece of cod liver. The stomachs and intestines

from cod, saithe (Pollachius virens) and haddock (Melanogrammus aeglefinus)

caught from the end of January until April are considered to be of good quality

for consumption in the northern Norway (RUBIN, 2002). In Japan the fresh

stomachs are consumed stewed with vegetables and spices. They are also used

after partial hydrolysis to prepare a dish (changi) with a characteristic flavour

and texture. High priced fish sauces are produced after full hydrolysis of fish

stomachs. The consumption of the Alaska pollock stomach is very popular in

Korea where it is called changran. In Taiwan the stomach of some sharks,

milkfish (Chanos chanos), and glassfish (Chanda spp.) are used for steaming

and sold as a delicacy. Stomach and intestines are also part of some traditional

Chinese dishes, usually steamed (Tùnsberg et al., 1996).

8.4.5 Fish maws

Dried fish maws means the dried swim bladder from different fish species. The

swim bladder is a part usually discarded in European fisheries, although dried


cod maw is available in some niche markets where it is considered a delicacy.

However, as reported by Clarke (2004) the foreign trade in fish maws to or

through Hong Kong has been very important for many decades. In the Far East

dried fish maws are consumed as a food, but it is also believed that they have

medicinal properties. Fish maws are produced from a variety of species (Nile

perch, Lates niloticus; croaker such as Bahaba taipingensis and Otolithoides

brunneus; jew fish, Pseudosciacna sp.; eel, Muraemesox talaboieds, among

others). The main characteristics looked for in the fish maws are shape, size,

colour (transparency), species and gender. The processing involves splitting

open the maw, washing and drying it in the sun. Fish maw is simply boiled with

other ingredients to prepare a soup or broth or is cooked with beans. In Hong

Kong the smaller fish maws are fried (dim sum) and consumed as a snack food,

especially for breakfast. Swim bladder is also a potential source of gelatin

(Regenstein, 2004).

8.4.6 Belly flap/trimmings

Fish bellies are frequently discarded, but in some markets they are considered a

delicacy. The belly of several species (salmon, tuna, milkfish, and blue marlin,

Makaira nigricans) is particularly appreciated and consumed in soups or grilled.

The reason for high preference of fish bellies is due to the high fat content and

softer texture. Canned tuna belly is an example of a softer and more succulent

product than the canned tuna flesh. At the beginning of the 20th century, the

production of salted salmon bellies was usual in some salteries but it was

forbidden under the Alaska fishing regulations because of the wastes resulting

from the production method followed (Jarvis, 1950). However, nowadays the

modern trimming machines can perform the most difficult trim cuts. In these

machines every single fillet is measured individually and trimmed according to

the operator's demand. The resulting cut offs may be used to prepare smoked

products, which present a good market in Japan. The belly flap of salmon and

salmon trout is popular in Taiwan where it is barbecued or fried (Tùnsberg et al.,

1996). The belly flaps of milkfish are sold in the Philippines as special parts,

which are more expensive than the fillets (Peralta, 2002). In the case of dry

salted cod the trimmings and small pieces are recovered and used in the

preparation of special dishes. The red muscle of tuna fish species used in the

canning industry is usually removed and reduced to fish meal. However, in some

markets it is sold as a salted product or used to produce low-priced canned fish.

8.4.7 Fish liver

The cod liver oil is a well known product, which was previously recommended

as a source of vitamin D and the actual recognition of the beneficial health

effects of !3 fatty acids gave a new interest for its consumption. Liver oil is still

the main product obtained from fish liver. According to Arason (2002), fish liver

oil (medicinal oil and ground fish oil) represented 85.6% of total production of


different products (4344 tonnes) from the liver of gadoid species in Iceland

(2001), whereas the other categories of products from fish liver were: canned

liver (5.0%), frozen and fresh liver (5.5%), and other liver products (3.9%). The

mean chemical composition of cod liver is 32% moisture, 55.1% fat, 4.6%

protein, and 3.6% ash. Fish liver is also consumed such as fish flesh. It is very

tender, tasty and highly nutritious but it should be extremely fresh. Hake

(Merluccius spp.), cod and monkfish are a few examples of fish species where

the liver is very much appreciated to prepare different dishes. Canned monkfish

liver (canned ankimo) is consumed in Japan as well as the pateÂ, which is

becoming very popular in United States.

8.4.8 Fish frames

Fish frames resulting from filleting as well as fish heads and tails present

considerable amounts of proteins, calcium and other minerals. As referred by

Gildberg et al. (2002) in the production of cod fillets about 60% of the whole fish

are by-products and the frames represent about 15% of the fish weight. Further-

more, the flesh attached to the frames is about 85% of the frame weight on a wet

basis. For the upgrading of these by-products several approaches have been tried.

The flesh can be removed by mechanical separation to afford frame mince,

which is much darker than the mince from headed and gutted fish. Frame mince

has much higher iron content due to the fish blood present and it is considered to

be of the lowest grade. It can be used to make a variety of fish products either for

human consumption or pet food manufacture (Arason, 2002). Kim et al. (1991)

evaluated the preparation of surimi from catfish frames resulting from filleting.

The results obtained indicated that surimi obtained had functional properties,

which were feasible for commercial production of different seafood products.

This raw material was also used by Hoke et al. (1994) to study the feasibility of

producing mince and methods of maintaining the overall quality of the mince

during frozen storage.

Another alternative for protein recovery of the attached flesh to the frames is

on the preparation of protein hydrolysates (Levin et al., 1990; Ferreira and

Hultin, 1994; Kim et al., 1997, Jeon et al., 2000; Liaset et al., 2000). The

enzymatic hydrolysis of proteins was also used to remove the flesh and recover

the fish bones (Kim et al., 2003). Fish bones are recognized as a good source of

calcium, especially for people with allergies or intolerance to milk or dairy

products, which are the most common source of calcium. Powdered fish calcium

produced from skipjack bones (Katsuwonus pelamis), was used in entreÂes as a

calcium supplement (Sada, 1984). Fish bones meal prepared from hake

(Merluccius sp.) or sole (Solea vulgaris) bone was also used as an ingredient

in baby food (Martinez et al., 1998). However, fish bones have traditionally

been used in soups, dried (eel) or fried as snack foods. In Japan the canned

tailbone of salmon (chum or sockeye salmon) or trout became popular. Canned

smoked salmon bone is another version of this product, which is relatively

recent in the Japanese market. Deep fried milkfish frames are sold as fish flakes


in Philippine restaurants (Peralta, 2002). Shark bone has a market in Hong Kong

where it is consumed as a medicine against cancer.

Fish bones have also been used as a source for the extraction of collagen.

Nagai and Suzuki (2000) used the bones from skipjack, Japanese sea-bass

(Lateolabrax japonicus), ayu (Plecoglosus altivelies), yellow sea bream (Dentex

tumifrons), and horse mackerel (Trachurus japonicus) for the extraction of

collagen. The yields achieved were between 40.1 and 53.6% and the denaturation

temperature ranged between 29.5 and 30.0ëC. According to these authors the

bones as well as the skin and fins from the different fish species studied have

potential in supplementing the skin of land vertebrates as a source of collagen.

The gelatin obtained by Gildberg et al. (2002) from cod bones generally had

relatively low molecular weight and as referred by the authors it could be used as

a nutraceutical. According to Morimura et al. (2002), the enzymatic hydrolysate

obtained from pre-treated fish bone exhibited both high anti-radical activity and

high potential for decreasing blood pressure, suggesting that it could be a useful

additive in food materials. The inhibitory activity of angiotensin I-converting

enzyme of fish peptides prepared from fish viscera or skin was also reported in

several works (Matsumura et al., 1993; Byun and Kim, 2001; Bordenave et al.,

2002). Antioxidant activity has been also measured in fish by-products and

gelatin, which opens good perspectives for their utilization in various seafood

products (Shahidi et al., 1995; Amarowicz and Shahidi, 1997; Amarowicz et al.,

1999; Kim et al., 2001; Sathivel et al., 2003). The quality of the Alaska pollock

bone gelatin obtained by Regenstein et al. (2003) was not as good as the gelatin

extracted from the skin but it may have some food applications.

8.4.9 Fish fins and fish skins

Fish fins are frequently dumped but are consumed in some countries such as in

Japan where the fins from some species are eaten as fried foods, called karaage,

and there is a certain market for dried blue shark. In fact, shark fins are much

appreciated in the Taiwanese, Chinese and Hong Kong markets. In the latter

market they are one of the most expensive seafood delicatessens where the price

depends on the grade. Salmon fins are also available in canned form as a snack

food (Tùnsberg et al., 1996).

Fish skins represent a significant fraction of fish wastes and are frequently

discarded but in some countries are converted to value-added products for direct

human consumption as snacks. However, several works on the utilization of fish

skin for the extraction of collagen or gelatine have been published (Gudmunds-

son and Hafsteinsson, 1997; Nagai and Suzuki, 2000; Montero and GoÂmez-

GuilleÂn, 2000; GoÂmez-GuilleÂn and Montero, 2001; FernaÂndez-DõÂaz et al., 2001;

GoÂmez-GuilleÂn et al., 2002; Regenstein et al., 2003; Haug et al., 2004).

8.4.10 Fish sauce

Fermented fish products are very common in tropical countries where the

traditional salting and drying preservation methods are prolonged primarily due


to the weather conditions. This process leads to the development of special

products enhancing the flavour or even masking some taste of food (Saisithi,

1994). This author classified these products into the three groups: (i) fish is

fermented by enzymes from fish and bacteria present in the fish/salt mixture; (ii)

fish and carbohydrate are fermented mostly by bacterial enzymes present in the

fish/salt carbohydrate mixture, and (iii) fish is fermented by fish enzymes and

the carbohydrate by yeasts and moulds added in starter cultures. Both small

whole fish or shrimp and dressed or whole medium or large fish are used to

produce fish sauce depending on the type of product. Fish sauce is the most well

known fermented fish product and not only in Southeast Asia region but also in

Europe and the United States. In this latter country the value of imported fish

sauce was $16.6 million in 2000 (Tungkawachara and Park, 2003). It is a clear

brown liquid with a salty taste and a mild fishy flavour, which is consumed on a

daily basis as a condiment by a large number of people (Raksakulthai et al.,

1986). Lopetcharat et al. (2001) give an overview of fish sauce manufacturing,

factors affecting quality, composition and estimation of the fish sauce quality.

The long production period required (6 to 12 months) has led to some efforts to

accelerate the fermentation period by adding external enzymes, heating or

adjusting the conditions to increase the enzyme activities (Gildberg, 2002). The

utilization of whole Pacific whiting or a mixture of surimi by-products and

whole fish for the production of fish sauce is described by Tungkawachara and

Park (2003). The sauce obtained had a positive consumer acceptance, good

quality and low cost and could potentially replace fish sauce from other sources.

Gawborisut et al. (2003) described the preparation of fish sauce from catfish

nuggets with bromelain added. Based on chemical results these authors claimed

that a catfish nugget sauce could be made in 14 days with 11% salt and a

maximum of 0.15% bromelain.

8.5 Future trends

An increasing number of new fish species has been introduced in the market in

recent years. It is expected that this tendency will continue as a result of the

market demand and the pressure on traditional fish stocks. There are also good

prospects of increasing the value of by-products from fisheries and aquaculture

as pointed out by Gildberg (2002) as was registered in recent years. On the other

hand, the legal requirements for fish waste disposal represent a challenge to find

alternatives for better utilization of seafood by-products. Another important

aspect is that fish and fish products will continue to be the most internationally

traded of all foodstuffs on the global market in the near future (Valdimarsson

and James, 2001).

The general trends mentioned above indicate that the introduction of manage-

ment measures to reduce discarding will be reinforced. Equally important is the

improvement of gear selectivity, which may represent a significant contribution

to reducing the by-catch. The improvement of sorting, handling and preservation


methods on board to improve the quality of fish and by-products when landing

will be in line with the advances recorded in last decade. A better knowledge of

the chemical composition, seasonal changes and technological characteristics of

unconventional or underutilized species and by-products is also needed. The

recent development of new equipment for fish processing and the increasing

demand for ready-to-eat and heat-and-eat products seem set to continue in the

future. The expanding international fish market also requires a reliable

traceability system in order that the entire story of fish along the value chain is

recorded and can be available. The development of the telemarket also opens new

perspectives for a global market of fish products and for increasing the revenue of

fishermen.


In recent years a number of books on fish processing, nutritional value of fish,

safety, and quality assurance have been published. In parallel, interest in the

upgrading of fish by-products has gained special relevance, particularly in the

area of biotechnology. The following recent books are also recommended:

2000. R. E. Martin, E. P. Carter, G. J. Flick Jr, G.J. and L. M. Davis.Marine and

Freshwater Products Handbook. Technomic Publishing Co., Lancaster,

USA, 963 pp.

2000. N. F. Haard and B. K. Simpson (eds), Seafood Enzymes Utilization and

Influence on Postharvest Seafood Quality. Marcel Dekker Ltd, New

York, USA, 681 pp.

2002. M. Fingerman and R. Nagabhushanam (eds), Recent Advances in Marine

Biotechnology. Vol. 7: Seafood Safety and Human Health. Science

Publishers Inc, Enfield, USA, 315 pp.

2002. C. Alasalvar and T. Taylor (eds). Seafoods ± Quality, Technology and

Nutraceutical Applications. Springer-Verlag, Berlin, Germany, 252 pp.

2002. H. A. Bremner (ed.), Safety and Quality Issues in Fish Processing.

Woodhead Publishing Limited, Cambridge, England, 520 pp.

2003. P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference

Proceedings. Alaska Sea Grant College Program, University of Alaska

Fairbanks, Fairbanks, 566 pp.

2004. M. Sakaguchi (ed.), More Efficient Utilization of Fish and Fisheries

Products. Elsevier Science Publishing Company, London, England, 464

pp.

2005. Y. Le Gal and R. Ulber (eds) Marine Biotechnology I. Advances in

Biochemical Engineering/Biotechnology. Vol. 96. Springer-Verlag,

Berlin, Germany, 288 pp.

However, the Internet has become a very rich and easy source of information

about this subject and the following websites are suggested:


www.fishbase.com, provides a vast amount of information on fish species

worldwide.

www.fao.org, is a solid base on fish and aquaculture statistics and diversified

information on fisheries in general.

www.nfi.org, this is a website with general information related to different

aspects on fish, aquaculture and fish consumption.

www.onefish.org, this website is a wide source of information of the fishing

sector, giving particular emphasis on developing countries.

8.7 Acknowledgement

The author would like to thank Dr Maria Leonor Nunes and Eng. Carlos Cardoso

for their help with the chapter and the fruitful discussions.

8.8 References

ALLAIN V, BISEAU A and KERGOAT B, 2003. Preliminary estimates of French deepwater

fishery discards in the Northeast Atlantic Ocean. Fish Res, 60: 185±192.

ALLSOPP W H L, 1982. Use of Fish By-Catch from Shrimp Trawling: Future Development.

In Fish by-catch ± bonus from the sea: report of a technical consultation on shrimp

by-catch utilization held in Georgetown, Guyana, 27±30 October 1981, pp. 29±36.

Ottawa, Ont., IDRC.

ALVERSON D L, FREEBERG M H, MURAWSKI S A and POPE J G, 1994. A global assessment of

fisheries bycatch and discards. FAO Fish Technical Paper, 339, 233 p.

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9.1 Introduction

In the past, fresh fish was purchased at retail market and prepared at home. Fresh

fish, however, undergoes rapid quality changes and when compared to other

muscle foods may need pre-treatment.1 In recent years, consumers have also

made major changes in their food purchasing habits. As social patterns changed,

consumers began to purchase processed seafood to avoid dealing with fish odor

and bones.2,3 However, the more the fish is processed for consumer con-

venience, the higher the price. The goal should then be to provide processed

seafood at a reasonable price.

Washed fish mince/surimi is an inexpensive intermediate material for

manufacturing various seafood products.4±6 Unwashed fish mince also offers

nutritional advantages (containing water-soluble vitamins, minerals, and lipids),

economic benefits (lower processing costs and higher yield of protein) as well as

functional advantages (exhibiting meat-like texture) compared to the other

intermediate materials.7,8 Higher yields in fish mince are especially important

now as many conventional fishery resources in the world continue to decline and

there is a shift toward higher utilization of harvest. Fish mince can also be

successfully used directly in various food systems and in a physically or

chemically altered form to produce an array of nutritional and functional

products.9

Most intermediate materials used for manufacturing various seafood products

are frozen to keep the quality good for a relatively long period. However, frozen

stability of fish mince is poor, particularly for cold water species like Alaska

pollock, due to the higher content of active enzyme systems (i.e., trimethylamine

9

Mince from seafood processing by-product and surimi as food ingredientsJ.-S. Kim, Gyeongsang National University, South Korea andJ. W. Park, Oregon State University, USA

oxidase),10,11 and their substrates,12 heme-containing compounds (imparting

undesired colour and catalyzing lipid oxidation),13±16 lipids containing n-3

polyunsaturated fatty acids,17 and trimethylamine.18,19

Consequently, the surimi processing method was developed20,21 in Japan to

extend the shelf life. For longer frozen shelf life of surimi, minced flesh is

repeatedly washed using chilled water (5±10ëC) which removes unnecessary

components that promote protein denaturation during frozen storage.22,23 Surimi

is commonly mixed with cryoprotectants (4% sugar, 4±5% sorbitol, and 0.2±0.3%

polyphosphates). Surimi has excellent functional properties, such as gelling,

emulsifying, and water-binding properties.24,25 The high concentration of

myofibrillar protein enables the product to gel upon heating to produce a chewy,

elastic texture.26,27 Under constant low temperature storage (below ÿ20ëC),frozen surimi can usually be stored up to 1±2 years without significant changes in

functional properties.23,28±30 Commercial surimi blocks are processed with the

label `good if used within 2 years'. Due to its unique characteristics, surimi is used

as an intermediate raw material for processing kamaboko31 and surimi seafood

products with various types and flavours of crabmeat,32,33 lobster meat,34,35 and

shrimp meat. In addition, its application has been extended beyond shellfish

analogs. These include fish nuggets,36 frankfurters,36 and fish patties.37,38

The ratio of supply to demand of fish in the world has decreased over recent

decades due to a rise in population, maximization of harvest limits, and a

positive change in consumer attitudes towards the consumption of seafood.39

Today's fishing industry, therefore, faces increasing demands for better

utilization of all the available raw materials. Unfortunately, seafood processors

producing fish fillets and surimi utilize only 15±30% of the harvest23,40,41 and

the remainder is discarded as processing by-product or waste.42 Improved

utilization of fish fillet or upgrade of surimi waste as food not only resolves

many of the environmental concerns that the fish processors face, it also serves

as a means of producing value-added products,43±46 and more profit.

This chapter will discuss manufacturing methods and machinery for fish

mince/surimi, characteristics of mince/surimi processing by-products, functional

and nutritional properties of mince/surimi, and its utilization.

9.2 Manufacturing of fish mince/surimi

Fish mince can be defined as deboned and unwashed fish flesh from fillets or

frames and is produced at the initial step of surimi manufacturing. When

compared to surimi, fish mince can be obtained at a significantly higher yield

with much less capital investment. Fish mince as a food ingredient also has a

functional advantage exhibiting a more meat-like texture than mechanically

deboned mammalian meat. However, it has inferior frozen stability due to the

higher content of active enzymes and their substrates,12 metals, lipids, and

trimethylamine. As a user-friendly ingredient, fish mince should be available in

a form that can be stored in a stable state for a reasonable period of time prior to

Mince from seafood processing by-product and surimi as food ingredients 197

use. In that sense, surimi was developed to improve frozen stability of fish mince

by washing and adding cryoprotectants. Surimi can, therefore, be considered

stabilized fish mince. Fish mince and surimi have their own advantages and

disadvantages based on the nature of processing and the subsequent functional

characteristics (Table 9.1).

9.2.1 Preparation of mince

Interest in white fish mince has increased in recent years and its production

volume has been similar to that of surimi. Fish mince is mechanically separated

fish flesh that has not been washed and, therefore, processed with a significantly

higher yield (Fig. 9.1). For the preparation of fish mince, H&G (headed and

gutted) fish is typically subjected to mechanical deboning, while some are

processed using fish fillets. In the mechanical deboner, the fish flesh is forced by

means of a rubber conveyor belt through a perforated drum. The skins and bones

from the fillets remain on the outside of the drum, while the fish mince is

collected from the inside. As shown in Fig. 9.1, fish mince can be processed

further into surimi using extensive washing, dewatering, refining, and screw

pressing consecutively.

9.2.2 Preparation of conventional surimi

For preparation of conventional surimi, fish mince is repeatedly washed with

chilled water (5±10ëC) until it becomes odorless and colorless. Washing cycles,

water volume, and water temperature will vary depending on fish species and

the condition of the fish upon processing. The washing cycles and total water to

meat ratios (v/w) commonly used are 2 to 4 times and 12:1 to 24:1 for on-shore

processors,20,47,48 respectively. However, with the recent efforts made by US

surimi processors, this ratio has decreased significantly to two washing cycles

Table 9.1 Definition and characteristics of fish mince/surimi

Items Mince Surimi

Definition · Deboned fish flesh separatedfrom fillets or fish framesthat have not been washed

· Deboned fish flesh that hasbeen washed with cold waterand mixed with cryoprotectants

Advantage · High yield · Good frozen stability· High nutritional property · High functional property· High functional property· Simple processing· Very low water consumption

Disadvantage · Poor frozen stability · Low yield· Complicated processing· Very high water consumption


and 2:1 to 3:1 for at-sea vessel processing and 5:1±10:1 ratio for on-shore

processing, respectively.23

Washing and dewatering removes lipids and water-soluble sarcoplasmic

proteins such as blood, enzymes, and heme compounds which cause lipid

oxidation. As a result, this process concentrates myofibrillar proteins. Washed

fish mince is then pumped to a refiner to remove connective tissues and small

pin bones. As a final dewatering step, washed meat is subjected to a screw press

before mixing with cryoprotectants. Cryoprotectants commonly used in a

commercial application for cold water species are 4% sugar, 4±5% sorbitol, and

0.2±0.3% polyphosphate with approximate moisture contents at 74±76%.23

However, for surimi from warm water species such as threadfin bream manu-

factured in SE Asia and India, only 6% sugar and 0.2±0.3% polyphosphates are

used without sorbitol. Even though fish proteins from warm water species have

better frozen stability, the addition of equal or similar amounts of cryo-

protectants is desired to maintain the consistency in sweetness and longer shelf

life. Finally surimi is stuffed into 10 kg plastic bags before subjecting to a plate

freezer. After freezing blocks with their core temperature at ÿ20ëC, two blocks

are packed in a carton box for frozen storage.

9.2.3 Preparation of surimi by new technology

In conventional surimi processing, washing and dewatering are repeatedly

required to concentrate myofibrillar proteins. Consequently, relatively lower

protein recovery and large quantities of wastewater are environmental and

economic challenges. Fish protein isolate processing by new technology, such as

acid- or alkali-aided method, demonstrates a high potential to provide good

protein recovery with acceptable functional properties. Unlike the conventional

Fig. 9.1 Processing flow of fish mince/surimi and waste.45


method, the new method utilizes protein charges and isolates the protein by

shifting pH prior to centrifugation. As a result, water-soluble proteins are

retained in the final products.

The solubility of fish mince at various pH values is very important for the

recovery of fish proteins by this new technology.49 The effect of pH on

solubility of Pacific whiting mince is shown in Fig. 9.2. The solubility of Pacific

whiting proteins was very low at pH 4 to 5 and increased as the pH is shifted to

acid or alkali. A sharp increase in solubility occurred at a pH from 9.5 to 10.5 in

the alkaline side and from 3.0 to 1.5 on the acidic side. These results show that

shifting pH away from the isoelectric point of fish muscle proteins, around pH

5.0, results in increased solubility. This indicates that for the surimi processing,

the fish muscle protein has to be solubilized at about pH 2.0 for the acid-aided

method and about pH 10.5±11.0 for the alkali-aided method, and then

precipitated at pH 5.0 for high recovery of protein.50±53

9.3 Machinery for preparation of fish mince/surimi

9.3.1 Fish header/gutter

The header/gutter is a machine for removing the head, viscera, and a major part of

the backbone from the raw material before filleting. Most of the microorganisms

and enzymes found in the viscera and gills are removed at this processing stage.

This step also influences both the quality and yield of fish mince. If the position of

the head cut is too far forward, the gills and heart remain and the product quality

decreases. If the position of the cut is too far to the rear, the yield decreases.

Fig. 9.2 Solubility of Pacific whiting mince as affected by pH (modified from Ref. 50).


9.3.2 V cut

The V cut is used for preparing a boneless fillet by removing the pin bones,

which generally accounts for 5±7% loss based on the original weight. The fillet

prepared by using a V cut is a very high-quality fillet material. Water knife or

laser cutting methods completely remove the bone portion.

9.3.3 Belt-drum type meat separator

A belt-drum type separator (Fig. 9.3) is the most common meat separation

technique for the mincing/deboning operation.23 For preparation of fish mince/

surimi, fish are fed into a hopper and transferred by a rubber conveyor belt to a

drum perforated with openings.54 Most mechanical deboners have perforated

holes of 3 or 5mm depending on the size of the fish. However, 5mm is probably

the most common size because it yields a more textured final product as a fish

mince compared to a smaller hole.23,55 Conversely, a larger hole may allow an

unacceptable level of bone. The size and texture of fish are also factors in

selecting the right meat separator for optimal recovery and quality. Fish of

smaller size or firmer texture would benefit from a smaller opening diameter. In

meat separation processing of smaller fish, the use of a large opening would

generate more bone fragments and/or broken skin in the resultant mince.23 The

higher the belt pressure, the higher the yield of minced fish forced through the

holes. However, elevated belt pressure can lead to lower frozen storage

stability.56 The effects of pressure, perforation size, and perforation area on bone

content, protein functionality, discoloration, and lipid stability can result in a

compromise for optimal deboner/mincer conditions.57

Fig. 9.3 Belt-drum type meat separator.


9.3.4 Rotary screen dehydrator

Fish mince mixed with wash water is strained through large rotary screens. The

rotary screen dehydrator is used for intermediate dewatering. The screens are

constructed with stainless steel mesh or punched plate and selected to obtain fine

meat particles. The holes in the screens are commonly about 0.5mm in diameter.

Decreasing the opening diameters of screens from the front end to the rear end

enhances dewatering action. However, fine particles lost through screens

account for about 8% of the starting mince meat weight. For these reasons, in

recent years, most surimi plants have employed a decanter centrifuge to recover

protein particles in the wash water. The moisture content of the material passed

through a rotary screen is commonly reduced from 99.0% to 93.3%.

9.3.5 Refiner

Before final dewatering under a screw press, a refiner is used to refine the

mince.23 The washed fish mince passes through a refiner to remove refiner

discharge, which consists of connective tissue, skin, bone and scale.58 The refiner

is commonly a rotating drum (Fig. 9.4). Running the refiner at a slower speed

with a smaller screen size will result in cleaner surimi with less recovery. On the

other hand, running the refiner at a faster speed with a larger screen size will

enhance recovery but with a risk of higher impurities. Screen sizes of 1.5±1.7mm

are commonly used in commercial applications.23 When washed mince is fed into

the machine, soft flesh is forced through the perforations under compressive force

generated by the rotor. The flesh emerges from the front part to the rear part. The

material passed through a refiner contains about 90% moisture.

9.3.6 Screw press

Screw press removes a significant amount of water at the final dewatering stage

before incorporating cryoprotectants and freezing in blocks. The length and

speed of the screw press, the volume reduction ratio, and the perforation of the

Fig. 9.4 Refiner and its close view.


screens determine the effectiveness of water removal. Screens with 0.5 to

1.5mm perforations are commonly used in the industry. In addition, screens

with smaller perforations are usually placed at the end section to preserve

recovery.23 The material passed through a screw press commonly contains about

80±84% moisture.

9.3.7 Decanter centrifuge

The use of a decanter centrifuge results in improvements in surimi yield

because of greater solid material (more than 80% insoluble solids) recovery

during centrifugation compared to traditional screen techniques.59 The main

part of decanter centrifuge is the rotating bowl, which consists of a cylindrical

part and a conical part.23 Inside the bowl a conveyor moves the solids toward

the solid discharge port. A stationary inlet tube is inserted into the centre of the

conveyor. The bowl is enclosed in a vessel with discharge arrangements and

mounted on a base frame. Three main factors influence the separation of mince

and water in a decanter centrifuge: the design of the decanter (geometrical

configuration, bowl diameter, length, and speed, differential speed of the

conveyor relative to the bowl, and conveyor type); the composition of the liquid

and particles to be separated (density, viscosity, size, distribution,

configuration, and concentration of the particles); and process-related aspects

(temperature and feed rate).23 A large bowl diameter increases the solids

handling capacity but also dictates a lower main speed, so as not to exceed

mechanical limitations. Increasing the bowl length generally improves the

liquid clarification as the residence time in the gravitational field increases. In

addition, the higher the bowl speed, the higher the gravitational force, which

results in better clarification and a drier solid cake. The differential speed

between the bowl and the conveyor also affects the separation of solid and

liquid and must be carefully adjusted.

9.3.8 Silent cutter/mixer

A silent cutter or ribbon blender is commonly used in surimi plants to incor-

porate cryoprotectants relatively uniformly in a short period of time. Uniform

mixing without generating heat will assure better quality throughout frozen

storage of mince or surimi.

9.3.9 Freezer

Surimi is frozen in rectangular blocks (10 kg) in a plate freezer. Before loading

the freezer, the freezer plates should be cleaned. The blocks should be loaded

evenly to maintain good contact between blocks and plates. Blocks are held for

approximately 2.5 h or until the core temperature reaches ÿ25ëC.23 To have

good shelf-life, frozen surimi (Fig. 9.5) should be kept with a minimum

temperature fluctuation in cold storage at ÿ18ëC.


9.4 Mince/surimi processing by-products

Fish mince/surimi manufacturing generates huge amounts of by-products, such

as surimi wash water and solid by-products.60,61 Fish mince/surimi processing

by-products include fish frame generated from the filleting step and surimi wash

water generated from the mince washing step, which contain a significant

amount of soluble proteins, including myofibrillar protein.62 Addition of

sarcoplasmic protein and myofibrillar protein recovered from fish mince/surimi

processing by-products back into fish mince/surimi is an important issue for the

fish mince/surimi processing industry. We briefly discuss the various kinds of

fish mince/surimi processing by-products, technologies for recovering soluble

protein, and myofibrillar protein from surimi wash water and fish frame, and

their application in this section.

9.4.1 Fish mince/surimi by-products

As shown in Table 9.2, fish mince/surimi manufacturing generates various by-

products, such as viscera in the gutting step, heads in the deheading step, frames

in the filleting step, skin and bone in the mincing step, surimi wash water in the

washing step, and refiner discharge in the refining step. The major components

of fish mince/surimi processing by-products are enzyme, lipid, and protein in

viscera, extractives, meat and bone in fish frame, collagen and mineral in skin

and bone, soluble protein in surimi wash water and collagen in refiner

discharges.

Fig. 9.5 Frozen surimi.


9.4.2 Protein recovery methods

Water-soluble protein

Myofibrillar proteins constitute approximately 66 to 77% of the total protein in

fish flesh. However, in a typical surimi operation, only 50 to 60% of the

myofibrillar proteins are retained through the washing and dewatering process.

Approximately 40 to 60% of the myofibrillar proteins are lost in the soluble or

insoluble forms due to factors such as changes in pH and ionic strength,

proteolysis, and mechanical forces in mincing, washing, screening, and screw

pressing.45 Surimi wash water can be treated by chemical and physical methods.

Chemical methods apply chemical agents to coagulate soluble and insoluble

materials from the waste water by shifting pH. Physical methods use screens,

membrane filters, and other mechanical actions to remove the solids.

Ultrafiltration can be a primary means for recovering protein from surimi wash

water,45,63,64 but it has never been successfully applied in a commercial scale

due to the nature of protein fouling. However, decanter technology has been

successfully applied and made a significant difference in surimi processing.

(a) pH-shifting method: Nishioka and Shimizu63 developed means of

precipitating proteins from surimi waste water by shifting pH. This method

is based on myogen-aggregation phenomenon, which occurs when a

solution of sarcoplasmic protein is acidified or alkalified beyond the critical

pH zone of 4±5 or 9±11, respectively, and then neutralized. The best

condition for pH setting is at first from pH 7 to <4 and then from <4 to 7±9.

The maximum amount of precipitation is obtained by changing the pH from

7 to <4 and then to a value between 7 and 9, or from 7 to >12 and then to a

value between 6 and 7. The precipitates are easily collected by

centrifugation at a low centrifugal force of 300 � g. This method is simple

and low cost for pretreating waste water but results in complete denaturation

of the proteins and no gelling properties.

(b) New fish protein isolate using pH shift: As briefly discussed earlier, this new

process is similar to Nishioka's method.63 However, there is a distinct

difference of eliminating lipids in the finished product. The old method

separated protein precipitates at 300� g, while the new method at 10 000� g,

Table 9.2 By-products generated during mince/surimi processing

Processing Processing Proportion of Valuablesteps by-products whole fish (%) components

Gutting Viscera 15±30 Enzyme, lipid, proteinDeheading Head 14±20 MineralFilleting Frame 17 Protein, mineralMincing Skin/bone 8±10 Collagen, mineralWashing Wash water 14±16 Soluble protein, enzymeRefining Connective tissue 4±8 Collagen


which removes membrane lipids.53 Fish protein isolates show good gel values

even though the protein is denatured during processing.

(c) Membrane filtration: Membrane filtration applies a positive pressure as a

driving force to push the liquid phase through membrane pores, without

involving a phase change.64 It is the most suitable method for recovering

and concentrating thermally sensitive components, such as protein, in the

food industry. When compared to other recovery methods, membrane

filtration has the advantage that no supplementary additives are used to aid

separation and enables recovery of pure soluble and insoluble proteins.

Functional properties of recovered soluble and insoluble proteins are

preserved and present utilization and new product opportunities for food

manufacturers. When wash water is pumped across the membrane, water,

and particles with sizes smaller than the membrane pores pass through it,

while particles with sizes larger than the membrane pores are retained. With

removal of water and smaller particles, solids larger than the membrane

pores are recovered.

(d) Centrifugation using decanter centrifuge: Decanter centrifuges have also

been adopted by the surimi industry to recover large particles from the

waste streams. Typically, a range of 1700 to 3000 � g centrifuge force is

used for recovering protein particles from surimi wastewater streams.

Swafford et al.65 reported the use of a decanter centrifuge to recover

insoluble solids from rinsing and dewatering wastewater. Up to 80%

insoluble solids were recovered. Surimi produced through a decanter is

assigned as recovery-grade surimi. Recovery-grade surimi possesses fairly

good color and low impurities, but is usually of lower gel strength.45

Fish frame protein

Fish frames are important by-products of the fillet industry because of their

significant volume. Frames consist of backbone, tail, and dorsal fins connected

by muscle not removed during filleting, which are usually discarded or rendered

into fish meal. However, a large volume of minced fish can be recovered during

the deboning step (Table 9.3). High levels of bones and parasites, bacterial

contamination and off-colors from skin, backbone and gut cavity limit the

appeal of frame mince prepared by mechanical deboning. It is considered low

quality when compared to minced fillets and fillet trimmings.15 Improved

methods for separating fish mince from frame are needed to produce frame

Table 9.3 Yields of mince from various fish frames

Frame source Yields (%) Reference

Pacific whiting 42.8 Wendel et al.41

Alaska pollock 31.4 Crapo and Himelbloom43

Channel catfish 57.4 Suvanich et al.44

Yield (%) = (unwashed mince weight/frame weight) � 100


mince with acceptable characteristics. Wendel66 reported that washing frame

meat or mince contaminated with kidney tissue can be more effective than

removing the kidney tissue before deboning to decrease textural degradation and

protein denaturation.

For effective utilization of frame mince, one approach is to develop

processing techniques, such as backbone removal, to reduce high defect levels.

If kidneys are removed from frames without contaminating meat, higher quality

mince can be obtained. Another approach is to blend minced frame flesh with

minced fillets and trimmings to produce an acceptable quality product. The

recovery methods of fish mince from frame are discussed in this section.

(a) Mechanical deboning: Mechanical deboning is the conventional method for

separating mince from frame. In mechanical deboning, fish frames are

deboned with a drum with 5 mm diameter perforations.

(b) Water jet deboning system: When compared to mechanical deboning, a

water jet deboning system is superior in mince quality, although inferior in

yield.66 The dewatering is more difficult in frame mince separated by water

jet deboning than that separated by mechanical deboning. It is probably

because particle size is smaller from the water jet deboning system than

from the mechanical deboning.67 Frames in water jet deboning system are

conveyed through high pressure oscillating water jets to remove the flesh.

The recovered flesh then goes to a rotary screen to remove broken bone and

skin particles. The system results in a white fish mince. However, it

consumes a large quantity of water and is difficult to dewater.

9.4.3 Characteristics of by-products recovered from surimi wash water

and frame

Surimi wash water

Insoluble particulates in surimi wash water are recovered by a rotary screen and

further concentrated by microfiltration. In the surimi industry, the microfiltration

method is applied for recovering protein from surimi wash water, reducing

wastewater impact on the environment, and exploring potential for recycling

process water. The moisture of discharged water is around 99%, and is reduced

to around 93% by the rotary screen and then to around 85% by the micro-

filtration system. The solids are increased by 6.7 fold and 15.5 fold, respectively.

As shown in Fig. 9.6, the microfiltration-treated solids are concentrated to

around 16% (protein: around 15%). However, that is slightly lower than around

20% of commercial surimi (protein: around 19%). The myosin heavy chain

content in rotary screen-treated protein and microfiltration system-treated

protein are 25% and 62%, respectively, as shown in Fig. 9.7. These results

indicate that most of the myosin lost in the form of particulate from the screw

press is recovered by the rotary screen. However, the myosin heavy chain

content of microfiltration system-treated protein is low when compared to

commercial surimi (100%).


Fig. 9.6 Moisture and crude protein contents of wash water, rotary screen-treatedprotein (RS), microfiltrated protein (MF), and commercial surimi (CS) (modified from

Ref. 62).

Fig. 9.7 Quantity comparison of myosin heavy chain from rotary screen-treated protein(RS), microfiltration-treated protein (MF), and commercial surimi (CS) (modified from

Ref. 62).


Frame mince

Mechanically deboned frame mince generally contains bone fragments and

reddish colored kidney juice, which contains high concentrations of the enzyme,

trimethylamine oxide (TMAO) demethylase. Therefore, the color and flavor

quality of mechanically deboned frame mince containing kidney is low

compared to commercial surimi.4 Mechanically deboned frame mince is also

low in salt extractable protein and high in bone, parasite and bacterial counts,

impurities, and off-flavor compared to commercial surimi. Frame mince is

generally much darker than commercial mince, as shown in Fig. 9.8. Color can

be improved by blending frame mince with commercial mince.

9.4.4 Utilization as a surimi resource

Water-insoluble proteins

Surimi wash water contains a significant amount of water-soluble and insoluble

protein. Lin et al.62 attempted to recover water-insoluble proteins using micro-

filtration and to use recovered proteins as an extending agent of surimi. The result

indicates that surimi with 10% replacement of microfiltration-treated protein has

the same gel quality as a commercial surimi with respect to gel hardness,

elasticity, water retention, and color. Recovered protein has a lower shear stress,

but the same shear strain values as a commercial surimi. Pedersen67 also reported

adding up to 10% protein recovered from surimi wash water resulted in no

measurable decrease in gel strength of surimi. The results indicate the recovered

Fig. 9.8 Lightness (L*) of Alaska pollock minces (modified from Ref. 43). 20/80 and10/90 indicate the mixture of 20% frame mince/80% commercial mince and 10% frame

mince/90% commercial mince.


protein from surimi wash water by microfiltration system can be used as an

extending agent of surimi. Lin et al.62 also reported that the surimi production

yield could be increased by 1.7% by adding microfiltration-treated protein back

into the production line without diminishing surimi functional property.

Fish frame protein

Fish frame generated in fillet processing contains a significant amount of flesh.

However, frame flesh has some defects, such as poor texture and color. The

defect of frame mince can also be improved by blending frame mince with

commercial mince, as shown in Fig. 9.9. Surimi gel texture prepared from

commercial mince has firmer texture than surimi prepared by frame mince.

Surimi gel texture prepared from blended minces (10/90 and 20/80) are as firm

as gels prepared from commercial mince. Two approaches can be proposed to

utilize frame meat: one is to develop processing techniques to reduce the high

defect levels and the other is to blend minced flesh with minced fillets and/or

trimmings to produce an acceptable quality product. Wendel66 also reported that

water jet deboning was a good method for separating flesh from frame and it can

avoid the low quality problem of conventional frame mince.

9.5 Functional properties of fish mince/surimi

Functional properties of proteins indicate their ability to make gels stronger,

holding more water or oil, or looking whiter. These include protein solubility,

water absorption capacity, water binding ability, viscosity, gelation, swelling

ability, emulsifying capacity and emulsion ability.68 Among them, the most

Fig. 9.9 Texture of cooked Alaska pollock minces (modified from Ref. 43). 20/80 and10/90 indicate the mixture of 20% frame mince/80% commercial mince and 10% frame

mince/90% commercial mince.


important functional property in fish mince/surimi is the gelling property.69

Gelation of fish mince is thermo-irreversible.8 Fish myofibrillar protein, which

is responsible for the gelation property, can form a strong and elastic gel upon

heating. Unlike myofibrillar proteins of land animals, fish myofibrillar proteins

can be heated to higher temperatures without sacrificing gel strength or water-

holding capacity.70 The functional property of fish mince/surimi can greatly be

affected through the biological characteristics of the fish, such as species,9,23,30,71

seasonality,23,72 sexual maturity,23 freshness/rigor71,72 and extrinsic factors,

such as harvesting and onboard handling, processing water,47,48,73 time/

temperature,15,74±76 washing cycle,21,77,78 pH,51,52 salinity,52,79 and functional

ingredients.19,28,37,80,81 A brief discussion will also be provided in this section on

factors affecting functional properties of both conventional surimi and fish

protein isolates prepared by new technology, such as acid- and alkali-aided

methods.

9.5.1 Fish species

In addition to Alaska pollock and Pacific whiting, there are a number of species

that are utilized as raw materials for commercial mince/surimi processing.

Functional properties of the final fish mince/surimi products vary with raw

material due to the different biological properties of fish, such as intrinsic

enzymes, lipid composition, the proportion of red flesh, and rigor status (Table

9.4). When fish mince/surimi from some fish, such as Pacific whiting,82

arrowtooth flounder,83 threadfin bream,84 lizardfish,23 Atlantic menhaden,85

white croaker, and oval filefish,86 are converted into mince/surimi-based sea-

food with functional properties, enzyme inhibitors are required or rapid cooking

is necessary to minimize functional deterioration caused by heat-stable enzymes.

Fish mince/surimi is also prepared from oily/red-fleshed fish. However, the

quality of surimi made from these species varies according to the whiteness,

trimethylamine oxide (TMAO) and fat contents of the fish.87 To make mince/

surimi with functional properties and storage stability from oily/red-fleshed fish,

such as mackerel, sardine, and salmon, certain steps must be applied to negate

the effects of the oil and heme proteins. Heme proteins, such as myoglobin and

hemoglobin, account for the red color of dark muscle. In addition, fat oxidation

in the dark muscle is promoted by heme proteins, which cause an offensive,

Table 9.4 Lipid, protein, and dark flesh composition in fish species

Lipid (%) Protein (%) Dark flesh (%)

Sardine 6.0 17.5 31.1Mackerel pike 8.4 20.0 23.3Mackerel 4.0 18.0 18.1Cod 0.6 16.6 <1Pacific whiting 1.5 16.8 <1


rancid odor.14 To utilize red fleshed-fish as a mince, vacuum packaging is used

or antioxidants can be added to prevent lipid oxidation. To utilize the red

fleshed-fish as a surimi source, 0.1±0.5% NaHCO3 is used in the first washing

solution and a decanter can be used to remove excess oil. The addition of 0.05±

0.1% sodium pyrophosphate and the use of vacuum during washing are also

recommended to remove heme proteins.

9.5.2 Seasonality and sexual maturity

Fish muscle is subjected to seasonal variations in parameters, such as pH, fat,

protein, and water content, which influence the functional properties of the final

products. The composition of Alaska pollock varies between seasons so protein

contents are highest (16.9%) in the fall and lowest (15.6%) in the spring,

whereas moisture contents are highest (82.7%) in the fall and lowest (80.9%) in

the spring.23 Bandarra et al.88 and Leu et al.89 reported seasonal changes of the

fat contents of sardines harvested off the Portuguese coast and mackerel

harvested in the Southern Rhode Island waters, Nantucket Sound, respectively.

The fat content of sardine was as high as about 18% in August±October and

lowest in March at 1.2%. The fat content of mackerel was as high as about

22.6% in November and lowest in March at 5.1%. Consequently, to manufacture

mince with good frozen storage stability from sardine harvested in summer and

mackerel harvested in winter, vacuum package technology must be applied

because of the higher fat content. To manufacture surimi with good functional

properties and whiteness from sardine harvested in summer and mackerel

harvested in winter, special technology using NaHCO3 and a decanter must be

applied because of the higher fat content.

Ingolfsdottir et al.72 reported on seasonal variations in functional properties

of cod mince. The results indicated that both hardness and cohesiveness showed

a drop from March to May and another drop was observed for cohesiveness

during the autumn months compared to mince prepared from cod harvested in

the winter. Surimi prepared from fish harvested in the feeding period commonly

has the highest functional properties. It is probably because fish muscle has the

lowest moisture content, as well as the highest total protein during the feeding

period.

9.5.3 Freshness or rigor

The functional properties of fish mince/surimi are influenced by freshness of

fish. It is probably because fish freshness affects biochemical properties of fish

muscle. Freshness of fish is primarily time/temperature-dependent. The

processing of Alaska pollock occurs within 12 h on at-sea vessels, whereas at

shore-side operations processing occurs within 24±48 h.23,59 However, due to

recent regulations of salmon by-catch and sea lion habitat, it is not uncommon to

see pollock processed 100 h after harvesting. Fish freshness is also affected by

harvesting conditions and methods used for capture, such as weather conditions


at sea, size of tow, length of tow, salt uptake, and temperature of fish after

capture, as well as on-board handling methods and vessel storage conditions.

Development of rigor mortis varies among fish species, and is dependent

upon post-mortem temperature, and ante-mortem conditions, such as stress,

method of capture, handling conditions, and seasons.90

9.5.4 Time and temperature of processing

Time and temperature for fish capture are the most important factors for the

functional properties of final mince/surimi. With prolonged storage/processing

time and increased storage/processing temperature, severe proteolysis could

occur in finished products, mince or surimi. If the fish is a proteolytic enzyme-

laden species and held for more than 24±48 h even at 5ëC, good functional

properties would not be expected due to proteolytic degradation. Consequently,

to prepare mince/surimi with good functional properties, fish should be

processed promptly on landing or kept at around 0ëC if holding is necessary.

Degradation of myosin heavy chain of Pacific whiting increased as the washing

cycles increased (Fig. 9.10).91 The trend of actin degradation was similar to that

of myosin heavy chain, but to a lesser extent.47,92 Actin degradation increased as

storage time and temperature increased. At 0ëC, actin was degraded, the extent

increased substantially with storage time. After 14 h at 0ëC, about 20% of actin

was degraded. Raw fish for mince/surimi preparation is commonly kept in

holding tanks (about 0ëC) up to 14 h before fish are subjected to processing.

Similar to myosin heavy chain degradation, actin degraded more rapidly at 5ëC

than at 0ëC. As temperatures and time increased further, degradation of actin

occurred more rapidly.23

Fig. 9.10 Loss of myosin heavy chain (MHC) at various washing cycles (modified fromRef. 91).


9.5.5 Effects of wash water and washing conditions

In conventional surimi making, washing is one of the most critical steps for

preparing surimi with good functional properties. A large amount of water is

used to remove sarcoplasmic proteins, blood, fat, and other nitrogenous

compounds from minced fish flesh. Functional properties of the final product are

greatly improved as the myofibrillar protein is concentrated by removal of

impurities during washing. The important factors associated with water for

preparing surimi with good functionality are temperature, mineral content, pH,

and salinity, quantity of water used and washing cycles.

Water must be refrigerated to a temperature below which the fish muscle

proteins can retain their maximum functional properties. Considering the change

in air temperature during processing, the recommended water temperature for

obtaining maximum quality is 5ëC or lower. Even though warm water species

are not as sensitive as cold water species, cold water temperature, in general,

results in better quality of finished products.

Soft water with minimum levels of minerals, such as Ca++, Mg++, and Fe++, is

recommended for washing. Hard water causes deterioration of texture and color

of fish mince/surimi during frozen storage. In addition, Ca++ and Mg++ are

responsible for gel texture property changes, whereas Fe++ is responsible for

color changes.59 The pH of water must be maintained at approximately that of

prerigor fish muscle tissue (pH 6.8±7.0) to obtain better functional property of

surimi. Before washing, the salinity of fish mince is approximately 0.7%. Too

much salt (>0.3%) in the wash water could cause solubilization of myofibrillar

proteins resulting in low yield, and accelerate denaturation during frozen

storage.

The degree of washing required to produce good functional surimi depends

on the type, composition, and freshness of the fish. Water to meat ratios ranging

from 4:1 to 8:1 are often used by processors. This washing process is often

repeated three or four times to ensure sufficient removal of sarcoplasmic

proteins. However, for the last 10 years, the US surimi industry has made an

effort to reduce the water to meat ratio to 2:1 and the washing cycle to 2,

resulting in a significant yield increase from 15% to 30%. Adu et al.76 reported

that most sarcoplasmic proteins are fairly soluble and removed during the initial

washing steps. Consequently, after the sarcoplasmic proteins are completely

removed, further washing causes a severe loss of myofibrillar proteins. Lin and

Park23,48 investigated minimizing water usage for leaching by reducing the

water/meat ratio and increasing the wash cycles and wash time. Increased wash

time did not enhance the removal of sarcoplasmic proteins once equilibrium was

reached. However, increased wash cycles continuously removed residual

sarcoplasmic proteins from the mince. At a low water to meat ratio (2:1 or

1:1), regardless of wash cycles and wash time, no significant loss of myofibrillar

proteins occurred. Myosin heavy chain content, water retention, and whiteness

of the washed mince, however, decreased when the water to meat ratio was

reduced. Increasing wash time and/or wash cycles, however, enhanced

functional properties but also resulted in higher moisture content.


9.5.6 Fish protein isolates by new technology

This new approach, initiated at the University of Massachusetts,49 has opened up

a new way of isolating functional fish protein isolate. New technology for

preparing fish protein isolates (acid- or alkali-aided methods) shows significant

potential as a new method for maximal protein recovery and results in surimi

with commercially acceptable (better with alkaline treatment) functional

properties. Unlike the conventional method of surimi manufacturing, no

washing or dewatering steps are continuously involved, which significantly

reduces waste and water consumption. Kim51 investigated the functional

properties of fish protein gels prepared after various treatments (Fig. 9.11). The

results indicated that the best functional properties are obtained from fish

proteins treated at pH 11 (alkali-aided) and pH 2 (acid-aided). Surface SH

content is critical for the formation of disulfide bonds. Alkali conditions,

especially pH 10.5 and pH 11.0, are favored for disulfide bond formation.

However, pH 10.5 treatment does not give high texture values. It is probably

because of the high cathepsin L-like activities, which interfere with gel forma-

tion.51 In addition, at pH 11, extensive thiol oxidation and disulfide interchange

reactions occur and more disulfide bonds contribute to strong gel formation.

Another interesting way of utilizing fish protein isolates from seafood

processing by-products has been explored at several universities.53 Using

primarily fish frame, fish protein can be isolated using the alkaline method

followed by centrifugation. This isolate is homogenized with water as a milky

Fig. 9.11 Textural properties of gels made from fish protein isolates after various pHtreatments (modified from Ref. 51). Samples were treated at various pH conditions duringprotein recovery, and then adjusted to pH 7.0. Different alphabetical letters indicate asignificant difference ( p < 0:05). Gels were prepared with 1.5% beef plasma protein.


slurry after mixing one part isolates and three parts water. This pure protein

slurry is injected into fillets like salmon, halibut, catfish, and other valuable

species. It enhances the yield and improves frozen stability of fish fillets. A

different attempt was also made to utilize fish proteins isolates from by-

products. When milky slurry was applied as a dipping solution for fish fingers

and patties before battering or breading, the quantity of oil absorbed in fried

products was significantly reduced. Fish protein isolate may form a protein film

and act as a fat blocker.93

9.6 Nutritional characteristics

Mince is flesh separated in a comminuted form from the skin, bones, scales, and

fins. Therefore, the nutritional properties of unwashed fish mince are similar to

those of the raw material, while superior to those of surimi. It is probably

because of the difference between washing and unwashing. Therefore, fish

mince contains a high level of water-soluble vitamins, mineral and fats com-

pared to surimi. Adu et al.76 also reported the effect of washing on the

nutritional characteristics of minced rockfish flesh. The results indicated that

unwashed rockfish mince was higher in ash and lipid contents based on dry

weight than washed rockfish mince. There was no difference in amino

composition between washed- and unwashed-rockfish minces.

There was a difference in mineral composition between washed- and

unwashed-rockfish minces. The mineral composition of the minced fish was

greatly changed by washing treatment. Phosphorus, potassium, and sodium

levels were reduced, while the iron, copper, zinc, and chromium levels increased

in the washed fish mince when compared to unwashed fish mince. Calcium

content is commonly high in fish mince compared to fish fillets, due to small

bone fragments in the mince as a result of the mechanical deboning.57 Babbitt

and Reppond33 compared trimethylamine oxide (TMAO) content between

washed mince and unwashed mince from Alaska pollock. Washing reduced the

TMAO content from 71.3 to 8.7mg/100 g. It is well known that fish oils, rich in

polyunsaturated fatty acid, have potential for prevention of heart disease, cancer

and other diseases. Therefore, surimi is lower in calories and oil including

omega-3 fatty acids. It is also bland in taste/odor and white in color due to

removal of water-soluble components by washing. However, the nutritional

values of surimi-based seafood can be controlled by combination with other

nutritious food and/or nutritional fortifiers.

Pedersen67 and Lin et al.62 reported the protein recovery of 80% solids in

surimi manufacture from Alaska pollock and Pacific whiting using membrane

filtration. Comparing ash contents of before (about 17% of the solid) and after

(about 3% of the solid) membrane filtration treatment of wash water, membrane

filtration served to remove mineral from the process water. There was no

difference in amino acid composition among raw materials, its primary product

(surimi) and the recovered protein concentrate from surimi wash water.


Ohshima et al.6 examined the chemical scores for essential amino acids in

surimi, beef, pork, chicken and turkey. The high level of nutritional quality in

surimi gel blended with recovered protein concentrate from surimi wash water

and commercial surimi matches that of land animal meats. Surimi gel, which is

blended with the recovered protein concentrate from surimi wash water and

commercial surimi, is higher in isoleucine, leucine, lysine, threonine and

tryptophan content than chicken, while lower in histidine and valine content.

The amino acid score of surimi is also that of a high quality protein, and is

similar to beef, chicken, and turkey.

9.7 Storage stability

Fish mince/surimi are commonly frozen for attaining longer shelf life. Utiliza-

tion of frozen fish also provides a more constant supply of raw material

independent of yearly variation and thereby facilitates consistent production

planning. Frozen storage stability of fish mince/surimi can be greatly affected

through biological characteristics of raw material (fish species, seasonality,

sexual maturity, and freshness), harvesting, onboard handling, and processing

conditions (water, time/temperature, washing cycle, pH, salinity, and functional

ingredients). A brief discussion of factors affecting frozen storage stability of

fish mince/surimi is provided in this section.

9.7.1 Factors affecting storage stability

Fish species

Various fish species are used as raw material for fish mince/surimi processing. The

frozen storage stability of fish mince/surimi varies with different biological factors

of fish, such as intrinsic enzymes, lipid composition, the proporation ratio of red-

flesh, and development of rigor mortis90 (Table 9.4). Usually, tough texture is

developed in gadoid fish muscle, such as cod, haddock, Pacific whiting, Alaska

pollock, during frozen storage. Such textural changes are caused by trimethylamine

oxide (TMAO) demethylase. Formaldehyde is hypothesized to be a cross-linking

agent in muscle proteins and may thus cause textural deterioration in frozen fish

mince.11,21,69 Dark-fleshed fish minces, such as mackerel, sardine, and salmon,

also undergo quality deterioration during frozen storage, mainly through lipid

oxidation. To improve frozen storage stability of mince/surimi from dark-fleshed

fish, certain steps must be applied to negate the effects of oil and heme proteins.

Heme proteins, such as myoglobin and hemoglobin, account for the red color of

dark muscle. In addition, fat oxidation in the dark muscle is promoted by heme

proteins, which causes denaturation of protein during frozen storage.14

Washing

Washing using chilled water of 5±10ëC before freezing removes denaturation

promoters, such as active enzymes and their substrates, metals, lipids containing


n-3 polyunsaturated fatty acids and trimethylamine oxide, from fish mince.21

This affects the stability of the remaining proteins (primarily myofibrillar

proteins) during frozen storage. If protein cross-linking induced by formaldehyde

is the main cause of textural hardening during frozen storage, the textural change

should be minimized by washing, since most trimethylamine oxide in fish mince

can be removed by washing. Consequently, to improve frozen stability in fish

mince, the formation of dimethylamine and formaldehyde should be reduced.

Functional ingredients

Washed fish mince is generally mixed with cryoprotectants, such as sugar,

sorbitol, and polyphosphates, in order to stabilize the fish proteins from freeze

denaturation. The use of high levels (>5%) of sugar made surimi too sweet and

caused a brown color during frozen storage. Therefore, sorbitol is used to reduce

the sugar content and subsequently, the level of sweetness and discoloration.

Sugar and sorbitol protect protein from frozen denaturation by solute exclusion

from the surface. As sugar or sorbitol is introduced to a system of proteins and

water, sugar/sorbitol is excluded from the protein-water system and destabilizes

water molecules. As water molecules are reoriented, the hydrophobic groups

hidden in the interior create stronger interactions, resulting in a stabilized protein

structure. Sugar/sorbitol also increases the surface tension of water. This

prevents withdrawal of water molecules from the proteins.4

Noguchi et al.94 reported that pentoses, such as xylose and ribose, have less

protective effect for protein denaturation than hexoses, such as glucose and

fructose. The difference of protective effect among the monosaccharides is

probably because of the difference in the number of OH groups on the

molecule. These compounds stabilize the native conformation of the proteins by

a solute exclusion mechanism.95,96 The cryoprotective effects of sugar and

sorbitol can be enhanced by adding phosphate. The effect of phosphate can be

explained based on two facts: one is to raise the pH and the other is to chelate

metal ions, which actively promotes oxidation. The average quantity of

cryoprotectants added to washed fish mince is approximately 4±6% sugar, 0±

5% sorbitol, and 0±0.3% phosphate.97 Under constant low temperature storage

(below ÿ20ëC), frozen surimi with cryoprotectants, such as sugar, sorbitol, and

phosphate, can usually be stored up to two years. However, it is important to

remember that cryoprotection does not stop, but rather minimizes the freeze

denaturation.

With its versatile characteristics, surimi can be used as an intermediate raw

material for preparing popular surimi seafood, which requires specific textural

attributes, such as kamaboko,31 crabstick,26,33,97 lobster analogs,34 shrimp

analogs,94 frankfurter,98 nugget,36 and fish patty.38

9.7.2 Comparison of storage stability between fish mince and surimi

Fish mince contains a significant amount of water-soluble components, such as

active enzymes and their substrates,99 metals, lipids, and trimethylamine. Many


problems of fish mince, such as color, texture, and odor change, are caused by

the water-soluble components. Therefore, surimi made after removing water-

soluble components is prepared to improve frozen storage stability of fish

mince. The storage stability of fish mince is, therefore, inferior to that of surimi.

Crawford et al.99 studied the stability of frozen Pacific whiting mince blocks

compared to fillets over a 12-month period and indicated higher levels of

oxidative rancidity in minced flesh due to textural deterioration. Regenstein100

examined the stability of frozen mince in cold storage and reported that many

problems caused by enzyme action can be minimized by maintaining the storage

temperature below ÿ30ëC. However, the storage stability of fish mince is a

continuing problem since most commercial storage conditions are well above

ÿ30ëC. Abdel-aal13 used antioxidants for extending the shelf life of frozen Nile

karmout (Claries lazera), which has many undesirable characteristics such as

rapid development of rancid off-flavor. Ascorbic acid and Na2EDTA could

retard rancidity development in the frozen karmout mince.

9.7.3 Storage stability of mince from surimi by-products

Fish mince from surimi by-products can be used as a surimi yield extender

because of its availability in large quantities. However, the unwashed mince

recovered from frame has numerous impurities from kidney and bone. If it is not

totally removed, residual kidney tissue may be incorporated into mince during

deboning. Since kidney tissue can not be totally removed from frame by the

conventional method, the chemical and textural changes of recovered fish mince

from frame during frozen storage are accelerated due to enzymatic and

microbial contamination from the kidney.68 To avoid quality problems

associated with trimethylamine oxide (TMAO) demethylase that accelerates

dimethylamine (DMA) and formaldehyde formation, Wendel et al.41 used a high

pressure water jet deboning system for recovering mince from frame and

reported that frozen storage stability of the mince was improved.

9.8 Utilization

Fish mince/surimi can be used as an ingredient in many composite products,

such as kamaboko, various surimi seafood (crabstick and other shellfish

analogs), seafood patty, seafood nuggets, and frankfurter analog. The mince/

surimi-based products are also very popular in the Asian diet. Due to their

healthy nutritional values, functional versatility, and competitive prices in

relation to their natural counterparts, the consumption of these products in the

West has reached almost 250 000 metric tonnes. Phenomenal growth has also

been experienced in the USA and Europe, including Russia, where sales have

grown from virtually zero in 1980 to 900 000 metric tonnes and 150 000 metric

tonnes in 2004.


9.8.1 Kamaboko

Kamaboko is the most typical surimi-based product in Japan. Surimi paste is

formed on a wood board before any thermal treatment. Sometimes its surface is

coated with colored paste for appearance. The shape and texture of kamaboko

varies depending on the geographical region. After its unique shape is formed,

the surimi paste is subjected to a low temperature setting process (20±40ëC for

30±60 min), depending on the species. During this process, the gel-forming

ability of solubilized myofibrillar proteins is highly enhanced, which yields a

strong gel.101 Cooking by either steaming or baking is carried out to complete

the gelation of fish proteins.

9.8.2 Crabstick

Crabstick is currently one of the most prevalent surimi-based fabricated seafood

products in the marketplace.102 The crabstick manufacturing flow is described in

Fig. 9.12. First, frozen surimi is either partially thawed or broken before surimi is

cut into small pieces. This step is required to reduce the size of particles by

avoiding a heavy load to blades or the shaft during chopping. Over-thawing which

may induce denaturation must be avoided. Salt is added first into surimi to extract

myofibrillar proteins. Then other ingredients (egg white, starch, sugar, sweet rice

wine, seasoning and flavors, natural coloring, and water) are commonly added to

salted surimi in the silent cutter. Chopping temperatures vary depending on

species between 0±5ëC for cold water species and 15±25ëC for warm water species

to produce higher gel functionality.101 The comminuted surimi paste is extruded

onto a conveyor belt of the cooking machine and then cooked.

A common sequence of heating is radiant heat, followed by steam heat, and

then radiant heat again. Regardless of the heating method, however, the ambient

temperature in the tunnel where the paste is exposed to radiant or steam heat is

90±95ëC. Total cooking time depends on the product specification. A thinly

extruded (1±2mm) sheet undergoes various heating procedures depending on

the machinery and production specifications, but in general this primary cooking

last 35±60 seconds. This short cooking process induces gelation of the surimi

proteins but is not sufficient to swell the starch or gel other protein additives.

Immediately on completion of the first cooking step, the product is cooled by air

at room temperature or below.

Fiberization is accomplished by elongated cuts running lengthwise on the

gelled sheet. Slitting is obtained by passing the sheet through two rollers with

slitters. The space between the slitters controls the number and width of

individual fibers. Bundling is a process that rolls the cooked product sheet

tightly into a rope shape. The rope is then passed through the rollers for a

conventional color application. A polyethylene plastic film, onto which colored

fresh paste is applied, wraps around the product rope, which is then cut to a

specified length. The colored paste is then set to a gel by cooking under steam

for 15±30 min, followed by rapid chilling. However, co-extruded color applica-

tion bypasses this laborious processing step.101


Fig. 9.12 A crabstick line with drum cooker (source: Courtesy of Young Nam Machinery, Korea).

Surimi crabmeat may be cut into different dimensions, such as flake, stick,

and chunk. The rope is cut diagonally at a 25±30ë angle in 5±8 cm long pieces,

tip to tip for flakes. Leg or stick shape is cut straight at a 90ë angle and usually

3, 5, or 8 inches long. Surimi crabmeat is packed in plastic films under full or

partial vacuum before going through metal detection followed by

pasteurization.

Throughout production processing temperatures are important in develop-

ment of gel structure and resulting textural characteristics of surimi crabmeat.

Finished product is strongly influenced by cooking temperature and time.25 The

pasteurization step adapted in surimi crabmeat must secure the microbiological

quality of products. However, the processing step is not sufficient to sterilize.

Pasteurization with proper heat treatment provides a greater advantage over

sterilization, which results in serious problems in sensory characteristics, with

regard to sensory attributes. The longer the cooking at a higher temperature, the

more negative the sensory attributes. For the safety of vacuum-packed surimi

seafood against Clostridium botulinum, the US government has recommended

the manufacturer to heat the package at 85ëC (internal) for a minimum 15 min,

while maintaining 2.4 water phase salt and storage temperature below 3.8ëC. A

European guideline of F90 � 10min is also approved for 6-log microbial reduc-

tion.101 The chilling must bring the product temperature to 4ëC within 30 min.

Refrigerated products are packaged after chilling to 5ëC.

9.8.3 Seafood patty

For preparation of seafood patty, frozen mince is either partially thawed or

broken before mixing. Various ingredients (85±87% minced fish, 2±3% soy

protein, 2±3% starch, 1.5±2.5% refined salt, a small amount of dried onion, dried

tomato, dried pepper, chilli powder, powdered garlic, and allspice, 4±8%

sorbitol, 1±2% chicken/beef/pork extracts) are added to thawed or broken mince,

and blended in a ribbon mixer. The temperature should be maintained at below

10ëC during mixing. The blend is held at ÿ20ëC for 1 h to facilitate easy forming

into patty, and to maintain about 0ëC during forming. Fish patties are formed and

dehydrated in a tunnel dryer for 10 h before vacuum packaging.

9.8.4 Seafood nuggets

For preparation of seafood nuggets, frozen mince is either partially thawed or

broken before mixing. Ingredients (salt, sugar, egg white, oil, and other

flavorings) are added to thawed or broken mince and blended in a ribbon mixer.

The temperature should be maintained at below 10ëC during mixing. The mixed

paste is stuffed, extruded and placed onto trays before chilling overnight. The

chilled strands are cut into lengths of about 2.0 cm. The cut product is

predusted, battered, and breaded. The resultant nuggets are deep-fried at 350ëC

for 45 seconds before cooling and packaging. Seafood nuggets are stored below

±20ëC.


9.8.5 Frankfurter analog or seafood sausage

For the preparation of frankfurter analog, frozen surimi is either partially thawed

or broken before surimi is comminuted. The thawed or broken surimi is placed

in a silent cutter and chopped for 2min at high speed with 2.5% NaCl. The

temperature should be maintained at below 10ëC during chopping (temperature

could vary depending on the species used). Additional ingredients are then

added. Chopping continues at high speed for 10min. The paste is stuffed into

casings. After standing at room temperature for 1 h, the links are heat processed

by immersion in a 90ëC water bath for 20min. They are then cooled by

immersion in a low temperature cooler (below 5ëC) until reaching an internal

temperature of below 30ëC and stored in a 2ëC cooler.

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10.1 Introduction

The proper utilization of limited aquatic resources has been a topic of great

interest for many decades. As discussed in more detail in other chapters in this

book, a major problem facing the seafood industry is that a substantial amount of

material is left behind after processing. Segments of the industry are also faced

with a tremendous amount of by-catch, which is not properly utilized. These

materials, which are often discarded or used for animal feed or fertilizer, are rich

in valuable and functional ingredients that can be recovered. Many years back it

was found that adding enzymes to this `waste' material, with the aid of other

processes such as filtration and centrifugation was an effective way to extract

and recover proteins from the material. The application of proteases to fish

processing waste leads to the hydrolysis of the proteins in the material, which

can then be separated from other constituents of the muscle. This process pro-

duces what is known as fish protein hydrolysates (FPH). A number of research

publications have demonstrated that using controlled enzymatic hydrolysis a

broad range of protein ingredients of good quality can be produced from

undesirable raw materials (e.g., Baek and Cadwallader, 1995; Shahidi et al.,

1995; Vieira et al., 1995; Onodenalore and Shahidi, 1996; Kristinsson and

Rasco, 2000a,b,c; 2002). On the commercial level, this promising technology

has, however, been faced with economic obstacles, lack of usability and often

low acceptance of the final products (Kristinsson and Rasco, 2000a). This is in

part related to the very complex nature of the raw material, which can often lead

to unstable, poorly functional and unacceptable final products. A few countries

and companies have, however, been successful in producing FPH on a

commercial level, and still are. The key to FPH success is their unique properties

10

Aquatic food protein hydrolysatesH. G. Kristinsson, University of Florida, USA

and the ability to produce FPH with very different properties from the same raw

material, as elaborated in this chapter.

10.2 The enzymatic hydrolysis process

Many different processes have been reported for recovering and hydrolyzing

proteins from aquatic food material with enzymes. The general principle of the

process is simple (Fig. 10.1) but there are many factors that need to be carefully

considered if the aim is to have good control over the process and produce a

final product of consistent and good quality. For most processes the raw material

is thoroughly minced and then added to water. A well homogenized raw material

in water is important to allow for good mixing and good enzyme access. Too

little water can significantly reduce the production/recovery of FPH (Slizyte et

al., 2003), likely in large part due to high viscosity which limits enzyme access

and would also hinder recovery attempts using filtration or centrifugation,

particularly if the aim is to produce FPH of limited hydrolysis. Generally the raw

materials for hydrolysis are a complex mixture which can present processing

challenges, as well as having a major influence on the quality of the final

product. Raw materials high in lipids and pro-oxidants (e.g., blood and heme

proteins) have a tendency to oxidize during processing and may lead to

significant lipid oxidation and color problems in the final material. To minimize

this problem, antioxidants can be added before hydrolysis, or raw material can

be washed to remove some of the heme proteins and lipids. Washing may,

however, lead to lower protein recovery as proteins can be washed out.

Fig. 10.1 Outline of the main steps in the production of fish protein hydrolysates.


Oxidation may also be enhanced at lower pH values and higher temperatures,

which can be avoided with proper enzyme selection. Many raw materials have

high levels of spoilage bacteria, which can be minimized by maintaining low

temperatures, low pH (which unfortunately will accelerate oxidation) and

applying proper enzyme inactivation temperatures, which also reduce the level

of microorganisms. Furthermore, components such as skin, bones and scales

may interfere with enzymatic hydrolysis, and ideally should be separated from

the material prior to hydrolysis, e.g. via mechanical deboning. Recent advances,

such as the advent of the acid- and alkali-aided solubilization/precipitation

process to recover proteins from byproducts (Hultin et al., 2005) (see Chapter 4)

now provide an economic way to start with a relatively pure protein material as

the starting material for fish proteins hydrolysis.

The next step in the process is to allow the homogenized mixture to reach the

temperature of interest. This is important, as enzymes may have markedly

different activities at different temperatures. The pH of the slurry is then

adjusted to the desired value and the enzyme of interest added to the mixture.

The choice of enzyme is a very important consideration when making FPH,

since different enzymes have different specificities and reaction rates, and thus

can yield very different products. The least expensive method is to utilize

proteolytic enzymes present in the fish itself, e.g. mixing visceral material with

the by-products. This autolytic process is still employed, but primarily for

animal feed or fertilizer applications. There are, however, products produced

with endogenous enzymes for human consumption, most notably fish sauce

which consists of extensively hydrolyzed fish proteins. The major problem with

using endogenous enzymes is that very little control is possible as visceral

material can vary substantially in activity and enzyme levels. This may afford

FPH that may be acceptable for animal or fertilizer applications, but typically

not for food use (with the exception of fish sauce) where consistency of the final

FPH is of great importance. Another problem is that endogenous enzymes have

been reported to give significantly lower recoveries than commercial

preparations (Hale, 1972; Shahidi et al., 1995). Kristinsson and Rasco (2000b)

did, however, report similarly good protein recovery for an enzyme preparation

made from fish pyloric ceca compared to commercial enzymes. The advantage

of using visceral material as a source of enzymes is, however, lower processing

cost. Another advantage is that endogenous fish enzymes often have high

activities at low temperatures, in contrast to most commercial preparations

(Kristinsson and Rasco, 2000d). This allows the reaction to operate at very low

temperatures, which minimizes microbial growth as well as quality and

functionality problems with the final product.

Where controlled hydrolysis is required, commercial enzymes are added. A

range of different commercial enzymes are available. Enzymes have either

endo- or exopeptidase activities. The former pertains to enzymes cleaving

peptide bonds within the protein, resulting in many peptides but relatively few

free amino acids. Exo activity refers to an attack on either end of the protein

polypeptide chain, thus giving many free amino acids and few large peptides.

Aquatic food protein hydrolysates 231

The two different enzyme activity forms can produce FPH with markedly

different properties. If limited hydrolysis and large peptides are the desired end

product, more specific enzymes have certain advantages. On the other hand,

extensive hydrolysis requires the use of less specific enzymes, and typically a

range of different enzymes. Normally a combination of enzymes with endo- and

exopeptidase activity is used in commercial hydrolysis. Many different

commercial enzyme preparations have been reported in the production of

FPH. Acid proteases, such as pepsin, were early on believed to be the enzymes

of choice, in large part since the low pH stabilized the reaction mixture with

respect to microbial growth (Hale, 1969). The acid pH can, however, have a

detrimental effect on the quality (mainly color and oxidative rancidity) and

functionality of the FPH. In recent years, the use of enzymes working under pH

conditions close to neutrality has been more common. The milder pH values

give a more functional and higher quality FPH. Several successes have been

reported with the enzyme preparations Alcalase, Flavourzyme and Protamex

from Novo Nordisk (e.g., Quaglia and Orban, 1987; Sugiyama et al., 1991;

Diniz and Martin, 1996; Benjakul and Morrissey, 1997; Kristinsson and Rasco,

2000b; Theodore and Kristinsson, 2005). The neutral proteases Corolase PN-L

and 7089 have also been found to produce FPH of good recoveries and qualities

(Kristinsson and Rasco, 2000b).

After adding the enzyme, it acts very fast initially on the proteins in the

mixture, significantly reducing the viscosity of the slurry. As more peptide

bonds are broken, the slower the hydrolysis gets and eventually the reaction

reaches its maximum level of hydrolysis (Fig. 10.2). The extent of the hydrolysis

can be followed using different methods (Kristinsson and Rasco, 2000a). One of

the most convenient methods is to follow the degree of hydrolysis (%DH) using

the pH-stat procedure (Adler-Nissen, 1986). With this method the %DH is

calculated from the volume and molarity of base or acid used to maintain

constant pH. The method defines the degree of hydrolysis as percent ratio of the

numbers of peptide bonds broken (h) to the total numbers of bonds per unit

weight (htot; meq/kg protein, calculated from the amino acid composition of the

substrate):

%DH � (h/htot) � 100 (10.1)

%DH can also be expanded to:

%DH � (C � NB � 100)/(� � htot � MP) (10.2)

where C is the acid or base consumption in ml (depending on the pH the

hydrolysis is operated at; base if operating at neutral to alkaline pH; acid if

operating at low pH), NB is the normality of the base (or acid), � is the average

degree of dissociation of the �-NH groups (if operating at neutral to alkaline pH)

or COOH groups (if operating at low pH), and MP = mass of protein in grams

(%N � 6.25). This equation works well at neutral to alkaline pH values, but is

difficult to use at acidic pH values, where other more appropriate methods are

more useful (e.g., Nielsen et al., 2001). Essentially, the more the protein is being


broken down by the enzyme, the more the %DH increases (Fig. 10.2). Typically,

the higher the %DH the higher the protein recovery (Kristinsson and Rasco,

2000b).

The choice of pH and temperature for the reaction is highly dependent on

what the operator wants to accomplish. Working at the enzyme optimal

conditions will lead to very rapid and often extensive hydrolysis. Additionally,

working close to the enzyme optima may in some cases compromise the stability

of certain enzymes if the reaction is long. If the goal is limited hydrolysis, it is

recommended to operate away from the enzyme optima, or at significantly lower

enzyme concentrations, to be able to arrest the reaction more effectively at lower

degrees of hydrolysis.

The termination of the enzyme reaction is accomplished by irreversible

denaturation of the enzyme. This can be done by heating the slurry typically to

temperatures above 85ëC for at least 10 minutes (depending on enzyme).

Another effective method is to use a combination of pH and temperature induced

denaturation, e.g. reducing temperature to a very low pH in which case a milder

heat treatment can be used. The proteins are then recovered by either filtration or

centrifugation where they are separated from other components of the raw

material (e.g., unhydrolyzed proteins, fat, bones, skin, scales, etc.) after which

they are concentrated or dried. In some cases the FPH is concentrated or dried

directly, if the raw material was a relatively clean source of protein. In many

cases it may be necessary to add stabilizing agents, e.g. antioxidants or

antimicrobials, or even introduce a deodorizing treatment.

Fig. 10.2 An enzymatic hydrolysis curve for salmon muscle hydrolyzed withFlavourzyme 1000L at two different enzymatic activities (AzU = Azocoll Units).


10.3 Properties of fish protein hydrolysates

Fish proteins have not only good functionality but also have a high nutritional

value. One way to modify and improve the properties of fish proteins is to use

proteases to produce a range of FPH. Most of the research in the past 50 years or

so with FPH has concentrated on investigating and optimizing conditions to

produce FPH of high protein yields as well as unraveling food functionality of

FPH (Kristinsson and Rasco, 2002). These studies have demonstrated that the

size and chemical properties of the peptides in FPH have a dramatic impact on

their function. Therefore, good control of enzyme specificity and degree of

hydrolysis (%DH) is critical if the goal is to produce hydrolysates with proper-

ties suitable for a wide array of products (Mullally et al., 1994; Kristinsson and

Rasco, 2000b). Recently interest in FPH has shifted more to their potential

bioactive properties rather than food functionality. Recent data on protein

hydrolysates (including FPH) has demonstrated that they may provide

significant physiological benefits to living systems. Some of the identified

bioactive properties may also find applications in stabilizing food products, e.g.

against lipid oxidation. However, very limited knowledge exists on the

molecular mechanisms behind these activities.

10.4 Role in food systems

Fish protein hydrolysates have been extensively investigated for their food

functionality and may be used in numerous applications in food products, as

summarized by Kristinsson and Rasco (2002). Food systems can be used as

vehicles for the consumption of bioactive FPH, but FPH can also be used to

improve function and quality of foods. Fish protein hydrolysates differ from

intact myofibrillar proteins in being readily soluble at a range of ionic strength

and pH (Shahidi et al., 1995; Vieira et al., 1995; Kristinsson and Rasco, 2000b),

while intact myofibrillar proteins have only high solubility at high and low pH

values (Fig. 10.3). The solubility reported for FPH has typically been between

90 and 100%. This is because upon hydrolysis the proteins are broken down into

many smaller units with effectively more exposed amino and carboxyl groups

than the parent protein, thus enabling more protein-water interactions

(Mahmoud et al., 1992). This is an advantage, as they can effectively be added

to food systems under very different conditions. The soluble nature of FPH has

sparked interest in its use as an injectable or mixable protein material to improve

water-binding in seafood products. Kristinsson and Rasco (2000b) found that

adding FPH from salmon to minced salmon muscle led to less drip loss on

thawing compared to adding no FPH. Studies by Varetzis et al. (1990) and

Shahidi et al. (1995) demonstrated a higher cook yield when FPH was added to

hamburgers and minced pork, respectively. Work in the author's laboratory has

shown that fish fillets can be tumbled or injected with solutions containing FPH

to increase product yield significantly on cooking, equal or better than injection/

tumbling fillets in phosphates (Fig. 10.4). The mechanism behind this functional


Fig. 10.3 Protein solubility of fish protein hydrolysate (salmon) compared to fishmuscle proteins (ground catfish muscle) as a function of pH.

Fig. 10.4 Weight increase and cook loss of catfish fillets injected with differentsolutions; NaCl, catfish FPH and salt, salt and phosphate, catfish FPH, salt and phosphate.The grey bars show the weight increase after injection. The white bars show weight lossafter cooking, calculated based on the original pre-injected weight of the fillets. For

example, a positive value refers to fillets that have a higher weight than the fillets hadbefore injection, while a negative value refers to fillets that has a weight below the pre-

injected weight of the fillets after cooking.


property of FPH is not well understood, but FPH could be a valuable natural

substitute for chemicals such as phosphates.

Work with FPH has demonstrated that they may possess cryoprotective

properties. Khan and coworkers (2003) reported that surimi containing FPH had

better gel-forming ability than surimi with no FPH added. Surimi with FPH also

demonstrated higher residual ATPase activity, which is a sign that it had a

protective effect on myosin during freezing and thawing. Several studies have

also demonstrated that FPH may protect proteins on drying (Zhang et al., 2002;

Hossain et al., 2003; Khan et al., 2003).

One problem during the production of FPH is that bitterness can develop.

This may greatly limit their food use. Furthermore, if FPH are produced from a

raw material that is susceptible to oxidation, rancid flavors may be associated

with the final product. Liu et al. (2000) demonstrated a good correlation between

the level of lipid oxidation and development of bitterness during autolytic

hydrolysis of frigate mackerel. In addition, Hoyle and Merritt (1994) showed

that defatted raw materials lead to less bitterness in the final isolate. Preventing

bitterness in FPH is a difficult task which requires extensive experimentation,

since certain functional properties may be compromised when the goal is to

produce a peptide profile of minimal bitterness. The cause of bitterness is

complicated and not well known. A high proportion of hydrophobic peptides in

hydrolysates may in part account for increased bitterness (Tamura et al., 1990).

There seems to be a sensitive balance between %DH and the extent of bitterness,

and results vary in the literature. In general, it appears that limited and extensive

hydrolysis leads to less bitterness rather than intermediate hydrolysis (Sugiyama

et al., 1991; Yu and Fazidah, 1994; Vieira et al., 1995; Kristinsson and Rasco,

2000a). A number of studies have also revealed that not only the degree of

hydrolysis but also the type of enzyme used can have a dramatic influence on the

level of bitterness (Sugiyama et al., 1991; Hoyle and Merritt, 1994). Several

enzyme preparations are now commercially available with the goal to greatly

limit the development of bitterness and at the same time obtain good functional

properties, e.g. Flavourzyme and Protamex from Novo Nordisk (Bagsvaerd,

Denmark). Certain chemical treatments can also reduce bitterness, such as

passing the FPH through activated charcoal (Shahidi et al., 1995), extraction

with chemical solvents (Lalasidis et al., 1978; Chakrabarti, 1983), or applying a

second hydrolysis step in the FPH with exopeptidases (Lalasidis et al., 1978;

Sugiyama et al., 1991). Fish protein hydrolysates which have been extensively

hydrolyzed or have a high proportion of small peptides (primarily di or

tripeptides) have been reported to have flavor enhancement effects similar to

MSG (Fujimaki et al., 1973; Noguchi et al., 1975; In, 1990; Imm and Lee,

1999). In fact several commercial seafood flavors made of or with FPH are on

the market and are used for a variety of different products, such as soups, sauces,

value-added seafood products, imitation seafood products, snacks and

seasonings.

Fish protein hydrolysates have been found to have excellent interfacial/

surface properties (Liceaga-Gesualdo and Li-Chan, 1999; Kristinsson and


Rasco, 2000b; Jeon et al., 1999) and thus may have potential use as emulsifying

and emulsion stabilizing ingredients in a variety of products (e.g., dressing,

margarine and meat batters) as well as aid in the formation and stability of foam-

based products (e.g., whipped cream, meringues and mousse). There are,

however, also studies reporting relatively poor interfacial/surface properties of

FPH (Miller and Groninger, 1976; Shahidi et al., 1995; Vieira et al., 1995;

Onodenalore and Shahidi, 1996; Sathivel et al., 2003). Many of those studies,

however, have not specified the level of hydrolysis which makes it very difficult

to interpret the data. The level of hydrolysis, i.e. peptide size, is very important

for interfacial/surface activity of the FPH (Jeon et al., 1999). Several studies

have shown that hydrolyzed fish muscle has an increased emulsification

formation and stability compared to unhydrolyzed muscle (Spinelli et al., 1972;

Liceaga-Gesualdo and Li-Chan, 1999). Limited hydrolysis (larger peptides)

generally leads to improved emulsification and foaming properties of fish

proteins, while extensive hydrolysis (small peptides) reduces these properties

(Quaglia and Orban, 1990; Liceaga-Gesualdo and Li-Chan, 1999; Kristinsson

and Rasco, 2000b; Jeon et al., 1999). Very small peptides don't have the ability

to form a good stable cohesive protein network around oil droplets or air

pockets. There is also evidence that as %DH increases (i.e. higher level of small

peptides), FPH exhibits less oil binding (Kristinsson and Rasco, 2000b). Enzyme

specificity is very important for interfacial/surface activity of FPH, since

different peptides are produced from different enzymes. Kristinsson and Rasco

(2000b) reported that different enzymes used to produce salmon FPH gave

different emulsification capacity and stability even at the same %DH.

Studies on a variety of different food proteins have shown that protein

hydrolysates have antioxidative properties, and as such may play a role in both

food and physiological systems in controlling peroxidative damage. Certain

peptides and amino acids, which can be found in muscle foods, have been found

to have excellent antioxidative properties (Karel et al., 1966; Chan and Decker,

1994). The action of these peptides and amino acids has been a subject of much

investigation lately. Some postulated mechanisms are that they can scavenge

radicals formed during peroxidation and chelate transition metals, which are

potent prooxidants. Fish protein hydrolysates, like other hydrolysates, are rich in

peptides and several studies have demonstrated their potential as a food

antioxidant. Shahidi and coworkers have performed a number of studies on the

antioxidative properties of FPH. Shahidi et al. (1995) reported that FPH made

from capelin effectively reduced lipid oxidation when added to ground pork by

up to 60.4%, as assessed by measuring thiobarbituric reactive substances

(TBARS). Amarowicz and Shahidi (1997) fractionated capelin FPH into four

fractions with gel filtration column chromatography, and found that different

fractions had different activities, some being prooxidative while others were

antioxidative. The fraction with the highest molecular weight was found to be

most effective. Other work has also demonstrated that larger peptides rather than

very small peptides are more effective. Work presented by Wu and coworkers

(2003) showed that intermediate peptides (1±1.5 kDa) more effectively


increased the lag time before oxidation when added to a linoleic acid emulsion

peroxidation system, compared to small molecular weight peptides. It was

proposed that this was due to the reducing ability of FPH as well as its ability to

chelate metals. Jeon et al. (1999) also demonstrated that different FPH fractions

representing different molecular weight ranges have different antioxidative

activities. It was reported that fractions below 5 kDa most effectively reduced

peroxidation of linoleic acid, and were as good as tocopherol. An extensive

study by Theodore and Kristinsson (Theodore, 2005; Theodore and Kristinsson,

2005) on the potential antioxidative mechanisms of FPH made from isolated

catfish muscle proteins at different %DH, demonstrated that the mechanism is

far from being a simple one. Both the whole FPH and the soluble fractions of

FPH were investigated for various different antioxidative tests. Both systems

were found to have high radical scavenging activity (as tested by two methods),

which increased as %DH increased (Fig. 10.5). Both systems were also found to

have good metal chelation ability, again higher as the %DH increased. In

addition, both systems exhibited reducing power, but in contrast to the other

results, the reducing power decreased as %DH increased. Interestingly, however,

when hydrolysates were tested in a linoleic acid peroxidation system they did

not display any antioxidative effect. The soluble fractions of the hydrolysates

(i.e. having smaller peptides), however, did display a strong antioxidant activity.

Many of the above studies employ systems where metal ions are the pro-

oxidants. Other prooxidants, such as heme proteins and lipoxygenases, are

present in muscle foods and work via different mechanisms than transition

metals. Chuang and coworkers (2000) demonstrated that FPH (from mackerel)

Fig. 10.5 The antioxidative activity (Trolox equivalence) of catfish protein hydrolysatesat different degrees of hydrolysis as assessed by the oxygen radical absorbance capacity

(ORAC) method (adapted from Theodore and Kristinsson, 2005). Control refers tounhydrolyzed catfish proteins.


had the ability to decrease hemoglobin-mediated lipid oxidation in a linoleic

acid system. Although no direct evidence was provided, the authors believed

that this was possibly due to dipeptides containing histidine, such as anserine

and carnosine, which are known to scavenge free radicals. Work done in the

authors' laboratory shows that FPH (channel catfish) and fractions of FPH may

have an antioxidative effect in a model fish muscle matrix system with hemo-

globin as the added prooxidant. There is also a possibility that the antioxidative

activity displayed by the FPH in the various studies presented above, is not

necessarily due to peptides formed during the hydrolysis process. There are a

number of compounds in fish muscle which display good antioxidative effects

which would be present in the FPH preparations. Undeland and coworkers

(2003) have demonstrated that the `press juice' of fish muscle (i.e. the aqueous

fraction of muscle after extensive ultracentrifugation) has strong antioxidative

activity on hemoglobin-mediated oxidation in a washed fish muscle matrix.

Work in the authors' laboratory has demonstrated that the soluble fraction of

catfish protein isolates has in some cases equal or better antioxidative activities

that FPH from the isolate, suggesting the action of small molecular weight

compounds, yet to be identified.

10.5 Physiological role in humans and animals

Much research in the past 50 years or so has shown that protein hydrolysates

have an excellent amino acid balance, good digestibility, rapid uptake and the

presence of certain bioactive peptide components. Hydrolyzed fish material is

widely used as feed for a variety of farmed animals as well as cultured food fish

(Dong et al., 1993; Gildberg, 1993; Kristinsson and Rasco, 2000a). Many

studies have demonstrated that FPH have excellent protein quality and are

readily utilized (Atia and Shekib, 1992; Sugiyama et al., 1991; Diniz and

Martin, 1996; Liaset et al., 2000; Abdul-Hamid et al., 2002; Bechtel et al.,

2003). Studies with laboratory rats show that adding FPH to rat diets leads to

more rapid growth and higher body weight compared to rats fed casein (Kienkas,

1974; Ballester et al., 1977; Atia and Shekib, 1992). Fish protein hydrolysates

present great promise for the aquaculture sector. Barrias and Olivia-Teles (2000)

pointed out that FPH containing fish diets lead to a better nitrogen retention and

have better feed conversion ratio than most commercial fishmeal feeds. Several

studies have also demonstrated that FPH may be particularly good in the early

stages of fish growth, as they increase the survival rate of young trout and

salmon (Lian and Lee, 2003; Gildberg, 2003). Refstie and coworkers (2004)

conducted an extensive study on post-smolt Atlantic salmon, by comparing the

inclusion of 0, 5, 10 and 15% FPH in fishmeal fed to salmon. It was found that

the higher the FPH inclusion the higher the feed consumption (FPH appeared to

act as a feeding stimulant) and the higher the growth. Fish protein hydrolysates

were also found to lead to increased protein retention and digestibility. Studies

by Gildberg and coworkers (Gildberg et al., 1996; Bogwald et al., 1996;


Gildberg, 2003) have shown that FPH may make fish more disease resistant and

may stimulate the immune system of fish. It should, however, also be pointed

out that several studies have shown no or a negative effect of feeding aquatic

species with FPH. In one study fishmeal was replaced by FPH which did not

lead to an increased growth or feed utilization of juvenile turbot (Olivia-Teles et

al., 2000). Fish protein hydrolysates from krill led to lower growth when fed to

shrimp, possibly due to an excessive amount of low-molecular-weight peptides

which may lead to an imbalance of amino acid absorption (Cordova-Murueta

and Garcia-Carreno, 2002).

The positive effects FPH may have on animals suggest that they may have

positive effects on humans as well. Just as FPH stimulates the immune system in

fish, peptides in fish sauce (essentially a version of liquid FPH) also stimulated

the proliferation of white blood cells in human subjects (Thongthai and

Gildberg, 2004). Fish protein hydrolysates have also been found to have a blood

thinning effect, i.e. increase flow of red blood cells (Chuang et al., 2000). A

small protein or protein fragment with strong anticoagulant and antiplatelet

properties has recently been isolated from yellowfin sole FPH (Rajapakse et al.,

2005). A recent study demonstrated that a commercial preparation of FPH from

Pacific whiting shows promise in improving the health and function of the

digestive system (Fitzgerald et al., 2005). When cultured rat epithelia intestinal

and colonic cells were given FPH, cell growth was increased and injury was

reduced significantly. It was also found that most of the active components,

glutamine containing di- and tripeptides, were soluble in ethanol and not water.

One of the most interesting effects FPH have on humans is their effect on the

neurological system. Studies in France have shown that FPH from cod and

mackerel may reduce anxiety as well as improve memory and learning in

humans (Dorman et al., 1995; Le Poncin, 1996a,b). Bernet et al. (2000) reported

that FPH administered to rats reduced their level of stress, in a similar fashion as

valium does. Just as FPH have been found to have antioxidative effects in vitro

and in food systems, they have been found to reduce peroxidation in vivo.

Boukortt et al. (2004) reported that overall antioxidant status was increased by

35% in hypertensive rats when they were fed fish protein hydrolysates,

compared to feeding them casein. Chuang et al. (2000) demonstrated that FPH

inhibited the activity of lipoxygenase, which is an enzyme implicated in low

density lipoprotein oxidation.

Recently significant interest has developed in the potential use of FPH to

reduce blood pressure. A number of studies show that FPH made from different

species have the ability to inactivate the angiotensin I converting enzyme (ACE)

(Kohama et al., 1991; Ukeda et al., 1992; Matsumura et al., 1993; Wako et al.,

1996). The compound angiotensin I is converted by ACE to angiotensin II which

has been connected to hypertension. Bordenave et al. (2002) found that FPH

from sardines and cod were able to inhibit almost 30% of ACE activity and

shrimp FPH inhibited 57% of ACE activity. Theodore and Kristinsson (2005)

found that FPH made from isolated catfish muscle proteins was able to inhibit

ACE activity in vitro by close to 90%. Interestingly, the intact fish proteins (i.e.


unhydrolyzed) also exhibited high ACE inactivation activity. Intact proteins

would, however, not play a role directly in ACE regulation, as they would be

hydrolyzed in the digestive system. Some studies have shown that the ACE

inhibition is sensitive to peptide size. The ability of cod FPH fractions to reduce

ACE activity was in the following order 3 kDa > 5 kDa > 10 kDa > 30 kDa,

suggesting small molecular weight peptides are more effective. Jung et al.

(2004) also found that lower molecular weight fractions (<5 kDa) of yellowfin

sole FPH more effectively inhibited ACE than high-molecular-weight fractions.

There is also evidence that FPH may play a role in regulating blood pressure in

vivo. A 9% drop in blood pressure was recorded when rats were fed feed con-

taining FPH compared to casein. This was believed to be due to the high

concentrations of cysteine, methionine and arginine in FPH, all known to have

an effect on hypertension. When hypertensive rats were fed a 300mg dose of

purified peptides from sea bream scale hydrolysates (i.e. collagen hydrolysates),

their blood pressure dropped significantly (Fahmi et al., 2004). These peptides

were found to have higher ACE inhibitory activity in vitro than the commercial

hypertension drug enalapril maleate. Related to cardiac disease, it has also been

demonstrated that FPH from salmon frames administered to rats reduced total

cholesterol and increased HDL cholesterol (Wergedahl et al., 2004).

In light of the many potential benefits FPH may provide to humans, it would

be useful if commercial products were available with functional and active

FPH. However, very few commercial products contain added FPH, for many

different reasons. Significant strides have been made to incorporate FPH into

the human diet, with mixed results. As has been mentioned before, seafood

flavors, many of which are essentially FPH, are available. It has been suggested

that to improve the protein quality of cereal products, they could be mixed with

FPH (Yanez et al., 1976; Morales de Leon et al., 1990). Enriching legume

products with FPH has also been reported, and it was found that the majority of

sensory panelists responded favorably to FPH supplemented products (Morales

de Leon et al., 1990). Some reports have shown that FPH can be successfully

incorporated into bakery products. Chevalier and Noel (1982) patented a biscuit

(a type of ènergy bar') which was made with dried skim milk (15%) and fish

protein hydrolysate (14%). Yu and Tan (1990) researched the incorporation of

tilapia FPH into crackers, which were later fried. The crackers were found to be

`highly acceptable' by panelists even at a 10% FPH addition level. It must be

kept in mind, however, that the acceptance of products containing FPH will

likely be greatly influenced by the culture and ethnic backgrounds the panelists

represent.

10.6 Role in plant growth and propagation

Humans have for a long time recognized the benefits of fish and fish waste as a

fertilizer for crops. The amino acid and peptide profile of FPH makes it an

excellent source of nitrogen for plants, and it is readily adsorbed and utilized.


Some commercial applications include fertilizer for cranberry and cherry trees

(George Pigott, personal communication, 1997), golf courses (Stephen D.

Kelleher, personal communication, 1999) and house plants. Shetty and co-

workers (Eguchi et al., 1997; Milazzo et al., 1999) have demonstrated that FPH

can have an effect beyond just basic nutrition for plants. It was reported that

FPH is able to stimulate somatic embryogenesis in anise (Pimpinella anisum)

better than proline, and has the potential to become a proline and amino acid

substitute for propagating plants. It has also been demonstrated that FPH can

stimulate plants to express high levels of bioactive compounds. Andarwulan and

Shetty (1999, 2000) found that FPH from mackerel stimulated the production of

phenolic compounds and rosemaric acid in oregano and phenolic compounds in

anise root cultures. Vattem and Shetty (2002) later reported that FPH increased

the level of phenolics in cranberry pomace. The presence of high levels of

glutamic acid and proline in the FPH is thought to be a possible reason for its

ability to stimulate the production of phenolic compounds (Andarwulan and

Shetty, 1999). Fish protein hydrolysates can therefore find a niche market where

they are used to express the production of potentially very valuable plant

compounds.

10.7 Role as growth media for microorganisms

An often overlooked application of FPH is as a specialty growth media for

microorganisms. Extensively hydrolyzed FPH are an excellent growth media for

a variety of microorganisms (Gildberg et al., 1989; de la Broise et al., 1998;

Gildberg 2003). It was reported that FPH was able to stimulate the growth of

lactic acid bacteria in skim milk (Yugushi, 1984). Fish protein hydrolysates have

reportedly performed better than commercial peptones, and have been a

successful media in cultivating fish pathogens (Gildberg, 2003). A very recent

study compared culture media with FPH from hake by-product to conventional

culture media and found that the FPH media was as good as a substrate to

support the growth of a variety of microorganisms (Martone et al., 2005).

10.8 Future trends

For many decades a number of devoted researchers and developers have worked

tirelessly to find ways to better utilize our limited aquatic resources. The

production and utilization of fish protein hydrolysates has come a long way, and

has received a surge of interest in recent years after a period of relatively slow

development. Future applications of FPH are now more likely to lie in specialty

products and markets, and not so much in the food functionality ingredient

market. Recent evidence shows that FPH have some unique properties such as

antioxidative activity, and as such may find use as a natural ingredient in several

food systems (likely seafood based) to improve product quality and shelf life.


The physiological benefits demonstrated in various studies suggest FPH may

find applications as a nutritional supplement, and products containing dried fish

proteins and FPH are now found on the market with various health claims which

require further investigation. Recent developments in the extraction and

isolation of proteins from by-products or underutilized species further increase

the possibility that very high grade and consistent FPH can be produced, since

the starting material would consist mainly of protein. The future of FPH is bright

and as more information is generated on its unique properties and how to

engineer FPH with specific unique properties it is likely that FPH will find a

strong market as an ingredient and supplement in the future.

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11.1 Introduction

In Japan and many other Asian countries there is much interest in marine-

derived ingredients; however, there is some demand for marine-derived func-

tional ingredients in North America. The projected functional food market for

North American is anticipated to grow by more than 20% per year for the next

several years (Barrow, 2005). In recent years, by-products from fishing

industries have been used as raw materials to produce some common bioactive

supplies for supplements or functional ingredients. The functional food market

in North American is currently valued at more than US$30 billion; however,

marine-derived ingredients account for only a small portion of this total. Marine

by-products that have potential as functional food ingredients include protein

hydrolysates from fish processing waste and underutilized fish species (Barrow,

2005).

Large amounts of protein-rich by-products from the seafood industry are

discarded or processed into fish meal. Novel processing methods are needed to

convert seafood by-commodities into marketable products. Many of these

protein-rich seafood by-products have a range of dynamic properties (Phillips et

al., 1994) and can potentially be used in foods as binders and emulsifiers. Soy

and milk proteins are widely used in many segments of the food industry, while

amino acids and peptides are gaining popularity for use in energy drinks and

other applications (O'Donnell and Dornblaser, 2002).

Proteins from fish processing by-products can be modified to improve their

quality and functional characteristics using enzymatic hydrolysis (Shahidi,

11

Engineering and functional properties ofpowders from underutilized marine fishand seafood productsS. Sathivel and P. J. Bechtel, University of Alaska Fairbanks, USA

1994). Utilizing proteolytic enzymes, fish protein hydrolysates (FPHs) can be

prepared with peptides having new and/or improved properties. Functional

properties can be defined as physical and chemical properties, which affect the

behavior of proteins in food systems during processing, storage, preparation, and

consumption (Kinsella, 1976). Functional properties of fish proteins are related

to their physical, chemical, and conformational properties. This chapter will

discuss engineering and functional properties of proteins and hydrolysates made

from marine fish and their by-products.

11.2 Fish protein powder as bio-active ingredients

A wide range of food products including protein supplements, infant formula,

formulas for the elderly and beverages contain protein hydrolysates as stabilizers

(Frokjaer, 1994; Kristinsson and Rasco, 2000). Some fish protein and protein

hydrolysates in addition to having good functional properties may also have

specific health benefits, including the ability to lower blood pressure, reduce the

risk of type-II diabetes (McCarty, 2003), and improve glucose tolerance and

insulin sensitivity (Lavigne et al., 2000). In Japan, sardine protein hydrolysate is

widely available in supplements. Sardine protein hydrolysate incorporated at a

dosage of 0.5 g into a vegetable drink was shown to lower systolic blood

pressure (Kawasaki et al., 2002). Protein hydrolysates derived from non-marine

food products such as soy and whey reduce blood pressure; however, marine-

derived hydrolysates may be effective at lower doses.

Hydrolysates from fish can be used in a number of food applications such as

enhancing water binding, and hydrolysates made from caplin and peptide were

shown by Shahidi et al. (1995) to inhibit oxidation, and peptides derived from

fish can also been shown to have antioxidant properties (Amarowicz and

Shahidi, 1997; Shahidi and Amarowicz, 1996). Antioxidants such as butylated

hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been

commonly used by the food industry to improve product quality during storage,

resulting in increased product shelf life. To meet consumer demands for safer

foods, numerous studies are currently focused on using natural ingredients to

enhance food quality and shelf life in order to avoid the use of synthetic

preservatives. Sathivel et al. (2003) reported that fish protein hydrolysates had

antioxidant properties. Arrowtooth flounder protein that was partially

hydrolyzed by endogenous enzymes was used to coat salmon fillets and

resulted in reduced lipid oxidation when compared to non-coated controls

(Sathivel, 2005).

11.3 Functional properties of fish protein powders

Amino acids and peptides are finding more uses in energy drinks and other

applications (O'Donnell and Dornblaser, 2002), and soy and milk protein, unlike


fish protein, are widely used in many segments of the food industry. Fish by-

products from coldwater marine species are good sources of high quality

proteins and there is an opportunity to use more fish processing by-products as

protein sources for food and feed ingredients and industrial applications. In

1993, the Association of Danish Fish Processing Industries and Exporters

commercially produced fish-based protein powders for use in frozen products to

enhance water binding and stability properties when frozen (Urch, 2001).

Phillips et al. (1994) reported that protein-rich seafood by-products had a range

of dynamic properties and could potentially be used in foods as binders and

emulsifiers. Sathivel et al. (2004) reported that protein powders from herring

and arrowtooth flounder were good sources of high quality fish protein with

many desirable functional properties.

Solubility is one of the most important properties of proteins (Kinsella, 1976;

Mahmoud et al., 1992). The nitrogen solubility values of hydrolysates from red

salmon ranged from 17.2% to 54.4% (Sathivel et al., 2005b) and solubility of the

soluble fraction of herring hydrolysates was 84.9% (Sathivel et al., 2003). High

solubility of fish protein hydrolysates is often due to cleavage of proteins into

smaller peptide units that usually have increased solubility (Shahidi, 1994),

which is mainly due to a reduction in the molecular weight and an increase in the

number of polar groups. Increased solubility is not only due to smaller peptide

size but also to the balance of hydrophilic and hydrophobic elements in the

peptides.

An emulsion is defined as a heterogeneous system consisting of at least two

immiscible liquid phases, one of which is dispersed in the other in the form of

droplets (Das and Kinsella, 1990). Gauthier et al. (1993) and Jost et al. (1977)

examined the role of peptide characteristics on emulsification properties. They

reported that hydrophobicity and peptide lengths influenced the emulsifying

properties. Peptides often have reduced emulsifying properties (Chobert et al.,

1988). A positive correlation between surface activity and peptide length was

reported by Jost et al. (1977), and Lee et al. (1987) reported that peptides should

have a minimum length of 20 residues to possess good emulsifying and inter-

facial properties. Sathivel et al. (2003) reported that emulsion stability of protein

powders was similar to a soy protein concentrate. Emulsifying stability of red

salmon head hydrolysates ranged from 66.9±100% (Sathivel et al., 2005b).

Emulsifying stability values of 52 to 61.0% and 48.5 to 54.2% were reported for

Atlantic salmon protein hydrolysates (Kristinsson and Rasco, 2000) and for

herring protein hydrolysates (Sathivel et al., 2003), respectively.

11.4 Flow properties analysis of emulsion containing fishprotein powders

Proteins extracted from fish are good sources of high quality proteins, have a

range of dynamic properties, and can potentially be used in foods as emulsifiers

(Sathivel et al., 2004). Sathivel et al. (2005a) reported that proteins extracted

Engineering and functional properties of powders from seafood products 251

from arrowtooth flounder provide desirable emulsifying properties in the

emulsion system that exhibits pseudoplastic and viscoelastic characteristics. It

may be possible to substitute egg yolk with fish protein powders in an oil-in-

water emulsion system.

The power law (Eq. 11.1) and the Casson equation (Eq. 11.2) can be used to

analyze the flow properties of the emulsion containing fish protein.

� � K n �11:1�where � � shear stress (Pa.s), � shear rate (sÿ1), K � consistency index

(Pa.sn), and n � flow behavior index. The logarithms are taken on both sides of

Equation 11.1, and a plot of log � versus log is constructed, and the magnitude

of K and n are determined from the resulting straight line intercepts for log K

and the slope for n values.

�0:5 � �o � K1 0:5 �11:2�

where K = constant and �o � yield stress (Pa). The square root of shear stress is

plotted against the square root of shear rate and �o and K1 are obtained from the

square of the intercept and the slope of the straight line, respectively.

The flow parameters of the flow index (n), consistency index (K), and the

Casson yield stress values of emulsion containing arrowtooth protein powders

are shown in Table 11.1. The flow index values for all mayonnaise samples were

less than 1.0, which indicated that they were pseudoplastic fluids (Paredes et al.,

1989). Values for n of 0.13 to 0.91 have been reported for some commercial

mayonnaises and model mayonnaise systems (Dickie and Kokini, 1983; Steffe,

1992). Higher K values of emulsion samples indicate a more viscous consistency

(Paredes et al., 1989). Values obtained for emulsions made with arrowtooth

flounder protein powder were within the range of yield stress values (9 to 91 Pa)

that Steffe (1992) reported for commercial mayonnaises. The wide range of n, K,

and �o values reported by Dickie and Kokini (1983) and Steffe (1992) was due

in part to different methods or use of different shear rate ranges.

Table 11.1 Flow parameters of mayonnaises containing fish protein powders

Samples n K (Pa.s) �o (Pa)

AFSPE1 0.5 � 0.0 5.6 � 0.8 40.5 � 1.0AFSPE2 0.6 � 0.02 4.2 � 0.4 30.5 � 1.8AFISPE3 0.9 � 0.02 0.2 � 0.01 0.7 � 0.3AFISPE4 0.8 � 0.01 0.3 � 0.4 1.6 � 0.1

n � flow index; K � consistency index; �o � yield stress.AFSPE1 contained 5% AFSP, 1% lemon juice, and 60.35% soybean oil.AFSPE2 contained 5% AFSP, 3% lemon juice, and 58.35% soybean oil.AFISPE3 contained 5% AFISP, 1% lemon juice, and 60.35% soybean oil.AFISPE4 contained 5% AFISP, 3% lemon juice, and 58.35% soybean oil.AFSP � arrowtooth flounder soluble protein powder: AFISP� arrowtooth flounder insoluble proteinpower. Data from Sathivel et al. (2005a).


11.5 Viscoelastic properties of emulsions containing fishprotein powders

Dynamic rheological tests can be used to characterize viscoelastic properties of

emulsions. Equations (11.3) and (11.4) can be used to define viscoelastic behavior:

G0 � �o o

� �cos � �11:3�

G00 � �o o

� �sin � �11:4�

where G0 is the storage modulus, G00 (Pa) is the loss modulus, and tan � is the losstangent. The storage modulus, G0, characterizes the rigidity of the sample and can

be viewed as the magnitude of the energy that is stored in the material per cycle of

deformation. The loss modulus, G00, characterizes the resistance of the sample to

flow, and is a measure of the energy that is lost through viscous dissipation per

cycle of deformation. For a perfectly elastic solid, all the energy is stored; that is,

G00 is zero and the stress and the strain will be in phase. In contrast, for a liquid

with no elastic properties, all the energy is dissipated as heat; that isG0 is zero andthe stress and strain will be out of phase by 90ë (Rao, 1999).

Dynamic rheological tests can be used to characterize viscoelastic properties of

emulsions. The G0 and G00 of the emulsion samples containing arrowtooth flounder

soluble protein powder and arrowtooth flounder insoluble protein powder were

determined as a function of frequency (!) at a fixed temperature of 25ëC (Fig. 11.1).

Fig. 11.1 Rheology properties of emulsions containing arrowtooth flounder proteinpowers. G0 (Pa) and G00 (Pa) indicate storage and loss modulus, respectively. Panel acontained arrowtooth flounder soluble protein powder (AFSPE1); panel b containedinsoluble arrowtooth protein powder (AFISPE4). Data from Sathivel et al. (2005b).


Emulsion containing arrowtooth flounder soluble protein powder showed a gradual

increase in both the loss modulus and the storage modulus and with increasing

frequency. For emulsion made with arrowtooth flounder insoluble protein powder

G0 crosses G00 as frequency increased. This behavior is typical of macromolecular

solution where the polymer molecules are mutually entangled. At low frequency

increases there is sufficient time for the entanglements to make and break. A high

viscosity occurred in the emulsion system due to entanglement but there are no

intermolecular cross-linkages (Oakenfull et al., 1997). Emulsions made with

arrowtooth flounder soluble protein powder had higher G0 than G00, which indicate aviscoelastic material with both G0 and G00 being independent of frequency. The

higher G0 and G00 values of emulsion made with arrowtooth flounder soluble protein

when compared to those made with arrowtooth flounder insoluble protein may be

due to the soluble protein content that increases interactions between the

neighboring droplets.

11.6 Thermal properties of fish protein powders

During extraction and preparation process, fish protein powders are subjected to

temperature changes, which may alter their physical state. The most commonly

occurring phase transition in protein is denaturation, which can alter the

properties of protein and thus the quality of final products. DSC thermograms of

the fish protein powder samples are shown in Fig. 11.2. The DSC thermogram

for (herring body protein powder) HBP and WHP (whole herring protein

powder) show a single small endothermic transition with total enthalpy, �Ht

Fig. 11.2 DSC thermograms of fish protein powders. Data from Sathivel et al. (2004).


values of 1.58 and 0.76 j/g. HGP (herring gonad protein powder), HHP (herring

head protein powder), and APP (� arrowtooth flounder protein powder) did not

show the sharp peaks. Although the results indicate a small transition in HBP

and WHP, all protein powders were subjected to a high degree of denaturation

that may have occurred during fish protein powder preparation.

Knowledge of thermal decomposition of the fish protein samples can be used

to improve their stability and functional properties. A thermal gravimetric (TG)

analyzer is a balance, which measures changes in weight as a function of

changing temperatures. A series of the TG thermogram of the fish protein

powder samples is shown in Fig. 11.3. Weight loss of FPP samples increased

with increasing heating temperatures between 0 and 600ëC and the mass losses

were slightly different among the protein powders. Four weight-loss temperature

regions were identified for a series of protein powders made from herring and

herring by-products.

The TG curves indicated the thermal stability in the following order: HGP >

HBP > WHP > HHP > AFP. Differences in thermal stability may be due to the

presence of components that interact with protein in powders such as phospho-

lipids, complexed metals (notably iron, calcium, and magnesium minerals), free

fatty acids, and peroxides and their breakdown products. The presence of those

components reduces the effectiveness of heat transfer to protein powders, and

thus the mass losses of protein powders.

Fig. 11.3 TG thermograms showing weight loss curve for fish protein powders. WHP =whole herring protein powder; HBP = herring body protein powder; HHP = herring headprotein powder; HGP = herring gonad protein powder; AFP = arrowtooth flounder protein

powder. Data from Sathivel et al. (2004).


11.7 Future trends

The functional food market for North America has been projected to grow by

more than 20% per year over the next several years. Protein powders and

hydrolysates made from fillets of underutilized species such as arrowtooth

flounder, or protein derived from fish processing by-products have good func-

tional properties that can be used as ingredients in both animal and human foods.

Protein hydrolysates and protein powder derived from marine fish can expect to

get into the North American functional food market in the next decade. In

addition to fish protein the small amounts of marine oils found in most of these

products are high in omega-3 fatty acids. The public is continuing to gain a

positive nutritional image of products containing the long chain omega-3 fatty

acids. However, to enhance utilization of fish protein hydrolysates and protein

powders, progress in processing and formulation will be needed to enhance

sensory, functional and nutritional properties and consumer education and

awareness will also be addressed.

11.8 References

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functional properties of peptides obtained from whey protein', J Dairy Sci, 76,

321±328.

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MATSUMOTO K (2002), Àntihypertensive effect and safety evaluation of vegetable

drink with peptides derived from sardine protein hydroysates on mild hypertensive,

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proteases', J Agric Food Chem, 48, 657±666.

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12.1 Introduction

Marine oils originate primarily from the body of fatty fish, the liver of white lean

fish, and the blubber of marine mammals such as seal. The main sources of fish

oils are pelagic species caught in large quantities, particularly those with oily

flesh, such as salmon, tuna, mackerel and herring or small fish such as anchovies

and capelin. The oily flesh is often used for the purpose of fish meal and oil

production, but fish oil can also be produced from offal from the fish processing

industry since there is a sizable and growing world market demand for high-

quality fish oils. Thus, commercial fish oil production can be quite profitable if

suitable raw materials are available (Bonnet et al., 1974).

Marine oils provide for excellent sources of long-chain omega-3 fatty acids

(Table 12.1). Both omega-3 and omega-6 fatty acids are essential poly-

unsaturated fatty acids (PUFA) that cannot be made in the human body (Din et

al., 2004). The Western diet is abundant in omega-6 fatty acids, mainly from

vegetable oils rich in linolenic acid (C18:2n-6). However, humans lack the

necessary enzymes to convert omega-6 fatty acids to their omega-3

counterparts, and the latter must be obtained from separate dietary sources

(Hulshof et al., 1999). The n-3 PUFA mainly include the essential fatty acid �-linolenic acid (ALA, C18:3n-3) and its long-chain metabolites eicosapentaenoic

acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3) and

docosahexaenoic acid (DHA, C22:6n-3) (Fig. 12.1). ALA is available from

certain plants such as the seeds and oils of flax or linseed, and to a lesser extent

perilla, soybean and canola (Newton and Snyder, 1997; Kamal-Eldin and

Yanishlievab, 2002). EPA and DHA, however, are derived marine products (i.e.

12

Marine oils from seafood wasteF. Shahidi, Memorial University of Newfoundland, Canada

fish and shellfish and algal species) (Newton and Snyder, 1997; (NDA)

Scientific Panel on Dietetic Products, Nutrition and Allergies, 2005; Watanabe

and Ackman, 1974; Ackman 1982; Myher et al., 1996; Mayzaud et al., 1999;

Tanabe et al., 1999; Arts et al., 2001; Oliveira and Bechtel, 2005), but DPA is

less abundant and found in less than 1% in most fish oils. Humans can

synthesize, up to approximately 5%, EPA and DHA, through desaturation and

elongation, from dietary ALA (Aliam, 2003). This pathway is an important

source of these long-chain n-3 PUFA in strict vegetarians, who do not consume

fish. Non-vegetarians can also obtain PUFA from a variety of food products

(Gebhardt and Thomas, 2002).

Table 12.2 shows fatty acid composition of lipid from several marine

organisms. The main components of marine lipids are monounsaturated fatty

Table 12.1 Dietary sources of various omega-3 polyunsaturated fatty acids (% of totalfatty acids)

Omega-3Alpha-linolenic acid (18:3) Eicosapentaenoic acid (20:5) Docosahexaenoic acid (22:6)

Freshwater fish (1±6%) Freshwater fish (5±13%) Freshwater fish (1±5%)Marine fish (~1%) Pacific anchovy (18%) Pacific anchovy (11%)Linseed (45±60%) Capelin (codfish) (9%) Capelin (codfish) (3%)Green leaves (56%) Mackerel (8%) Mackerel (8%)Rapeseed (10±11%) Herring (3±5%) Herring (2±3%)

Sardine (3%) Sardine (9±13%)

Source: Newton and Snyder (1997).

Fig. 12.1 The omega-3 fatty acid family.

Marine oils from seafood waste 259

Table 12.2 Distribution of selected FA (% wet weight) in various marine organisms

Component Atlantic Common oyster Northern krill Atlantic salmonmenhaden (Crassostrea (Meganyctiphanes (Salmo

oil1 virginica)2 norvegica)3 gairdneri)4

14:0 7.3 3.5 4.6 5.5C16:0 19.0 25.9 18.4 10.2C18:0 4.2 3.6 2.2 2.7SFA 30.5 36.0 25.2 18.4C16:1 9.1 4.2 3.5 8.1C18:1 13.2 8.2 14.7 16.9C20:1 2.0 5.0 2.3 15.1C22:1 0.6 0.3 0.9 14.4MUFA 24.9 17.7 21.4 54.5C18:2n-6 1.3 2.0 1.8 4.5C18:3n-3 1.3 3.3 1.3 0.9C18:4n-3 2.8 2.6 3.1 1.7C20:4n-6 0.2 2.3 1.0 0.6C20:5n-3 11.0 11.2 10.8 6.2C22:5n-3 1.9 Ð 1.0 1.8C22:6n-3 9.1 9.7 28.6 9.1PUFA 27.6 31.1 45.8 24.8Other 17.0 15.2 7.6 2.3

Component Sardine Anchovy Cod Skipjack(Sardine (Engraulis (Gadus tuna8

pilchardus)5 encrasicholus)6 morhua)7

14:0 5.7 10.2 0.8 8.2C16:0 17.8 23.5 17.2 26.5C18:0 3.1 3.4 4.0 11.9SFA 27.2 40.4 22.2 38.4C16:1 6.8 10.6 3.0 8.2C18:1 7.8 10.3 13.4 6.8C20:1 Ð 5.0 0.4 0.5C22:1 5.1 0.7 0.0 ÐMUFA 19.7 28.4 16.9 7.3C18:3n-3 1.9 0.8 0.4 0.0C18:4n-3 Ð Ð 0.6 1.3C20:5n-3 8.4 8.2 16.5 11.1C22:5n-3 Ð 0.9 0.9 ÐC22:6n-3 15.5 15.2 36.9 29.1C18:2n-6 2.6 1.6 1.3 1.1C20:4n-6 Ð 0.7 3.0 3.1PUFA 28.4 29.4 61.0 45.7Other 25.0 1.8 Ð Ð

1 Ackman (1982); 2 Watanabe and Ackman (1974); 3 Mayzaud et al. (1999); 4 Oliveira and Bechtel(2005); 5 Newton and Snyder (1997); 6 Kalogeropoulos et al. (2004); 7 Hyvonen and Koivistoinen(1994); 8 Tanabe et al. (1999).


acids along with PUFA and some saturated fatty acids that are present in

different proportions (Watanabe and Ackman, 1974; Ackman 1982; Myher et

al., 1996; Mayzaud et al., 1999; Tanabe et al., 1999; Arts et al., 2001; Oliveira

and Bechtel, 2005). Among these, the ratio of DHA to EPA and possible

presence of DPA in modest amounts are most important (Shahidi, 2002).

Although EPA and DHA are found abundantly in different marine oils, DPA is

present in significant amounts only in seal blubber oil (Wanasundara et al.,

1998; Aidos et al., 2002; Sathivel et al., 2003; Jayasinghe et al., 2003).

The spatial distribution of fatty acids in triacylglycerols in fish and marine

mammal oils differ in that fish oils contain long-chain PUFA mainly in the sn-2

position of triacylglycerols whereas marine mammal lipids have them

predominantly in the sn-1 and sn-3 positions. These factors greatly influence

the metabolism and potential health benefits of marine lipids (Shahidi, 1998).

12.2 Oil from fish processing by-products

The fish industry produces a considerable amount of by-catch as well as fish

species that are primarily harvested for the fish meal industry with oil being a

by-product. One example is menhaden oil. Menhaden (Brevoortin tyrannus) is a

filter feeder fish that is available near the shores of Atlantic coast of the United

States and Gulf of Mexico. Menhaden is used for production of meal for

aquaculture and land-based animal feed. Menhaden oil, a by-product of

menhaden fish meal industry, contains 18% EPA and nearly 10% DHA. The

major fatty acids of menhaden oil are given in Table 12.3.

Capelin (Mytilus edulis) is a small marine fish that is also widely processed

for fish meal as well as whole body oil. It is a major prey species that is often

used as a bait in some fisheries. Capelin contains 7±10% oil, mainly

triacylglycerols and about 20% PUFA as shown in Table 12.3. The oil from

capelin is used in aquaculture feed formulation as well as other applications.

By-products from gutting, filleting, and other processing operations are

good raw materials for fish meal and oil production. One can obtain oil from

different parts of fish with diverse nutritional composition. Composition, lipid

content and fatty acid profile of individual by-products is of increasing

importance, as different by-products are being segregated and used for

different end products.

Shark liver is the principal site of lipid storage. The oil content and fatty

acid composition in shark liver are influenced by the gender, season and

species of shark. Table 12.4 presents the fatty acid profile of liver oil from

male and female blue shark. In addition to fatty acids, shark liver oil contains

high amounts of squalene, low-density lipids (diacylglyceryl ethers) and

vitamin A (Jayasinghe et al., 2003). In recent years, scientific evidence has

emerged in support of the therapeutic value of shark products, particularly

shark fins, cartilage, and liver oil as a good source of n-3 PUFA. At present, in


the market cod liver oil is dominant and it contains high levels of vitamins A

and D (Engelhardt and Walker, 1974). Here it should be noted that cod as well

as halibut liver oils, although rich in n-3 PUFA, are used primarily as a source

of vitamins A and D.

Salmon deposits oil mainly in its head, which contains approximately 15±

18% lipids. However, salmon oils can also be produced from viscera, whole fish

(down-graded) and filleting by-products (heads, trimmings such as belly-flaps,

as well as skin, and frame bones). Significant differences were found in the lipid

and fatty acid content and composition of salmon by-products. Pink salmon

heads had the highest lipid content and viscera the lowest. The n-3/n-6 ratios for

pink salmon samples ranged from 7.7 to 10.5 and only viscera values were

statistically different (Wanasundara et al., 1998; Aidos et al., 2002; Sathivel et

al., 2003; Jayasinghe et al., 2003). The fatty acid composition of salmon oil

depends on the composition of the raw material used (Table 12.5). The average

content of n-3 PUFA in salmon oil is in the medium range compared with other

Table 12.3 Major fatty acids of menhaden oil1 and capelin oil2

Fatty acid Menhaden (%) Capelin (%)

14:0 7.30 5.915:0 0.65 0.216:0 19.45 8.716:1 9.05 10.516:2 n-7 0.50 Ð16:2 n-4 1.55 Ð16:3 n-4 1.70 0.816:4 n-1 2.60 2.017:0 1.05 Ð18:0 4.45 0.618:1 10.40 6.018:2 n-6 1.30 0.518:2 n-4 0.50 0.118-3 n-3 0.65 0.218:4 n-3 2.65 1.220:1 1.45 17.620:2 n-6 0.30 Ð20:3 n-3 1.00 Ð20:4 n-3 0.80 Ð20:5 n-3 18.30 9.321:5 n-3 0.90 Ð22:1 1.55 27.822:4 n-3 0.60 Ð22:5 n-3 1.80 0.722:6 n-3 9.60 4.1

1 Ackman (2005); 2 Copeman and Parrish (2004).


fish oils. Even though the by-products from most other fish-processing indus-

tries tend to vary with season, both in quality and quantity, salmon slaughter-

houses generate high-quality offal at a relatively constant rate (Park et al., 2004;

Rora et al., 2005).

The by-products of catfish processing consist of heads, frames, skin, and

viscera, which often end up in landfills or rendering plants. Producing edible oil

from viscera may add value to catfish viscera. The total unsaturated fatty acids

in the purified oil from catfish viscera was 67.7% (Table 12.5). The combined n-

3 fatty acids of the purified catfish viscera oil was only 4.6 mg/g of oil (Sathivel

et al., 2003).

Herring oil is produced from three different types of by-products, only heads,

mixed, and headless by-products are of interest (Sathivel et al., 2003). Even

though by-products from heads and their oil have the highest oxidation levels

and the lowest �-tocopherol content, heads contain the lowest PUFA and the

highest amount of saturated fatty acids (Table 12.6). No significant differences

were found between the fatty acid composition of the mixed and the headless

by-products or their oil (Aidos et al., 2002).

Table 12.4 Fatty acid composition in total lipids from Blueshark (P. glauca) liver (w/w %)1

Fatty acid Blue shark liver

Male Female

14:0 2.3 2.816:0 22.0 16.818:0 4.9 3.9Total SFA 36.0 30.316:1 3.9 5.318:1 16.0 27.620:1 3.0 4.922:1 0.0 0.024:1 0.8 0.9Total MUFA 23.6 38.718:2n-6 0.8 0.718:3 0.2 0.218:4n-3 0.4 0.420:5n-3 4.5 2.722:5n-3 2.1 2.522:6n-3 23.2 18.4Total PUFA 39.2 30.2

1 Jayasinghe et al. (2003).


Table 12.5 Fatty acid profile of different by-product oil from Alaska pink salmon (Oncorhynchus gorbuscha), catfish (Ictalurus punctatus), cod(Gadus morhua) and of their respective whole fish oil (w/w %)

Fatty acid Alaska pink salmon1 Catfish2 Cod3

Whole fish Head Viscera Whole wild fish Viscera Whole fish Liver (crude) Liver (RBD)

14:0 3.6 4.12 3.44 3.04 3.45 2.60 2.01 3.3316:0 12.04 12.39 12.8 20.77 12.91 16.85 9.42 11.118:0 3.25 2.87 4.45 4.30 15.75 7.31 3.33 3.89Total SFA 22.49 19.38 20.69 33.68 32.90 28.75 15.28 19.1716:1 3.66 3.96 3.82 7.38 10.56 2.99 6.19 7.8518:1 16.04 14.75 16.83 22.45 21.62 14.18 22.13 21.1620:1 9.66 10.68 6.36 1.14 6.26 3.13 11.3 10.422:1 9.05 11.43 6.82 Ð 0.00 0.09 7.81 9.0724:1 0.77 0.91 0.00 Ð 0.00 Ð 0.00 0.00Total MUFA 39.18 41.73 33.83 32.68 38.44 20.97 48.06 49.0718:2n-6 1.73 1.67 1.38 0.27 20.30 0.24 0.81 0.7418:3n-3 1.34 1.27 1.16 0.51 3.30 Ð 0.57 0.4618:4n-3 2.71 2.95 1.32 4.02 0.00 0.17 0.65 0.6120:5n-3 7.70 7.56 10.93 0.73 0.00 8.32 13.9 11.222:5n-3 2.75 2.33 2.83 3.69 Ð 1.57 1.43 1.1422:6n-3 15.78 11.77 17.32 4.65 1.18 28.59 16.9 14.8Total PUFA 32.53 31.38 40.35 26.00 28.62 46.93 34.66 29.29

RBD refers to crude and refined-bleached and deodorized (RBD) oils.1 Oliveira and Bechtel (2005); 2 Bonnet et al. (1974); Sathivel et al. (2003); 3 Bonnet et al. (1974); Wanasundara et al. (1998).

12.3 Marine mammal oils

Marine mammals are unique in that they possess a layer of insulating fat under

their skin, known as blubber, which allows their survival in the cold waters of the

Arctic and Antarctic (Holmer, 1989). Blubber also helps the animals with their

movement and buoyancy. The blubber may vary in thickness, depending on a

number of variables, but is on average about 5 cm thick for seals. Seal blubber oil

is a by-product of seal meat and seal industries. The oils from marine mammals

contain various lipid classes, including triacylglycerols, diacylglycerols,

monoacylglycerols, free fatty acids, wax esters, cholesterol, cholesterol esters,

hydrocarbons, vitamins, and ether lipids. Triacylglycerols (TAG) of seal blubber

oils are the main component of neutral lipids which contain a variety of lipid

classes. Neutral lipids account for 98.9% of blubber in contrast to intramuscular

lipids (78.8% neutral and 21.1% polar lipids) (Shahidi et al., 1994).

In addition to TAG, wax esters (long-chain alcohols esterified to fatty acids)

are another important group of neutral lipids found in marine mammals. Most

species of marine mammals have C32, C34, C36, and C38 (total of alcohol plus

acid) as major components (Lee and Patton, 1989). Whale oils are especially

interesting because some contain fatty acids that are largely in the form of wax

esters (Gruger, 1967). The oils from the blubber of the Physeteridae may consist

mainly of wax esters. The sperm whale blubber oil consists of a mixture of about

79% wax esters and 21% TAG (Hansen and Cheah, 1969). The dwarf sperm

whale (K. simus) blubber oils consist of 42% wax esters and 58% TAG

Table 12.6 Fatty acid profile in extracted oils from different by-products of herring(Clupea harengus) and of whole body herring oil (w/w %)1

Fatty acid Herring heads Herring headless Herring mixed Whole fish oil

14:0 9.4 8.8 9.0 5.0416:0 14.5 13.8 14.2 19.9218:0 2.3 2.0 2.2 1.52Total SFA 26.2 24.6 25.4 28.1916:1 5.6 5.4 5.5 6.3618:1 7.1 7.2 7.5 26.1520:1 9.9 10.3 10.3 1.2922:1 17.8 17.7 17.9 0.7124:1 0.0 0.0 0.0 1.39Total MUFA 40.4 40.6 41.2 36.3918:2n-6 1.4 1.3 1.2 5.1018:3n-3 1.4 1.4 1.2 2.5218:4n-3 3.0 3.2 3.2 1.4920:5n-3 8.7 8.8 9.0 7.3322:5n-3 Ð Ð Ð 0.5522:6n-3 8.0 8.1 8.5 15.56Total PUFA 22.5 22.8 23.1 35.42

1 Aidos et al. (2002).


(Litchfield et al., 1975). The blubber fat of beaked whales (Berardius,

Hyperoodon, and Ziphius) is composed almost entirely of wax esters (94±99%)

along with low levels of TAG (2±6%) (Litchfield et al., 1976). A number of

possible functions for wax esters in marine mammals has been proposed; these

include their role as a reserve energy store, buoyancy, metabolic water, thermal

insulation, and biosonar (Nevenzel, 1970; Sargent et al., 1976; Sargent, 1978).

Among unsaponifiable matters, hydrocarbons, especially long-chain hydro-

carbons, are found in detectable amounts in marine mammal oils. Some marine

oils contain less than 0.1% hydrocarbons, while others contain as much as 90%

(Heller et al., 1957). In the liver of the seal, Arctocephalus (Pinnipedia), liver

squalene was 0.50% of the oil (Karnovsky and Rapson, 1947). High squalene

contents (90, 91 and 92.8%) occur in shark liver oils (Karnovsky and Rapson,

1947; Heller et al., 1957). Total hydrocarbons were present at 0.3% of dry

matter weight of the blubber, 1.6% in liver, and 1.3% in the muscle (Bottino,

1978). Among cetaceans, limited data for two dolphins have been published: in

Delphinus longirostris liver very long-chain hydrocarbons (C44) were detected;

and zamene was present in Langenorynchus acutus (Blumer and Thomas, 1965).

The fatty acid composition of marine lipids varies significantly, but all

contain a large proportion of long-chain highly unsaturated fatty acids, similar to

fish oils. However, the proportion of fatty acids in fish and marine mammals

varies considerably (Shahidi, 1998).

A marine oil typically contains some 40 different fatty acids with carbon

numbers varying from 10 to 24, resulting in a large number of different TAG

with the same carbon number, but with different levels of unsaturation (Shahidi

et al., 1994; Borch-Jensen and Mollerup, 1996). Even-numbered carbon fatty

acids make up about 97% of the total fatty acids, with a few notable exceptions

(Hansen and Cheah, 1969). Some fatty acids with odd-numbered carbon chain

such as C15:0 and C17:0, along with traces of C13:0 and C19:0 have also been

found in marine oils (Ackman, 1989). Besides, monomethyl branched fatty acids

have been isolated from marine oils, such as 3-methyldodecanoic acid from

blubber of the sperm whale physeter catodon (Ackman, 1989).

In contrast to relatively small amounts of saturated fatty acids, marine

mammal oils have been characterized by high amounts of monounsaturated fatty

acids (MUFA) and n-3 PUFA (Bang et al., 1976, 1980). For instance, the

content of MUFA in neutral and polar lipids in seal blubber are more than 60

and 46%, respectively (Shahidi et al., 1996). Most of these fatty acids are long

chain with 20 to 22 carbon atoms and have !3 configurations. Ackman et al.

(1965) have pointed out that the total C20 and C22 MUFA and PUFA in each

layer of whale blubber is nearly constant, but the ratios of the monounsaturated

to polyunsaturated fatty acids change very significantly. The most common long

chain PUFA in marine lipids are EPA and DHA as well as a smaller amount of

DPA, all of which belong to the !3 family (Wanasundara, 1997). The high

content of !3 fatty acids in marine lipids is suggested to be a consequence of

cold temperature adaptation, because at lower habitat temperatures, !3 PUFA

remain liquid and oppose any tendency to crystallize (Ackman, 1989). Most of


the long chain PUFA are formed in unicellular phytoplankton and multicellular

sea algae and eventually pass through the food web and become incorporated

into the body of fish and other higher marine species, including marine

mammals which often eat fish (Yongmanichai and Ward, 1989). The fatty acid

composition of oils from most species of marine mammals has been summarized

(Ackman and Lamothe, 1989). Seal oils, due to the increasing interest in seal

fishery and product development, have been in focus and frequently studied by

researchers. The fatty acid composition of oils from different species of seal has

been reviewed (Shahidi, 1998). Table 12.7 shows the fatty acids and their

contents in blubber lipid from six species of seals.

The fatty acid composition of blubber of marine mammals such as seals is

regulated by their diet (Grahl-Nielsen and Mjaavatten, 1991), location (West et

al., 1979a,b), season as well as physiological conditions such as age (Engelhardt

and Walker, 1974) and sex (West et al., 1979a,b) of the animal. In some marine

mammals, the depot fats are largely dietary fatty acids laid down with a

minimum change, but the fatty acids of the lipids of the essential organs have

terrestrial characteristics (Ackman and Lamothe, 1989). Fatty acid composition

also depends on tissue and species of the animal. However, differences are most

apparent among tissues. Seal blubber, for example, had a high content of

monounsaturated fatty acids, but was low in arachidonic acid, dimethyl acetals

and DHA. Lung tissue lipids were high in palmitic acid and heart tissue lipids

had a higher content of linolenic acid. The proportions and fatty acid

Table 12.7 Fatty acid composition (g/100g) of blubber of various species of seal1

Fatty acid Bearded Gray Harbor Harp Hooded Ringed

14:0 3.05 3.83�0.03 4.52�0.13 4.66�0.49 4.40�0.38 3.36�0.6616:0 DMA ND ND ND ND ND ND

16:0 10.14 6.61�0.08 8.03�0.38 6.24�0.44 9.81�1.57 4.82�2.0716:1 !7 17.77 12.77�0.09 19.26�0.53 14.93�0.46 10.09�0.35 23.12�0.1818:0 DMA ND ND ND ND ND ND

18:1 !9 DMA ND ND ND ND ND ND

18:1 !7 DMA ND 0.45�0.01 ND 0.46�0.00 ND ND

18:0 2.15 0.94�0.02 0.85�0.02 0.95�0.03 1.83�0.31 0.42�0.1918:1 !9 16.76 24.50�0.44 18.61�0.55 18.59�1.01 22.77�2.66 19.72�1.3318:1 !7 9.49 4.95�0.09 5.16�0.44 3.57�0.36 3.75�0.47 5.03�0.4618:2 !6 2.30 1.28�0.00 1.27�0.04 1.36�0.20 1.63�0.20 2.58�0.0220:1 !9 5.08 12.50�0.43 9.06�0.33 12.56�2.92 13.00�1.86 6.71�2.1720:4 !6 0.94 0.51�0.00 0.44�0.00 0.36�0.96 0.31�0.03 0.30�0.0220:5 !3 8.28 4.85�0.13 9.31�0.21 6.82�0.69 5.21�1.65 8.72�1.0622:0 0.63 <0.3 1.19�0.02 <0.3 <0.3 0.75�0.6722:1 !11 0.27 0.62�0.03 0.31�0.01 0.77�0.61 0.86�0.33 0.34�0.0122:5 !3 4.26 5.06�0.05 4.22�0.14 4.78�0.25 2.29�0.08 5.46�0.4722:6 !3 7.22 8.91�0.29 7.76�0.98 10.48�1.98 9.56�2.36 9.45�1.74

DMA: dimethyl acetal.ND: not detected.1 Durnford and Shahidi (2002); Durnford et al. (2003).


constituents in different tissues are different, most probably due to their varying

functional requirements (Durnford and Shahidi, 2002). The lipids of vital organs

of seals and whales contain high proportions of fatty acids of the !6 family,

similar to those of terrestrial animals. The distinction between the fatty acids of

functional organs such as liver, heart, and other organs with depot fat has been

discussed (Ackman et al., 1972; Durnford and Shahidi, 2002).

As explained earlier, the fatty acid distribution in the TAG molecules in

blubber oil are different from fish oil and the omega-3 fatty acids are located

primarily in the sn-1 and sn-3 positions of TAG (Table 12.8), while in fish oils

they are located abundantly in the sn-2 position of TAG (Wanasundara and

Shahidi, 1997). Mag (2000) has reported that the different distribution of fatty

acids may influence the metabolism and potential health benefits of marine

lipids, and moreover, may account for the higher oxidative stability of marine

mammal oils compared to fish oils.

12.4 Algal oil

As an alternative to usual marine oils, PUFA can be obtained from micro-

organisms. Microorganisms, in particular the marine algae, are widely used in

the aquaculture industry, mostly to feed fish, crustaceans, and bivalves directly.

They are also used indirectly, although to a much lesser extent, via feeding of

rotifers, copepods, and brine shrimps, which are in turn used as live feed.

Therefore, they are thought to be the primary producers of n-3 PUFAs in the

marine food chain (Coutteau et al., 1997; Wikfors et al., 2001). Although marine

fish and mammals appear to have some capacity for in vivo biosynthesis of n-3

PUFAs, the majority of the PUFAs in their body originate from their diet

(Ackman et al., 1964).

Depending on the microbial species and environmental conditions, the lipid

content of microorganisms may vary between a few percent to over 80% of the

Table 12.8 Fatty acid distribution in different positions of triacylglycerols of harp sealblubber oil1

Fatty acid sn-1 sn-2 sn-3

Total saturates 6.34 25.56 4.32Total unsaturates 90.51 73.25 94.32Total monounsaturates 62.91 65.98 51.09Total polyunsaturates 27.60 7.27 43.23Eicosapentaenoic acid (EPA) 8.36 1.60 11.21Docosapentaenoic acid (DPA) 3.99 0.79 8.21Docosahexaenoic acid (DHA) 10.52 2.27 17.91Total omega-3 25.65 5.56 38.87Total omega-6 0.75 1.58 3.34

1 Shahidi (1998).


biomass on a dry weight basis (Ratledge, 1993; Leman, 1997). To make some

kind of a distinction, the term oleaginous has been introduced and these micro-

organisms accumulate over 20±25% lipid on a dry biomass basis (Ratledge and

Evans, 1989). Oleaginous microorganisms store lipids mainly in the form of

triacylglycerols. Various eukaryotes can accumulate large amounts of triacyl-

glycerols (Ratledge, 1993).

Microbial oil or single cell oil (SCO) production via fermentation is a

relatively new concept, first proposed in the twentieth century (Ratwan, 1991;

Ratledge, 2001). In SCO processes, microorganisms that are able to produce the

desired oil are cultivated in a bioreactor (Sijtsma and de Swaaf, 2004). In the

industrial-scale fermentation, cells are harvested at maximum volumetric

productivity followed by drying and further processing of the oil (Kyle, 1997).

Since heterotrophic cultivation is independent of light, this production system

possesses many advantages such as axenical operation, optimal controlled

conditions, increased reproducibility, higher biomass concentrations, and

straightforward scale-up of the fermentation process (Chen, 1996). High levels

of DHA are found in heterotrophic marine algae including Traustochitrium,

Schizochytrium and Crypthecodinium cohnii species, except for Amphidinium

sp. Phototrophic alga contains a relatively higher level of EPA (Table 12.9)

(Myher et al., 1996; Sijtsma and de Swaaf, 2004; Molina et al., 1993; Viso and

Marty, 1993; Servel et al., 1994; Singh and Ward, 1996; Vazhappilly and Chen,

1998; Shields et al., 1999; Meireles et al., 2002). A major drawback of

Schizochytrium sp. for producing DHA is the presence of DPA (n-6) in the

microbial oils, in addition to DHA (Sijtsma and de Swaaf, 2004).

Algal oils have many benefits in functional foods due to their high n-3 fatty

acids and the lack of environmental toxins. Furthermore, algae usually contain

one specific PUFA rather than a mixture of various PUFA. This gives the algal

oils an added advantage compared to fish oils, which contain mixtures of PUFA.

In addition, PUFA can be purified more easily (and thus more economically)

from oils, which contain one PUFA instead of a mixture of PUFA (Kendrick and

Ratledge, 1992).

12.5 Marine oil manufacturing process

Crude oils contain varying amounts of substances that may impart undesirable

flavor and color, including free fatty acids, phospholipids, proteins, water,

pigments, and fat oxidation products. Therefore, crude oils are subjected to a

number of commercial refining processes designed to remove undesirable

materials (Bell et al., 2003). Fish oil refining steps include extraction of the

crude oil, degumming, neutralizing, bleaching, and deodorizing. Both insoluble

and soluble impurities are removed through degumming, if necessary;

neutralization of crude oil with caustic soda removes free fatty acids. Bleaching

removes soap, trace metals, sulphurous compounds, and part of the more stable

pigments and pigment-breakdown products. This process also degrades


Table 12.9 Fatty acid profile of selected algal oils1

Organism 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 18:4 20:4 n-6 20:5 n-3 22:5 22:6 n-3

Thraustochytrium aureum(H,76) 3 8 16 2 2 3 52Schizochytrium sp. (H,77) 18 38 6 1 5 1 1 1 9 18Crypthecodinium cohnii (H,33) 15 15 2 2 15 1 6 37Amphidinium carterae (H,78) 8 30 15 5 3 5 6 17 4 4 2Isochrysis galbana (P,79) 12 15 11 1 3 2 11 25 11Skeletonema costatum (P,80) 17 10 11 2 1 6 41 7Amphidinium sp. (P,81) 5 17 18 17 2 2 8 17Pavlova lutheri (P,82) 14 27 10 1 3 4 12 7

H: heterotrophic growth, P: phototrophic growth.1 Sijtsma and de Swaaf (2004).

hydroperoxides to their respective aldehydes, ketones and other products.

Finally, deodorization is carried out in order to remove residual free fatty acids,

aldehydes, and ketones, which are responsible for unacceptable odor and flavor

of the oil (Aidos et al., 2003; Dauksas et al., 2005).

Even though fish and fish oils are the main sources of PUFA, the quality of

fish oil, however, is variable and depends on fish species, season and location of

catch (Moffat, 1995; He and Daviglus, 2005). Marine fish oils may contain

environmental pollutants and problems associated with the typical fishy smell

and unpleasant taste may exist (Sijtsma and de Swaaf, 2004). Fish oils also

contain EPA which is undesirable for use in infant formulas because it leads to

reduced arachidonic acid levels and hence reduced rates of infant weight gain

(Carlson, 1996; Heird and Lapillonne, 2005). In order to meet the rapidly

growing demand for PUFA in human nutrition, fish feeds for aquaculture

operations and pharmaceutical applications, and to circumvent the drawbacks of

fish oils, alternative production processes for PUFA have been developed. These

include the development of novel refining techniques of fish oils (Shahidi and

Wanasundara, 1998; Carvalho and Malcata, 2005) as well as production of

concentrates with varying proportions of EPA and DHA. In addition, exploita-

tion of microbial PUFA has taken place (Ishihara et al., 2000; Meireles et al.,

2002).

12.6 Health effects of PUFA

Recognition of the health benefits associated with consumption of seafoods (n-3

fatty acids) is one of the most promising developments in human nutrition and

disease prevention research in the past three decades. According to the current

knowledge, long-chain n-3 PUFA play an important role in the prevention and

treatment of coronary artery disease (Alexander, 1998), hypertension (Howe,

1997), diabetes (Krishna Mohan and Das, 2001), arthritis and other inflam-

matory (Babcock et al., 2000), and autoimmune disorders (Kelly, 2001), as well

as cancer (Rose and Connolly, 1999; Akihisa et al., 2004) and are essential for

normal growth and development, especially for the brain and retina (Anderson et

al., 1990). The most direct and complete source of n-3 oils is found in fish oils

and the blubber of certain marine mammals, especially harp seal. Among its

advantages is that the body's absorption of n-3 fatty acids from marine mammal

blubber may be faster and more thorough than is the case with flaxseed and fish

oils (Mag, 2000). Since marine mammal oils contain a high concentration of

monounsaturated fatty acids (MUFA), it is possible that some of their beneficial

effects may be ascribed to their MUFA or to the combined effect of MUFA and

n-3 PUFA (Hansen et al., 1994). A pilot study indicated that a low dose of seal

oil supplementation can reduce atherogenic risk indices in young healthy

individuals, and the effects are strongly dependent on the integrated n-3 fatty

acids dose (Deutch et al., 2000; Bonefeld Jorgensen et al., 2001). The essential

fatty acids found in seal oil include a high level of DPA (up to ten times that of


fish oils). There is growing evidence that DPA is the most important of fatty

acids that keep artery walls soft and plaque-free (Mag, 2000). Marine oils are

also attractive from a nutritional point of view because they are thought to

provide specific physiological functions against thrombosis, cholesterol build-up

and allergies (Kimoto et al., 1994). Oils from the blubbers of seal and whale

have beneficial effects on selected parameters that play a role in cardiovascular

disease; it has been hypothesized that the effect of whale oil is not mediated by

its n-3 fatty acids alone (Osterud et al., 1995). The difference in the beneficial

effects of whale and seal oils on cardiovascular disease may argue against the

distribution of n-3 fatty acids in TAG as being relevant to the superiority of

whale oil, since the n-3 fatty acids are mainly in the sn-1 and sn-3 positions of

both of these oils. The effect of whale oil is probably not mediated by n-3 fatty

acids alone as the content of these fatty acids is relatively low in whale oil. Thus,

in addition to !3 fatty acids, other dietary factors may play a role in the

protective effects against atherosclerosis and thrombosis in Greenland Eskimos

(Osterud et al., 1995).

The beneficial effects of PUFA have also been ascribed to their ability to

lower serum TAG, to increase membrane fluidity and to reduce thrombosis by

conversion to eicosanoids (Kinsella, 1986). Both EPA and DHA, induced

increases in the serum concentrations of the corresponding fatty acids as well as

their relative contents in platelets (Vognild et al., 1998). However, distribution

of n-3 PUFA in TAG molecules influences glycerolipid metabolism and

arachidonic acid contents of serum and liver phospholipids, as well as throm-

boxane (TX) A2 production. In rats that were fed marine oils, for instance,

plasma and liver TAG concentrations were more effectively reduced by dietary

seal oil than by fish oil. Furthermore, dietary seal oil reduced arachidonic acid

content in liver phosphatidylcholine and phosphatidylethanolamine, and serum

phosphatidylcholine more effectively than fish oil. Activities of fatty acid

synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH) and the malic

enzyme were significantly lowered when hamsters were fed seal oil (Yoshida et

al., 2001). The predominant effect of seal oil was due to the suppression of fatty

acid synthesis in the liver (Yoshida et al., 1999). In addition, reduction of TX A2

production of platelets and whole blood platelet aggregation by seal oil has been

observed (Ikeda et al., 1998; Brox et al., 2001). Benefits of DPA in health have

been described (Rissanen et al., 2000; Yazawa, 2001).

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YOSHIDA, H., IKEDA, I., TOMOOKA, M., MAWATARI, M., IMAIZUMI, K., SETO, A. and TSUJI, H.

(2001). Effect of dietary seal and fish oils on lipid metabolism in hamsters. J. Nutr.

Sci. Vitaminol., 47, 242±247.


13.1 Introduction

Collagen is one of the major structural components of both vertebrates and

invertebrates, and is found widely in skin, bone and other connective tissues

(Balian and Bowes, 1977). Gelatin, on the other hand, is a class of protein

fractions that have no existence in nature, but are derived from the parent protein

collagen, by procedures involving the destruction of cross-linkages between

polypeptide chains of collagen along with some level of breakage of polypeptide

bonds. Most commercial collagen and gelatin are obtained from mammals,

mainly from bovine bone, bovine hide, and porcine skin. In recent years studies

on collagen and gelatin obtained from seafood processing by-products have

drawn extensive interest, in part due to the requirements for kosher and halal

food product development and consumers' concern about bovine spongiform

encephalopathy (BSE, `mad cow disease') in products from mammals. This

chapter provides a brief review of the literature for the following three main

topics, namely, manufacture of collagen and gelatin from seafood processing by-

products; chemical, physical and nutritional properties of marine collagen and

gelatin; and food and non-food applications of collagen and gelatin.

13.2 Key drivers of marine collagen and gelatin

At present, most of the world's production of collagen and gelatin comes from

porcine skin, bovine hide or bovine bone (Table 13.1, Gelatine Manufacturers of

Europe, 2004). Unfortunately, these sources of collagen and gelatin present

religious and safety-orientated concerns for various consumer communities.

13

Collagen and gelatin from marineby-productsJ. M. Regenstein and P. Zhou, Cornell University, USA

13.2.1 Requirement of kosher and halal product development

Kosher and halal certifications are important components of the food business.

Kosher food, in particular, has become a major part of modern food production.

Many companies in the food industry would prefer to operate all of their

processing lines as kosher because of the complications and cost involved in

switching back and forth between non-kosher and kosher. On the other hand, the

number of Muslims in the world is more than 1.3 billion people, and trade in

halal products is currently about 150 billion US dollars (Regenstein et al., 2003).

The absence of any widely accepted collagen and gelatin for both Jewish and

Muslim groups is probably the most significant factor holding up further

expansion of kosher and halal food products. Although some lenient rabbis permit

some types of traditional collagen and gelatin as kosher, the mainstream Orthodox

kosher supervision agencies, such as the OU, OK, Star-K or Kof-K, only certify

collagen and gelatin from kosher fish (fish with fins and removable scales) and

kosher slaughtered animals (Regenstein et al., 1996). Similarly, for most Muslims,

pork gelatin is forbidden and gelatin from non-religiously slaughtered cattle is not

desirable; but most fish collagen and gelatin can be certified as halal.

13.2.2 Consumers' safety concerns

BSE, which has been spread in Europe but is also found in many other countries,

raised a serious health concern with respect to collagen and gelatin. In many

countries such as the United States, the government agencies issued specific

rules to regulate the source and processing of collagen and gelatin to reduce the

potential risk posed by BSE. Although the scientific evidence did not show that

collagen and gelatin were BSE carrier materials, many consumers still have

concerns about the safety of these products.

13.2.3 Economic drivers from the fishing industry's point of view

The waste from fish processing after filleting can account for as much as 75% of

the total catch weight (Shahidi, 1995). It includes the heads, skin and scales, guts/

Table 13.1 World gelatin market data from 2001 to 2003

2001 2002 2003

Production Percentage Production Percentage Production Percentage(metric ton) (metric ton) (metric ton)

Pork skin 110 400 41.0 113 600 41.7 117 950 42.4Bovine hides 77 200 28.6 77 500 28.4 81 650 29.3Bones 80 800 30.0 79 600 29.2 76 750 27.6Fish and others 1 000 0.4 1 800 0.7 1 950 0.7Total 269 400 272 500 278 300

Source: Research by Gelatin Manufacturers of Europe, 2004.


internal organs, frames (bone rack with adhering meat), and trim (pieces cut from

the fillets during processing) (Regenstein, 2004). About 30% of such waste consists

of skin, bone and scale with high collagen content that could be used to produce

collagen and gelatin (Young and Lorimer, 1960; GoÂmez-GuilleÂn et al., 2002;

Sadowska et al., 2003). Thus, preparation of collagen and gelatin from marine by-

products can not only satisfy the kosher and halal requirement and consumers'

concern for BSE, but can also increase the economic return for the fishing industry.

13.3 Sources of marine collagen and gelatin

Collagen and gelatin can be obtained from various marine sources. The marine

species available for collagen and gelatin manufacture can be roughly divided

into three categories: marine invertebrates, sea mammals and fishes (Table

13.2). Fish, based on their living environments, are usually subdivided into four

groups: hot-water fish, warm-water fish, cold-water fish (Eastoe and Leach,

1977), and ice-water fish. Cold-water fishes, such as pollock, cod and salmon,

account for a large part of commercial fish capture (FAO, 2002). They are often

processed into the form of skinned and boned fillets, leaving large amounts of

fish skin, scales and bones as waste. These by-products, especially the skin,

usually contain a large amount of protein, most of which is collagens (Young

and Lorimer, 1960). Warm-water fish account for most fresh-water fish aqua-

culture production (FAO, 2002), and currently, many commercial fish gelatins

come from fish in this category. In the following sections the studies of collagen

and gelatin from both marine sources and some fresh water fish species will be

reviewed to examine the variation among different sources.

13.4 Manufacture of marine collagen and gelatin

The yield and properties of collagen and gelatin would be influenced by the

source of the raw material, the nature and concentration of acid or alkali used

Table 13.2 Marine collagen and gelatin sources

Species Tissues

Invertebrate Cuttlefish, octopus and squid Outer skin and cartilaginous tissuesJellyfish Exumbrella and mesogelaStarfish Body wallSea urchin Test (or shell)Sea cucumber Body wall

Sea mammal Seal and whale Skin

Fish Jawless fish Skin, notochord and cartilaginous tissuesCartilaginous fish Skin and cartilageBony fish Skin, scale. bone, and swim bladder

Collagen and gelatin from marine by-products 281

during pretreatment, the temperature and time of pretreatment and extraction,

and other process variables depending on the details of the process selected.

13.4.1 Manufacture of collagen

Collagens, based on the different preparation processes, can be divided into salt

soluble collagen, acid soluble collagen (ASC), and pepsin soluble collagen

(PSC). Although a few studies applied cold neutral salt solutions for collagen

extraction (Young and Lorimer, 1960, 1961), the soluble fraction obtained by

this method has a very low collagen yield and contains a large portion of non-

collagenous proteins. So, in this section only the acid process and the enzymatic

method will be covered (Fig. 13.1).

Pretreatment

Since collagen is only one of the constituents of the raw materials, before

collagen extraction, one or several pretreatments might be applied to remove the

contaminants and increase the purity of the final extract.

Non-collagenous proteins, pigments, and lipids are usually treated as

contaminants during collagen extraction. Alkaline pretreatment can be used to

remove non-collagenous proteins and pigments (Nagai et al., 2002), and a

diluted NaOH solution is widely used. To remove lipids, the raw materials can

be treated with 10% butyl alcohol, and washed with distilled water (Nagai and

Suzuki, 2000); a diluted alkaline solution can also partly satisfy this function.

Some raw materials, such as fish bones (Nagai and Suzuki, 2000), fish scales

(Kimura et al., 1991; Nomura et al., 1996), the testes of sea urchin (Omura et al.,

Fig. 13.1 Manufacturing procedures for marine collagen.


1996) and the body wall of starfish (Kimura et al., 1993) are highly calcified.

Two different solvents can be used for demineralization: 0.05M ethylenedi-

aminetetraacetic acid (EDTA) in neutral buffer (Nagai and Suzuki, 2000) or HCl

solution (Nomura et al., 1996). Both solvents can remove most of the Ca in the

raw materials; but loss of protein is higher in treatments using HCl solution

compared to those using 0.05M EDTA. In addition, collagen may undergo a

partial hydrolytic deterioration during the demineralization under acid condition.

Thus, the demineralization is better done with an EDTA treatment (Nomura et

al., 1996).

Acid extraction process

The acid extraction process has been widely used for collagen preparations. The

most common acid extraction condition uses a 0.5M acetic acid solution

(Kimura and Ohno, 1987; Kimura et al., 1991; Nomura et al., 1996; Sivakumar

and Chandrakasan, 1998; Nagai and Suzuki, 2000; Sadowska et al., 2003;

Muyonga et al., 2004a). In some other cases, acid processes with citric acid

solutions are applied (0.1M cold citrate buffer at pH 3.5, in Young and Lorimer,

1960; 0.5M citric acid, in Sadowska et al., 2003). The collagen obtained by an

acid extraction process is usually called acid soluble collagen.

During the acid process, the collagen yield may be influenced by many

factors, such as acid concentration, the ratio of raw material to acid solution,

incubation temperature and incubation time. Increases in acetic acid

concentration from 0.1M to 0.5M result in slight increases in cod skin collagen

yield (from 52% to 59% for minced cod skin, Table 13.3). Increasing the acid

solution portion in an extraction mixture could also increase the yield (Sadowska

et al., 2003); however, because this change may require extra effort to con-

centrate in the subsequent preparation steps, it is generally not technically useful

to do so. In addition, most of the acid extractions are done at 4ëC. Although an

increase in incubation temperature can offer a higher collagen yield (Muyonga

et al., 2004a), it may also cause the degradation of the peptide chains of collagen

(especially for the cold-water fish), which is not desirable for the end product.

The incubation time for acid extraction is usually 24 h or longer (Table 13.3).

Compared to simply prolonging the extraction time, applying several

consecutive extractions may get better ASC yield (Sadowska et al., 2003).

The yield of ASC varies not only with process conditions, but also with raw

materials based on species and tissues (Table 13.3). In addition, Sadowska and

coworkers (2003) suggested that minced cod skin could give a higher yield of

ASC compared to the whole skin (Table 13.3); and some other researchers also

prepared smaller pieces of the raw materials to facilitate the collagen extraction

(Kimura and Ohno, 1987).

By using acid extraction, especially with the consecutive process, a relatively

high collagen yield could be obtained from fish skin and bone. However, for

some marine species, particularly marine invertebrates, the yield of ASC is low

(Table 13.3). Thus, a further extraction process with a partial enzymatic

digestion is often applied to improve collagen yield.


Table 13.3 Extraction conditions and resultant yields of manufactured marine collagens

Raw materials Extraction conditions Yield# Reference

Solution Tempera- Timeture

Octopus, outer skin 0.5M acetic acid 4ëC 2�24 h 5% Nagai and Suzuki (2002)0.5M acetic acid with pepsin 4ëC 48 h 50% Nagai and Suzuki (2002)

Squid, cranial cartilage 0.5M acetic acid 4ëC ± 12% Sivakumar and Chandrakasan (1998)0.5M acetic acid with pepsin 4ëC ± 60% Sivakumar and Chandrakasan (1998)

Cod, skin (minced) 0.1M acetic acidb 4ëC 24 h 52% Sadowska et al. (2003)0.25M acetic acidb 4ëC 24 h 54% Sadowska et al. (2003)0.5M acetic acidb 4ëC 24 h 59% Sadowska et al. (2003)

Cod, skin (whole) 0.5M acetic acidb 4ëC 24 h 20% Sadowska et al. (2003)0.5M citric acidc 4ëC 24 h 10±25% Sadowska et al. (2003)0.5M citric acidc 4ëC 3�24 h 70±90% Sadowska et al. (2003)

Carpa, bones 0.5M acetic acid 4ëC 24 h 20% Kimura et al. (1991)Carpa, scales 0.5M acetic acid 4ëC 24 h 7% Kimura et al. (1991)Japanese sea bass, bone 0.5M acetic acid 4ëC 72 h+48 h 41% Nagai and Suzuki (2000)Japanese sea bass, skin 0.5M acetic acid 4ëC 72 h+48 h 51% Nagai and Suzuki (2000)Nile percha (adult), skin 0.5M acetic acid 15ëC 16 h 59% Muyonga et al. (2004a)Nile percha (young), skin 0.5M acetic acid 15ëC 16 h 63% Muyonga et al. (2004a)Ocellate puffer fish, skin 0.5M acetic acid 4ëC 72 h 11% Nagai et al. (2002)

0.5M acetic acid with pepsin 4ëC 48 h 45% Nagai et al. (2002)Sardine, scales 0.5M acetic acid 4ëC ± 5% Nomura et al. (1996)

0.5M acetic acid with pepsin 4ëC 96 h 14% Nomura et al. (1996)0.5M acetic acid with pepsin 15ëC 96 h 71% Nomura et al. (1996)

Shark, cartilage 0.5M acetic acid 4ëC ± 20% Sivakumar and Chandrakasan (1998)0.5M acetic acid with pepsin 4ëC ± 54% Sivakumar and Chandrakasan (1998)

#, The yield was on the basis of the dry weight of skin.a, Fresh water fish species; b, the ratio of skin : acid solution was 1:6; c, the ratio of skin : acid solution was in the range of 1:4 to 1:20.

Enzymatic method

To increase the yield of collagen extracted, various enzymes, such as trypsin and

�-amylase, have been used to facilitate the solubilization of collagen (Johns and

Courts, 1977). For marine collagen extraction, extraction with limited pepsin

proteolysis is widely used, and it can be applied alone or right after the acid

extraction process.

Pepsin digestion of collagen is usually done in 0.5M acetic acid at low

temperature, and collagen obtained by this method is called pepsin soluble

collagen. As mentioned before, this method is often applied to those raw

materials where collagen is hard to extract with the acid process alone. By using

this method, the collagen yield from cuttlefish outer skin (Nagai et al., 2001),

octopus outer skin (Nagai and Suzuki, 2002), and fish skin and scales (Nomura

et al., 1996; Nagai et al., 2002) has been significantly improved.

Nomura and coworkers (1996) suggested that the yield and the properties of

the resultant soluble collagen were highly influenced by the incubation tempera-

ture and time. Although increases in temperature and time can increase the yield

significantly, they may also result in a significant amount of low molecular

weight components. Therefore, most of the preparation steps are done at low

temperature (usually 4ëC) and a reasonable time period (24 to 96 h).

13.4.2 Manufacture of gelatin

Most manufacturers produce gelatin, instead of collagen, owing to the wider

industrial uses of gelatin. The ultimate aim in gelatin production is the conver-

sion of collagen into gelatin with a maximum yield and good physicochemical

properties. The yield and qualities of gelatin are influenced not only by the

species or tissue from which it is extracted, but also by the manufacturing

process.

In gelatin manufacture two methods are usually used: the acid process and the

alkaline process. The gelatin prepared by the acid process is called type A

gelatin, while that prepared by the alkaline process is called type B gelatin

(Hinterwaldner, 1977). Although the properties of marine raw materials are

different from those of mammals and avian species, the marine gelatin extrac-

tion processes may still be divided into these two categories: an acid process and

an alkaline process (Table 13.4).

Acid process

For gelatin extraction, the acid process refers to a pretreatment of raw materials

with an acid solution followed by an extraction that is carried out in an acid

medium (Devictor et al., 1995; GoÂmez-GuilleÂn and Montero, 2001; Gilbert et

al., 2002; Arnesen and Gildberg, 2002; Muyonga et al., 2004b). During the acid

pretreatment, the breakage of some inter-chain cross-linkages occurs, which

facilitates the following extraction process. On the other hand, the acid

pretreatment can partly exclude the degradation of collagen by endogenous


Table 13.4 Pretreatment and extraction conditions and the resultant yields of manufactured marine gelatin

Raw materials Pretreatment Water extraction Yield# Reference

Pretreatment steps Temperature

Nile perch* (adult), skin 0.01M H2SO4, 16 h 20±25ëC 50, 60, 70 and 95ëC, each 5 h 16% Muyonga et al. (2004b)Nile perch* (adult), bones 3% HCl, 9±12 d 20±25ëC 50, 60, 70 and 95ëC, each 5 h 2.4% Muyonga et al. (2004b)Nile perch* (young), skin 0.01M H2SO4, 16 h 20±25ëC 50, 60, 70 and 95ëC, each 5 h 12% Muyonga et al. (2004b)Nile perch* (young), bones 3% HCl, 9±12 d 20±25ëC 50, 60, 70 and 95ëC, each 5 h 1.3% Muyonga et al. (2004b)Megrim, skin 0.2M NaOH, 1.5 h; 5ëC; room 45ëC, overnight 7.4% GoÂmez-GuilleÂn et al. (2002)

0.05M acetic acid, 3 hSole, skin 0.2M NaOH, 1.5 h; 5ëC; room 45ëC, overnight 8.3% GoÂmez-GuilleÂn et al. (2002)

0.05M acetic acid, 3 hTilapia*, skin 0.2% NaOH, 2 h; All 15±27ëC 40±50ëC, overnight 15% Grossman and Bergman (1992)

0.2% H2SO4, 2 h;then 1% citric acid, 2 h

Cod, skin 0.1±0.4% NaOH, 2 h; All room 45ëC, overnight 11±14% Gudmundsson and0.1±0.4% H2SO4, 2 h; Hafsteinsson (1997)then 0.4±1.4% citric acid, 2 h

Cod, skin 0.2M NaOH, 1.5 h; 5ëC; room 45ëC, overnight 7.2% GoÂmez-GuilleÂn et al. (2002)0.05M acetic acid, 3 h

Hake, skin 0.2M NaOH, 1.5 h; 5ëC; room 45ëC, overnight 6.5% GoÂmez-GuilleÂn et al. (2002)0.05M acetic acid, 3 h

Lumpfish, skin 0.1M NaOH, 24 h; All 5ëC 55ëC, 1 h 14% Osborne et al. (1990)0.1M HCl, 20 h

Pollock, skin 0.12M Ca(OH)2, 2 h; All 2ëC 50ëC, 3 h 18% Zhou and Regenstein (2004)then 0.1M acetic acid, 1 h

# The yield was on the basis of the wet weight of skin; * Fresh water fish species.

proteases and minimize the enzymatic breakage of intra-chain peptide bonds of

collagen during extraction (Zhou and Regenstein, 2005).

The period of acid pretreatment varies with raw materials, but is usually

within one day. The pretreatment temperature is critical for the acid process;

increasing the temperature may facilitate the pretreatment, but it will cause the

loss of collagen. For sea mammals and warm-water fish, the pretreatment can be

done at room temperature, while for cold-water fish gelatin extractions, the

optimal temperature is lower than 10ëC (Zhou and Regenstein, 2004).

Alkaline process

The alkaline process refers to a pretreatment of raw materials with an alkaline

solution, in most cases followed by the neutralization with an acid solution, so

the extraction may be carried out in an alkaline, neutral or acid medium. One

advantage of this process is that the pretreatment with an alkaline solution can

remove considerable amounts of non-collagenous materials (Johns and Courts,

1977; Zhou and Regenstein, 2005). The alkaline pretreatment also breaks some

inter-chain cross-linkages and excludes the effects of proteases on collagen.

The neutralization with an acid solution provides an optimal weak acid

extraction medium, which guarantees a high extraction yield with good gel

quality.

During all the pretreatments, the temperature should be controlled within a

certain optimal range. For cold-water fish, this temperature is very critical, and

should be kept below 10ëC to avoid the extensive loss of collagen during pre-

treatment processes. During the studies on pollock skin gelatin, Zhou and

Regenstein (2005) also suggested that during the alkaline pretreatment, the type

of alkali does not make a significant difference, but the concentration of alkali is

critical. The acid type and concentration during the acid neutralization process

determines the final pH of the extraction medium, and affects the yield and gel

quality of the gelatin extracts.

13.4.3 Stabilizing marine collagen and gelatin

Marine collagens and gelatins, especially those from fish, have lower melting

points than mammalian gelatins, and care is needed in their preparation and

storage because they are extremely susceptible to microbiological attack and

thermal hydrolysis (Jones, 1977). Fish collagens and gelatins are commercially

available in both concentrated liquid or solid form. The liquid form usually

requires a mixture of methyl and propyl hydroxybenzoates or other preservatives

to prevent bacterial attack (Norland, 1990). The solid collagen and gelatin

usually contain less than 15% moisture. In most situations, collagen and gelatin

are dried and processed into a powder or sheet, and the quality of the final

products is influenced by the methods applied. It has been reported that freeze-

dried gelatins have better gel properties than air-dried gelatins (Gudmundsson

and Hafsteinsson, 1997; FernaÂndez-DõÂaz et al., 2001).


13.5 Properties of marine collagen and gelatin

13.5.1 Chemical properties

The amino acid composition of gelatin is very close to its parent collagen,

and no amino acid sequence rearrangements occur during the collagen-gelatin

conversion. Glycine (Gly), alanine (Ala), proline (Pro) and hydroxyproline

(Hyp) are four of the most abundant amino acids in collagen and gelatin; and

the frequency of occurrence of Gly is about 1 out of every 3 amino acids

(Eastoe and Leach, 1977). The amino acid sequence is characterized by the

repeating sequence of Gly-X-Y triplets, where X is mostly Pro and Y is

mostly Hyp. The presence of Gly at every three residues is a critical

requirement for the collagen super-helix structure. Gly contains no side chain,

which allows it to come into the center of the super helix without any steric

problems to form a close packing structure (te Nijenhuis, 1997). The super-

helix structure is further stabilized by the steric restrictions that are imposed

by the pyrrolidine residues and the hydrogen bonds that are formed between

amino acid residues.

The polypeptide forming collagen is an � chain, which contains about 1000

amino acids and has a MW of around 100 kD depending on the source. The �chain forms a left-handed helix by itself, and three � chains together form a

right-handed super triple helix. The conversion of collagen to gelatin yields

molecules of varying mass: each is a fragment of the collagen chain from which

it is cleaved. Therefore, gelatin is a mixture of fractions varying in MW from 15

to 400 kD (Gelatin Manufacturers Institute of America, 1993). By optimizing

the pretreatment and extraction conditions, a gelatin extract with a higher

molecular weight distribution can be obtained, where certain inter-chain cross-

linkages present in the collagen are destroyed but with less breakage of peptide

bonds.

The amino acid composition of collagen and gelatin may vary, depending

mainly on the source (Tables 13.5 to 13.10). Except for those from sea mam-

mals, marine collagens and gelatins vary according to the living environments of

their sources, particularly with respect to water temperature. In general, those

collagens and gelatins from warm-water fish have a lower amino acid content

than those from mammals, but a higher imino acid content than those from cold-

water fish and ice fish; while those from marine invertebrates are much less

consistent and differ even between similar species. In addition, the amino acid

composition may vary among collagens and gelatins prepared from different

tissues and/or with different methods. During the studies on a fresh water fish,

Nile perch, Muyonga and coworkers (2004a,b) suggested that the maturation

stage of the source could also influence the amino acid composition of the

collagen and gelatin extracts.

13.5.2 Physical properties

The formation of thermo-reversible gels in water is one of gelatin's charac-

teristics. When an aqueous solution of gelatin with a concentration greater than a


critical point is cooled down, it may form a gel. The critical gelation

concentration and temperature depend mainly on the amino acid composition

and the molecular weight distribution of the gelatin.

Gel strength is the major physical property of gelatin gels, and the

commercial value of gelatins is principally based on their gel strength. Besides

the influence of amino acid composition and molecular weight distribution of

the gelatin itself, the strength of a gelatin gel also varies with gelatin con-

centration, thermal history (gel maturation temperature and time), pH, and the

presence of any additives (Choi and Regenstein, 2000). In addition, the size and

shape of the container used to form the gelatin gel and the parameters of the

instrument applied in the determination of gel strength will also affect the final

value. Thus, bloom strength, the gel strength determined by the standard bloom

method at 10ëC with certain well-defined requirements (Wainewright, 1977)

was developed to standardize the test and it has become the most critical

standard used to commercially assess the grade and quality of a gelatin. A

detailed review of gel strength and its determination can be found in Waine-

wright (1977). The gel strength of most commercial gelatin varies from less than

100 bloom to more than 300 bloom. Although warm-water fish gelatin gels can

Table 13.5 Amino acid composition of collagen from ice fish

R. glacialis T. eulepiodotus T. leonnbergiiSkin Skin SkinASC ASC ASC

Ala 83 99 103Arg 49 47 47Asx 61 63 57Cys ± ± ±Glx 77 73 70Gly 332 339 350His 7 8 8Hyl 3 5 5Hyp 47 45 47Ile 15 13 11Leu 45 30 28Lys 23 32 32Met 11 12 10Phe 16 15 15Pro 104 98 100Ser 70 67 65Thr 30 28 26Trp ± ± ±Tyr 2 3 4Val 25 23 22Amino acids 151 143 147Total 1000 1000 1000Reference Rigby (1968) Rigby (1968) Rigby (1968)

ASC, acid soluble collagen.


reach a bloom strength of 300, some cold-water fish gelatin solutions remain

liquid at 10ëC (Norland, 1990).

As a thermo-reversible gel, a gelatin gel will start melting when the

temperature increases above a certain point. This point is called the melting

point and is usually lower than the human body temperature. This melt-in-the-

mouth property has become one of the most important characteristics of gelatin

gels, and is widely applied in the food and pharmaceutical industries. No other

biopolymer has this unique property, although many efforts have been made to

find a substitute for gelatin. The melting point of gelatin from cow and pig

ranges from 30 to 33ëC, but fish gelatins show lower melting temperatures than

mammalian gelatin, due to their lower imino acid content. The melting point for

warm-water fish gelatin usually ranges from 23 to 29ëC, while cold-water fish

gelatin may melt around 10ëC.

Viscosity is the other commercially important property of gelatin samples.

Low viscosity gives short, brittle gelatin gels; while high viscosity gives tougher

Table 13.6 Amino acid composition of collagen and gelatin from cold-water fish

Alaska pollock Cod Hake Lumpfish SalmonSkin Swim Skin Skin Skin Skin

bladderASC GB ASC ASC GB GB GB ASC

Ala 108 114 114 107 96 119 100 106Arg 51 51 51 51 56 54 54 52Asx 51 54 52 52 52 49 53 52Cys ± ± ± ± ± ± 3 ±Glx 74 75 75 75 78 74 62 77Gly 358 365 348 345 344 331 333 361His 8 6 8 8 8 10 7 10Hyl 6 0 8 6 6 5 8 8Hyp 55 59 52 53 50 59 65 58Ile 11 9 12 11 11 9 11 10Leu 20 19 22 23 22 23 24 19Lys 26 27 25 25 29 28 28 25Met 16 12 17 13 17 15 12 16Phe 12 10 13 13 16 15 17 12Pro 95 96 94 102 106 114 112 104Ser 63 65 65 69 64 49 58 51Thr 25 24 24 25 25 22 23 22Trp ± ± ± ± ± ± ± ±Tyr 3 2 4 5 3 4 3 2Val 18 11 16 19 18 19 27 15Imino acids 150 155 146 155 156 173 177 162Total Aa 1000 1000 1000 1002 1001 999 1000 1000Reference a b a c d d e f

ASC, acid soluble collagen; GB, alkaline processed gelatin.References: a, Kimura and Ohno, 1987; b, Zhou and Regenstein, 2005; c, Rigby, 1968; d, GoÂmez-GuilleÂn et al., 2002; e, Osborne et al., 1990; f, Matsui et al., 1991.


and extensible gels. For many applications, gelatins of high viscosity are

preferred and command higher prices, given that other properties are equal

(Wainewright, 1977). Besides the chemical properties of the gelatin itself, the

viscosity of gelatin depends on its concentration, temperature, pH and the

presence of additives. Molecular weight distribution appears to play a more

important role in the effect on viscosity than it does on gel strength and melting

point. The viscosity of gelatin is normally measured at 60ëC and a 6.6% gelatin

concentration, in both Europe and North America. Most commercial gelatins

have a viscosity between 15 and 75mPs (Gelatin Manufacturers Institute of

America, 1993; Poppe, 1997).

It is worth noting that no single physical property can adequately convey the

texture and sensory appreciation of a gel by a consumer, and an overall

evaluation of gelatin combining all the critical physical properties may be

necessary.

Table 13.7 Amino acid composition of collagen and gelatin from warm-water fish andhot-water fish

Warm-water fish Hot-water fishMackerel Megrim Puffer fish Sole Tilapia* Lungfish*Skin Skin Skin Skin Skin SkinASC GB PSC GB GB G

Ala 124 123 106 122 123 126Arg 55 54 54 55 47 54Asx 49 48 50 48 48 44Cys ± ± 2 ± ± ±Glx 72 72 87 72 69 77Gly 334 350 351 352 347 327His 6 8 8 8 6 5Hyl 6 5 5 8 6Hyp 66 60 67 61 79 78Ile 9 8 12 8 8 10Leu 23 21 23 21 23 20Lys 26 27 19 27 25 24Met 14 13 14 10 9 3Phe 14 14 10 14 13 14Por 108 115 103 113 119 129Ser 44 41 48 44 35 42Thr 27 20 25 20 24 24Trp ± ± ± ± ± ±Tyr 3 3 4 3 2 1Val 20 18 17 17 15 18Imino acids 174 175 170 174 198 207Total Aa 1000 1000 1000 1000 1000 1002Reference a b c b d e

* Fresh water fish species.ASC, acid soluble collagen; PSC, pepsin soluble collagen; GB, alkaline processed gelatin; G, gelatin.References: a, Kimura, 1983; b, GoÂmez-GuilleÂn et al., 2002; c, Nagai et al., 2002; d, Sarabia et al.,2000; e, Eastoe and Leach, 1977.


13.5.3 Nutritional properties

Since the nutritional quality of proteins can vary greatly and is affected by many

factors, it is important to have standards for evaluating quality. Quick

assessment of a protein's nutritive value can be obtained by determining its

content of amino acids, and comparing it with the essential amino acid pattern of

an ideal reference protein (Fennema, 1996). The ideal pattern of essential amino

acids in proteins (reference protein) for preschool children (2±5 years) is used as

the standard for all age groups except infants (Table 13.11). Collagen or gelatin

completely lacks the essential amino acid Trp and is deficient in several others

(Table 13.11), and it has a chemical score of 0 out of 100. Therefore, gelatin

alone is of very low nutritive value. Many studies performed to evaluate the

nutritional value of collagen/gelatin attest to the inadequacies of dietary

collagen/gelatin as a nutritional protein source. It has been suggested that diets

Table 13.8 Amino acid composition of collagen and gelatin from jawless fish andcartilaginous fish

Jawless fish Cartilaginous fishHagfish Shark DogfishSkin Skin SkinASC ASC PSC G ASC

Ala 108 123 124 119 106Arg 52 54 54 50 51Asx 46 41 41 43 43Cys ± 0 0 ± ±Glx 58 75 74 66 68Gly 367 321 323 333 338His 8 9 9 7 13Hyl 5 9 9 5 6Hyp 59 67 69 79 60Ile 13 19 19 19 15Leu 16 25 24 24 25Lys 19 25 24 24 27Met 16 16 16 10 18Phe 10 14 13 14 13Pro 98 111 111 113 106Ser 67 43 42 45 61Thr 31 22 23 26 23Trp ± ± ± ± ±Tyr 4 3 1 1 3Val 23 23 24 22 25Imino acids 157 178 180 192 166Total Ala 1000 1000 1000 1000 1001Reference a b b c d

ASC, acid soluble collagen; PSC, pepsin soluble collage; G, gelatin.References: a, Kimura and Matsui, 1990; b, Yoshimura et al., 2000; c, Eastoe and Leach, 1977; d,Piez et al., 1963.


containing inadequate amounts of essential amino acids might threaten the

health of adults and depress the normal growth of children (Fennema, 1996). In

addition, over-consumption of any particular amino acid can lead to an àmino

acid antagonism' or toxicity. Excessive intake of one amino acid often results in

an increased requirement for other essential amino acids. This is due to

competition among amino acids for absorption sites on the intestinal mucosa

(Fennema, 1996).

The nutritional quality of a protein that is deficient in an essential amino acid

can be improved by mixing it with another protein that is rich in that essential

amino acid, or by supplementing it with essential free amino acids that are

under-represented (Fennema, 1996). However, in the case of collagen/gelatin as

a major protein in the diet, it will be very difficult to achieve a satisfactory

improvement, because collagen/gelatin not only contains no Trp but also is

deficient in several other essential amino acids.

Although gelatin alone cannot serve as a main dietary protein, as a supple-

mentary protein, it may have some advantages. Gelatin hydrolysates are used

Table 13.9 Amino acid composition of gelatin from mammals

Harp seal Minke whale Pork CattleSkin Skin Skin SkinGA GA GA GB

Ala 108 104 112 112Arg 52 53 49 46Asx 45 48 46 46Cys 0 0 ± ±Glx 76 80 72 71Gly 316 302 330 333His 6 6 4 5Hyl 7 9 6 6Hyp 101 85 91 98Ile 10 12 10 12Leu 25 28 24 23Lys 26 30 27 28Met 5 6 4 6Phe 14 15 14 12Pro 120 126 132 129Ser 38 40 35 37Thr 23 29 18 17Trp 0 0 ± ±Tyr 4 5 3 2Val 23 22 26 20Imino acids 221 211 223 227Total Aa 999 1000 1003 1003Reference a a b b

GA, acid processed gelatin; GB, alkaline processed gelatin.References: a, Arnesen and Gildberg, 2002; b, Eastoe and Leach, 1977.


Table 13.10 Amino acid composition of collagen from marine invertebrates

Cuttlefish Jellyfish Sea cucumber Sea urchin Starfish

S. lycidas S. officinalis S. meleagris S. japonicus A. ijimai A. amurensis A. pectinifera A. planci

Outer skin Cartilage Cornea Exumbrella Body wall Test Body wall Body wall Body wallPSC PSC PSC PSC PSC PSC PSC PSC PSC

Ala 83 65 65 82 99 78 113 123 128Arg 56 47 46 52 52 59 55 59 55Asx 64 105 102 79 74 61 59 57 66Cys 2 ± ± ± ± ± 0 0 1Glx 92 72 67 98 112 94 85 86 75Gly 318 326 348 309 278 325 342 346 342His 18 5 4 2 7 5 2 2 2Hyl 19 14 27 9 9 5 4 6Hyp 90 126 127 40 48 84 60 67 76Ile 21 16 15 22 24 11 17 11 15Leu 29 23 21 34 30 28 17 13 13Lys 13 8 7 38 17 7 14 14 13Met 1 10 10 4 11 12 13 14 10Phe 10 8 7 10 18 4 3 3 2Pro 98 96 94 82 85 97 94 90 100Ser 48 28 27 45 48 56 69 48 39Thr 29 20 18 35 42 41 25 33 28Trp ± ± ± 0 ± ± ± ± ±Tyr 5 2 2 6 14 6 5 6 6Val 23 18 17 35 32 23 22 24 23Imino acids 188 222 221 122 133 181 154 157 176Total Aa 1000 994 991 1000 1000 1000 1000 1000 1000Reference a b b c d e f f f

PSC, pepsin soluble collagen.References: a, Nagai et al., 2001; b, Sivakumar et al., 2003; c, Nagai et al., 1999; d, Saito et al., 2002; e, Omura et al., 1996; f, Kimura et al., 1993.

widely as nutritional supplements due to their high protein content, availability,

ease of preparation, ease of addition, and low cost.

13.6 Food applications

Collagen and gelatin are usually used as functional ingredients rather than

nutritional ingredients in food applications.

13.6.1 Food applications of collagen

Collagen can be used to produce edible casing for sausages. Sausage casings

were originally derived from the gastrointestinal tract of cattle, sheep and pig.

However, with the rapid growth in demand for sausage products, the collagen

casings were developed, and they had some advantages over the natural gut

casings, due to the convenience, economic efficiency and uniformity of the

regenerated collagen casings (Hood, 1987). Collagen can also be used as a

clarification agent to remove the colloidal suspensions during production of

alcoholic drinks and fruit juices (Courts, 1977).

13.6.2 Food applications of gelatin

Water desserts

Gelatin's largest single use is in water gel desserts. Gelatin desserts can be

traced back to 1845 when a US patent was issued for `portable gelatin' in

Table 13.11 Recommended essential amino acid pattern for food proteins and theessential amino acid pattern of gelatin powder

Essential Aa Recommended pattern1 Pork skin Cod skin Tilapia skin(mg/g protein) gelatin2 gelatin2 gelatin3

Infant Preschoolchild

His 26 19 6 8 6Ile 46 28 12 11 8Leu 93 66 30 22 23Lys 66 58 38 29 25Met + Cys 42 25 5 27 9Phe + Tyr 72 63 27 19 15Thr 43 34 20 25 24Trp 17 11 0 0 0Val 55 35 28 18 15Total 434 320 165 159 125

1 The ideal pattern of essential amino acids in proteins (reference protein) for preschool children isused as the standard for all age groups except infants, Fennema, 1996; 2 Sarabia et al., 2000;3 GoÂmez-GuilleÂn et al., 2002.


desserts. The current US market for gelatin desserts exceeds 100 million pounds

(approximately 45 000 tons) annually.

Gelatin desserts consist of mixtures of gelatin powder, sweeteners, acids and

compounds to offer the desirable flavor and color. Some other biopolymers,

such as agar and carrageenan, can also form thermally reversible gels with

water. However, the main difference between gelatin and the polymers from red

seaweed is the low melting point of gelatin gels, which is usually lower than the

body temperature and makes gelatin gels melt-in-the-mouth. The former study

from our laboratory has suggested that gelatin dessert made of gelatin from

warm-water fish, which has a lower melting point than mammalian gelatin,

showed a better release of aroma and gave a stronger flavor than one made of

pork gelatin (Choi and Regenstein, 2000). Furthermore, gelatins of high

viscosity give chewier jellies than gelatins of low viscosity, which are more

brittle (Jones, 1977).

Dairy products

Gelatin is used in dairy products as a stabilizer and a texturing agent. It is widely

used in yogurt, ice cream and other dairy products. Gelatin is added to yogurt to

reduce syneresis and to increase firmness. Gelatin is an ingredient compatible

with milk proteins, and gives a fat-like sensory perception because of its unique

property of melt-in-the-mouth. It also masks the product flavor less than some

other gums (Jones, 1977). Potentially, the use of different concentrations of

gelatin would make it possible to obtain a wide range of textures, from the

creamy, slightly gelled texture of yogurt to the firm, `moldable' gel of curd. In

ice cream, gelatin is used to prevent the formation of coarse ice crystals, and to

give body and a firm smooth texture (Jones, 1977). The gelatin concentration

required for ice cream depends on its bloom strength and other factors such as

melting point (Jones, 1977).

Although gelatin has unique characteristics and is widely used in dairy

products, there are continuing attempts to replace it with food polysaccharides.

This is because the gelatin used in current dairy products is mainly from pig or

non-religious slaughtered beef and is unacceptable to Jewish and Muslim

consumers. Based on the gelatin status, the dairy products in the market can be

divided into two main groups: those where gelatin is replaced with pectin,

modified starch, carrageenen, carob bean gum, or locus bean gum; and the other

where it does contain a gelatin that is not permitted by any of the major US

certifying agencies. Gelatins from kosher and halal fish species, which include

both warm-water fish and cold-water fish, may be a promising solution. Some

dairy products using fish gelatins have appeared in the market place. Furthermore,

fish gelatins can have a broad range of melting points, which may contribute more

choices in designing the texture and melting properties of dairy products.

Other food products

Gelatin has been used in confectionery products such as gummy-type products

and marshmallows. Gummy-type products contain gelatin as the main gelling


agent, because it offers the right texture and mouth feel. Marshmallow usually

contains about 2±3% gelatin, in which it serves as a stabilizer and whipping

agent (Jones, 1977). In recent years, marshmallows made from fish gelatin have

become commercially available. Gelatin can also be applied in wine fining and

juice clarification, in meat products to absorb meat juices, and to give form and

structure to products that would otherwise fall apart (Gelatin Manufacturers

Institute of America, 1993). Further information about food applications can be

obtained from the reviews by Jones (1977), Johnston-Banks (1990), and Poppe

(1997).

13.7 Non-food applications

The non-food uses of collagen and gelatin are in the pharmaceutical industry,

photographic industry, and other technical fields such as paper manufacture and

printing processes (Gelatin Manufacturers Institute of America, 1993).

13.7.1 Pharmaceutical applications

Pharmaceutical gelatin accounts for a significant proportion of the total

production (Wood, 1977), and it is used in the manufacture of capsules, tablets

and pastilles. The use of gelatin to produce capsules accounts for the largest

usage in pharmaceutical applications. Hard gelatin capsules are usually made

from high bloom strength gelatin with a small quantity of edible dyes; while soft

gelatin capsules are made from a medium grade gelatin with added plasticizers

such as propylene glycol, sorbitol, glycerin and other approved mixtures (Wood,

1977; Gelatin Manufacturers Institute of America, 1993). Gelatin can act as a

binding agent in tablets; and it is also used for tablet coating to reduce dusting,

mask unpleasant tastes, and allow for printing and color coatings for product

identification (Gelatin Manufacturers Institute of America, 1993). Glycerinated

gelatin or gelatin/gum arabic is used as a base for pastilles, serving as a binder of

the ingredients (Wood, 1977). In addition, gelatin is used to produce micro-

encapsulated oils for various pharmaceutical applications; to form a sterile and

water insoluble sponge to control bleeding during surgery; and to serve as an

adhesive in protective dressings.

13.7.2 Photographic applications

Gelatin has been used for photographic emulsions for more than 100 years, and

is still the principal constituent of the binder in most commercial photographic

films and papers (Kragh, 1977). During the preparation of photographic products

that are based on silver halide technology, gelatin prevents flocculation of silver

halide crystals in solutions of high ionic strength, facilitates washing of the

emulsion, controls the digestion process so that high photographic speeds can be

obtained with low fog, and serves many other functions (Kragh, 1977).


13.8 Improving the quality of collagen and gelatin

13.8.1 Extraction process optimization

As mentioned earlier in the manufacture section, the quality of collagen and

gelatin is influenced not only by the species or tissue from which it is extracted,

but also by the extraction process. For each specific source, an optimization of

the extraction procedure would be necessary to improve the quality of extracts.

One important step for process optimization is to determine the critical con-

trol variables. In an earlier study on cod skin gelatin extraction, Gudmundsson

and Hafsteinsson (1997) suggested that the alkaline and acid concentrations

during pretreatments would affect gelatin quality. In a subsequent study on

megrim, GoÂmez-GuilleÂn and Montero (2001) also suggested that the type of

acids used in the extraction might affect the gel properties of gelatin from

megrim skin. With a fractional factorial design, Zhou and Regenstein (2004)

further confirmed the importance of the pretreatment alkaline and acid concen-

trations on the quality of gelatin extracts. To obtain a high quality gelatin

extract, the alkaline concentration should be high enough to remove the non-

collagenous proteins and exclude the effect of proteases, and the acid concen-

tration should be in a proper range to offer an optimal weak acid extraction

medium (Zhou and Regenstein, 2004, 2005). Too strong an alkaline or acid

extraction medium can cause significant degradation of the peptide chains, and

result in an extract with poor quality. To guarantee a reasonable yield, the

pretreatment temperature and the extraction temperature should also be

controlled within a proper range.

13.8.2 Enzymatic modification

Collagen and gelatin from fish, especially the cold-water fish, have low gelling

temperatures and melting points. However, many applications require gelatin to

gel and keep its gel form at room temperature. A possible way of improving the

characteristics of a given fish gelatin is to use transglutaminase (TGase). The

enzyme TGase can cross-link gelatin chains by catalyzing the reaction between

a lysine residue and a glutamine residue, and make covalent chemical cross-

linkages within the gelatin network (Babin and Dickinson, 2001).

Several studies have been done on the effects of TGase on fish gelatins from

megrim, cod or hake (GoÂmez-GuilleÂn et al., 2001; FernaÂndez-DõÂaz et al., 2001;

Kolodziejska et al., 2004). The significance of TGase influence varies between

different gelatins, and also depends on the enzyme quantity, gelatin concen-

tration, and whether cross-linking occurs predominantly before or after the

gelatin gel develops. After covalent cross-linking by TGase, the gelatin gel's

characteristic thermo-reversible character is partly or completely lost. To obtain

the enhanced gel strength for the final product, TGase should not be added over

an appropriate enzyme concentration, and to get the desired cross-linking

generally requires that the reaction occurs during or after gel development.

Adding a high concentration of TGase or cross-linking before gelation would be

detrimental to gel strength (Babin and Dickinson, 2001). Kolodziejska and


coworkers (2004) also suggested that gelatin solutions with a high protein

concentration could form better gels using TGase modification than those with a

low concentration. Thus, modification with TGase under certain conditions,

could improve fish gelatin's properties and produce a gelatin gel at room tem-

perature, even for the cold-water fish gelatin. But for each gelatin, a thorough

study on the enzyme activity and optimizing the reaction is necessary.

13.8.3 Removing impurities

The extracted marine collagen and gelatin may contain some impurities, such as

insoluble particles, inorganic salts, pigments, and compounds responsible for the

unpleasant fish flavors. The insoluble particles can be removed by centrifugation

and/or filtration; inorganic salts are traditionally removed by ion exchange

technology (Gelatin Manufacturers Institute of America, 1993), but they can

also be removed by ultrafiltration, which concentrates the extract at the same

time (Chakravorty and Singh, 1990; Simon et al., 2002); and pigments and some

of the compounds causing the unpleasant fish flavors might be removed by

activated charcoal.


This chapter only covers a limited introduction to collagen and gelatin from

marine by-products. For a more thorough understanding of collagen and gelatin,

additional references should be consulted. The oldest review on gelatin was a

book entitled Glue and Gelatin by Alexander (1923). Half a century later, Ward

and Courts (1977) edited an excellent book of contributed chapters, The Science

and Technology of Gelatin, which covers almost every area related to gelatin

and is still a very useful source of information for current collagen and gelatin

producers and users, and for researchers. The book edited by Pearson, Dutson

and Bailey (1987), on the other hand, gives a solid introduction to collagen. The

book by Veis (1964) mostly focuses on the chemical properties of collagen and

gelatin. For those who are interested in the rheological properties of gelatin, the

review article by te Nijenhuis (1997) would be very helpful. There are also

several brief reviews on gelatin, including those by Johnston-Banks (1990) and

Poppe (1997). In addition, the article prepared by the Gelatin Manufacturers

Institute of America (1993) would give a good introduction to gelatin from the

point of view of the gelatin manufacturers.

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14.1 Introduction

In recent years, there has been a significant decrease in fishery resources

worldwide due mainly to overfishing coupled with poor management. In

response, serious efforts have been made to utilize underexploited resources and

processing by-products. In the last few years, several conferences (Keller, 1990;

Bechtel, 2002) have been held in the United States to review the progress and

developments in the seafood processing by-product utilization. The following is

a product list of commercial importance or in development.

Finfish:

· Fish meal, oil and bone meal

· Fish hydrolysate (produced by acid or enzyme)

± Aquaculture feeds

± Silage for animal feeds

± Flavour extracts

· Pet foods

· Hydrolyzed fish protein (HFP)

± Functional food ingredients

± Aqua-feed for immatured digestion system of juvenile.

Shellfish:

· Shell meal (e.g., squid, shrimp) for aqua-feeds

· Chitin and chitosan from shell

· Enzymatic hydrolysate for flavor extracts

· Mechanical recovery of shell meat.

14

Seafood flavor from processingby-productsC. M. Lee, University of Rhode Island, USA

The utilization strategy should take several important factors into consideration.

They include resource availability at a low cost, market demand for end

products, affordable production cost, and technology availability.

In general, the processing by-products make up from a half to two-thirds of

the incoming raw material of finfish to shellfish. One of the viable approaches to

convert these by-products into commercially profitable products is `seafood

flavor manufacturing' since flavor is considered a high value product and good

quality seafood flavors are in high demand. The production of natural seafood

flavor extracts from the process by-products has been an industrial practice in

France and Japan. As a common industrial practice, the natural seafood flavors

are reformulated by adding other ingredients and artificial flavors for flavor

characteristics desired. Seafood flavors are being used in seafood sauces,

chowders, soups, bisques, instant noodles, snacks and surimi seafoods. In

seafood entreÂe, the use of good flavored sauce is critical to the acceptability of

the meal. An analogy is one may not eat salad without dressing of one's choice.

Generally, there are three basic process routes for making seafood flavors

from finfish and shellfish processing by-products, namely, aqueous extraction,

fermentation, and enzymatic hydrolysis.

14.2 Aqueous extraction

This process has been widely employed since it is simple, generates a good

quality flavor, and is often done as part of the existing process.

Raw material! (homogenize)! cooking! (press)! raw extract! concentration

The steps in parenthesis can be omitted depending upon the process employed.

The cooking can be done under either atmospheric or elevated pressure. The good

examples of aqueous extraction process are extracts which are generated during

cooking of clam, crab or lobster. The drawbacks of this process are a low yield, its

dependency on cooking juice, and its high salt level upon concentration.

However, it does provide the highest level of flavor and aroma retention of the

original material. Ochi (1980) stressed that the freshness of the raw material is the

most important requirement in ensuring consistent and high quality finished

products. Preparation and product characterization of water-extracted flavors in

the form of cooking juice or processing wash water have been reported for clam

(Joh and Hood, 1979; Burnette et al., 1983; Reddy et al., 1989).

14.3 Fermentation

Fermentation has been a traditional practice of producing various fish sauces in

Asia (Lee et al., 1993). The process involves enzymatic hydrolysis by endogen-

ous enzymes with some level of flavor-producing microbial activity. This makes

the flavors derived from fermentation different in flavor characteristics from

Seafood flavor from processing by-products 305

other flavor production processes. It requires a very high level of salt; as high as

30% to control the growth of any pathogenic microorganisms (Gildberg, 1993).

salt/pH adjustment/starter culture

#Raw material ! autolysis (proteolytic digestion) ! (press) ! raw extract

! filtration/clarification

A special interest has been taken to shorten the maturation period from 6±12

months to 1±2 months by lowering pH and salt concentration which hasten

proteolysis as well as microbial fermentation. To vary the flavor characteristics,

starter cultures of interest can be added at the beginning of fermentation. The

resulting extract from the low salt fermentation can be stabilized by concen-

tration or dehydration and used as an umami-giving seafood flavor, not as fish

sauce for soup and sauce applications.

14.4 Enzymatic hydrolysis

Enzymatic hydrolysis is a process which is being employed for the production of

most commercial natural seafood flavor extracts.

enzyme (protease)

#Raw stock ! homogenize (pasteurize) ! hydrolysis ! enzyme inactivation by

heating ! filtration for raw extract ! concentration/dehydration

As more high-performance commercial enzymes become available, highly

acceptable seafood flavors can be mass-produced through hydrolysis of various

seafood processing by-products. Most successful products in the market are

clam, crab, shrimp, and lobster extracts. Also available are flavors from various

fish species such as tuna, bonito, salmon and mackerel.

The type of enzyme and hydrolysis conditions used for the fish protein

hydrolysate and seafood flavor production may differ from each other.

Endoprotease, such as Alcalase (Novozymes North America, Franklinton, NC)

prepared from Bacillus spp, is widely accepted for the production of fish protein

hydrolysate which prefers a high degree of hydrolysis (DH) for high solubility

and digestibility. The technical information on the use of enzyme for the

crustacean and fish flavor production is limited. According to the seafood flavor

production at Isnard Lyraz (Queven, France) (In, 1990a,b), the process consists

of liquefaction, separation, and concentration. Liquefaction is essentially an

enzymatic hydrolysis which releases naturally occurring flavoring substances

from the raw material and facilitates a separation process by solubilizing

insoluble proteins, the main constituent of the seafood process by-products.

Separation is to remove shells and bones and is done by filtration, but sometimes

accompanied by chromatography which is employed to remove undesirable

components such as off-flavors and pigments (In, 1990b). Both endoprotease


and exopeptidase have to be considered in flavor-producing enzymatic

hydrolysis. Enzymatic hydrolysis changes the original amino acid profile and

concentration. It also generates peptides of different molecular weights. Amino

acids and peptides can further react with reducing sugars or other aldehydes and

carbonyl groups in the solution and contribute many novel volatile compounds

to the hydrolysate. Thus, enzymatic hydrolysis has a significant effect on both

taste and aroma.

Enzyme selection and hydrolysis optimization based on the degree of

hydrolysis (DH) were studied in the crayfish (Baek and Cadwallader, 1995) and

crab processing by-product (Baek and Cadwallader, 1999). Production of protein

hydrolysate for flavor from lobster body was studied by Vieira et al. (1995a,b).

The tyrosine yield by enzymatic hydrolysis was used as a response in evaluating

the effect of different hydrolysis conditions, which included reaction time,

enzyme type and enzyme concentration. They concluded that the use of

enzymatically-produced hydrolysates as flavorants was potentially promising.

Overall, the available technical information on crustacean flavor production

from the processing by-products is limited despite ongoing industrial

production. The advantages of enzymatic hydrolysis over other methods are

high yield, yet good quality with less off-flavor generated, and control of flavor

characteristics through variation of enzyme reactions. The key consideration is

the selection of the right type of enzyme for a given raw material. In the past, the

main problem with enzymatic production of seafood flavor was the formation of

the bitter flavor. Now, with new generation enzyme system that comes with both

endoprotease and exopeptidase, a flavor can be enzymatically produced without

bitterness. Several seafood flavor manufacturers have been successfully pro-

ducing seafood flavors through enzymatic hydrolysis. Among them are Isnard

Lyraz (France), Hasegawa (Japan-USA), Takasago (Japan-USA), and Givoudan

Roure (USA).

14.4.1 Source of enzymes for hydrolysate production

After evaluation of several commercial proteases for their performance in terms

of the organoleptic quality of produced hydrolysates and the rate of hydrolysis,

Protamex and Flavourzyme (Novozyme North America, Franklinton, NC) were

chosen for further study (Yang and Lee, 2000). Others tested were Alcalase,

Neutrase (Novozyme North America, Franklinton, NC), Optimase, and HT-

Proteolytic 200 (Solvay Enzymes, Elkhart, IN). Protamex is a Bacillus protease

complex developed for hydrolysis of food proteins. In contrast to most other

endoproteases, Protamex is claimed to produce non-bitter protein hydrolysates.

It had declared activity of 1.5 Anson units per gram (AU/g) and optimal reaction

conditions at pH 5.5±7.5 and 40±60ëC. Protamex can also be used together with

Flavourzyme for extensive protein hydrolysis. For this purpose, the dosage of

Protamex suggested by the enzyme supplier is 6±15 kg per ton of protein (0.6±

1.5%) at 55ëC for 15±30 min before Flavourzyme is added. Flavourzyme is

produced by a strain of Aspergillus oryzae and consists of several enzymes, both


endoproteases and exopeptidases, each with different activities and pH optima.

The exopeptidase activities remove terminal amino acids that may cause bitter-

ness. Flavourzyme is best used under neutral or slightly acidic conditions, and its

optimal pH and temperature are in the range of 5.0±7.0 and 50±55ëC, respec-

tively. It has a declared activity of 1,000 leucine aminopepeptidase units per

gram (LAPU/g), and its suggested usage level is 5±10 LAPU/g protein (0.5±

1.0%) for flavor generation. For extensive protein hydrolysis, a dosage of 10±50

LAPU/g (1±5%) is recommended.

14.4.2 Bitterness generation from enzymatic hydrolysis

Flavor production by enzymatic hydrolysis comes with a problem associated with

generation of bitterness which is known to be caused by the exposure of the

hydrophobic peptides and amino acids. Some type of exopeptidase such as

Flavourzyme can break up bitter-causing peptides. To be effective in hydrolyzing

and debittering, the enzyme should contain both endoprotease for hydroysis of

protein and exopeptidase for breaking down bitter peptides (In, 1990b).

14.4.3 Source of raw material

The sources of raw material for enzymatic production of seafood flavor are:

Finfish whole or process by-products

· white lean fish: fish frames or underutilized species

· dark flesh fish: tuna, bonito, mackerel, anchovy, and others

Shellfish process by-products

· crab, shrimp, clam and lobster

The raw material should be of food grade and its quality has to be carefully

monitored not to produce inferior finished products. Freshness should be main-

tained from the point of collection through processing with low microbial counts

and histamine content by handling the raw material in the same manner as fresh

fish is handled. For this very reason, the term `by-product' rather than `waste' is

more appropriate.

14.5 Enzyme-assisted seafood flavors from processingby-products

At the University of Rhode Island, a process development was initiated for

seafood flavor production from processing by-products including lobster body,

red hake frame mince and clam belly, which are available in the New England

region. Other species under consideration are crab and shrimp processing by-

products and other underutilized species such as low fat herring and mackerel.


The process development work focused on seafood flavor production from

lobster bodies, red hake frames, and clam processing by-products along with

process schemes and variables used for process optimization. The general pro-

cess consisted of removal of organs and bones, homogenization, enzymatic

hydrolysis, filtration to remove unhydrolyzed fractions, and concentration or

dehydration.

14.5.1 Lobster flavor extracts from lobster bodies

More than 160 million pounds (approximately 73 000 tons) of American lobsters

(Homarus americanus) are being landed each year in the United States and

Canada combined, of which about 40 million pounds (approximately 18 000

tons) are used to produce canned lobster products (Richardson, 1993; Holliday

and O'Bannon, 1997). After removal of the tail and claw meat (56.2%) for

canning, the rest (lobster body) constituting 43.8% is usually discarded as

process waste. An alternative way to utilize the valuable components remaining

in the lobster body is to convert the body meat to hydrolysate as a flavor extract

employing enzymatic hydrolysis.

As for lobster bodies, 100 kg lobsters yield 44 kg bodies after removal of

claws and tails or 21 kg cleaned bodies after removal of carapaces, gills and

internal organs. The process of flavor extraction from cleaned bodies consists of

grinding to homogenate, proteolytic hydrolysis to liberate flavor-giving free

amino acids, enzyme inactivation, filtering, and concentration or dehydration

(Fig. 14.1). Variables used for process optimization were selection of a suitable

enzyme system, the reaction condition, the degree of hydrolysis, and yield. The

flavor quality of the hydrolysate was assessed using free amino acid profile, the

ratio of hydrophilic (Gly + Arg + Ala + Pro) to hydrophobic amino acids (Val +

Met + Leu + Ile), umami, sweetness, and bitterness. The ratio of hydrophilic to

hydrophobic free amino acids was determined as a characteristic free amino acid

profile related to the flavor property. Those hydrophilic free amino acids are

associated with the characteristic lobster flavor (Yang and Lee, 2000). When

applied singly, Flavourzyme (Novozyme North America) generated more free

amino acids, higher hydrophilic to hydrophobic amino acid ratio, and less bitter

short-chain peptides with a higher overall acceptability than other enzyme

systems evaluated. A desirable lobster flavor was produced by pre-extracting the

cooking juice and then combining it with the hydrolysate of the juice-removed

body homogenate prepared with 0.5% Flavourzyme (on a homogenate weight

basis) at 55ëC for 3±5 h. The enzyme used had a declared activity of 1,000

LAPU/g with a suggested usage level of 5±10 LAPU/g protein in substrate

(equiv. to 0.5±1.0%).

The predominant free amino acids in the hydrolysate of juice-removed body

homogenate were Gly, Ala, Arg, and Leu, while those in unhydrolyzed, juice-

retaining body homogenate were Arg, Gly, Ala and Pro (>100mg/100ml)

(Table 14.1). The GMP (guanosine 50-monophosphate) content was about

0.9mg/100 g, while small amount of AMP (adenosine 50-monophosphate) and


IMP (inosine 50-monophosphate) were detected in the concentrated product (0.2

and 0.1mg/100 g, respectively; ~30% solids).

No significant differences were found in the desirability of the hydrolysates

prepared by 0.15, 0.5 and 0.83% Flavourzyme, except that the saltiness increased

with an increase in the enzyme concentration due to the presence of NaCl in the

enzyme preparation. While the hydrolysate prepared with 0.15% Flavourzyme had

a significantly chalky mouthfeel, probably due to insufficient hydrolysis at a low

enzyme concentration, the one prepared with either 0.5 or 0.83% Flavourzyme did

not. Since there was no clear difference in flavor quality between hydrolysates

prepared with 0.5 and 0.83%, the 0.5% level of Flavourzyme was chosen for

economic reasons. On the other hand, the levels between 0.15 and 0.5% were not

examined. It is conceivable that some levels between 0.15 and 0.5% may be as

good as 0.5%. For this reason, a further screening could be necessary.

Hydrolysis time beyond 5 h decreased the flavor quality. The 9 h hydrolysate

at any enzyme concentration had lower acceptability with detectable off-flavor,

while 5 h hydrolysate did not have any detectable off-flavor. From the results, it

was concluded that the 5 h hydrolysis at 0.5% Flavourzyme and 55ëC gave the

highest acceptability. The yield of hydrolysate from body homogenate was

37.8% on a dry weight basis or 84.8% in the protein recovery. The low yield was

due to the presence of remaining shell and other insoluble matters.

Fig. 14.1 A general scheme of enzymatic production of lobster flavor extract andpowder. The optimum hydrolysis condititon: 0.5% Flavourzyme (homogenate weight

basis) at 55ëC for 5 h.


Enzymatic hydrolysis released flavor-imparting free amino acids and short-

chain peptides. The extent of release and the type of products are determined by

the type of enzyme and hydrolysis conditions used. Even though aqueous

extraction provides the highest flavor and aroma retention of the original

material, its protein recovery is very low without enzymatic hydrolysis, meaning

a low product yield. Among factors studied, type and concentration of enzyme

had the most significant effects on the free amino acid profile and the TCA

(trichloroacetic acid) soluble peptide concentration. The hydrolysate prepared

by Flavourzyme generated more free amino acids with a higher hydrophilic to

hydrophobic free amino acid ratio and a better acceptability than other

commercial enzymes evaluated. The lobster flavor with good quality and high

yield was produced by incorporation of the lobster body cooking juice into the

hydrolysate from the juice-removed body homogenate (Table 14.2). It was

thought that the enzymatic hydrolysis done on the whole homogenate (body

meat plus juice) may lead to an adverse change in the flavor profile of the juice

during the prolonged hydrolysis process at an elevated temperature, resulting in

a flavor quality inferior to that prepared by combining the hydrolysate of juice-

removed body meat with juice.

In an effort to bring the free amino acid profile close to that of cooking juice,

the appropriate amounts of hydrophilic free amino acids (Gly, Arg, Ala and Pro)

were supplemented to the hydrolysate. Such a compensation significantly

increased the sweetness and the overall desirability. Sensory scores of the free

amino acid-compensated hydrolysate and the non-compensated one are

Table 14.1 Free amino acid concentration (mg/100 g) of lobster body homogenatecooking juice and the 5 h hydrolysate of juice-removed lobster body homogenate

Amino acids Cooking juice Hydrolysate ofjuice-removed meat

Asp 6.9 24.22Glu 33.8 78.43Ser 15.1 50.93Gly 234.5 145.53His 15.5 38.01Arg 431.4 112.81Thr 15.6 55.43Ala 147.8 114.56Pro 119.7 45.94Tyr 18.2 52.36Val 22.3 58.83Met 21.1 31.26Ile 12.6 50.98Leu 31.3 106.64Phe 11.3 66.66Lys 19.5 93.27

The cooking juice was prepared from lobster body homogenate without enzyme treatment. Thehydrolysate was prepared with 0.5% Flavourzyme at 55 ëC for 5 h (n � 2).


compared in Table 14.3. The same data is presented in a schematic flavor profile

(Fig. 14.2).

The same processing technique can be adopted for other crustaceans such as

crab and shrimp. As for flavor extract from the crab processing by-product, the

same processing steps can be employed using Flavourzyme under the similar

conditions.

14.5.2 Fish flavor from white fish frames

The production of seafood flavor from underutilized fish species through protein

hydrolysis is somewhat challenging due to the difficulty of ensuring high

organoleptic quality. The hydrolysis of protein often accompanies flavor defects

such as bitterness and off-flavor along with the formation of desirable flavor

Table 14.2 Comparison of sensory properties of cooking juice, juice-retaining andremoved hydrolysate, and juice added hydrolysate of lobster body homogenate

Sweetness Bitterness Umami Lobster Off-flavor Desirabilitytaste

Cooking juice 5.9a 1.8a 4.3a 6.9a 1.0a 7.2a

Juice-retaininghydrolysate 4.7b 2.3b 4.3a 4.8b 1.9b 5.6b

Juice-removedhydrolysate 2.6c 1.2a 2.6b 4.8b 1.2a 4.7c

Juice-removedhydrolysate +cooking juice (1:1) 6.5a 1.0a 6.4c 7.4a 1.0a 7.6a

All samples were freeze-dried and prepared with water at 5% concentration before serving. Thehydrolysate was prepared with 0.5% Flavourzyme. The intensity or hedonic score was 1 to 9 in which1 was very weak or very poor and 9 was very strong or excellent. The number of panelists was 8.Different superscripts in the same column indicate significant differences (p < 0:05).

Table 14.3 Comparison of sensory properties of lobster body hydrolysate, hydrolysatecompensated with Gly, Arg, Ala and Pro, and cooking juice


Hydrolysate 4.4a 2.3 4.3 4.6a 1.9a 5.6a

Compensatedhydrolysate 5.7b 1.8 4.3 5.0a 1.5a 6.6b

Cooking juice 5.9b 1.8 4.3 6.9b 1.0b 7.2b

The hydrolysate was prepared from the cooked lobster body homogenate (juice retaining). Allsamples were freeze-dried and prepared with water at 5% concentration before serving. Thehydrolysate was prepared with 0.15% Flavourzyme. The intensity or hedonic score was 1 to 9 inwhich 1 was very weak or very poor and 9 was very strong or excellent. The number of panelists was16. Different superscripts in the same column indicate significant differences (p < 0:05).


(Kilara, 1985). Among lean fish species, red hake (Urophycis chuss) is found to

be a kind of species that produces a uniquely mild pleasant flavor with low fat

(0.8%, on a wet weight basis) (Imm and Lee, 1999). Red hake is one of the

underutilized fish species found in Northern and Mid-Atlantic coasts of the

United States and it is usually marketed only in the fresh form (Gendron, 1980)

because of the development of extensive texture hardening during frozen

storage. The use of filleting by-products (frame mince) would potentiate a

commercial prospect of flavor manufacturing from red hake.

Flavor quality of hydrolysates depends on several parameters. The use of

fresh raw material is crucial in ensuring good quality flavor, as stressed pre-

viously. Fatty fish species are not desirable because of their high susceptibility

to lipid oxidation and high cost of removing excess fat (Ritchie and Mackie,

1982). However, the lipid content in fatty fish species varies greatly with season.

The fish with a low lipid content (< 5%) prior to spawning can be used. The

extent of hydrolysis also determines sensory quality and is dependent upon the

specificity of protease, level of enzyme, water-to-substrate ratio, pH and

temperature. These parameters affect not only the quality of flavor, but also the

yield of hydrolysate. Currently, several commercial proteases are available for

the production of protein hydrolysates, and their optimum processing conditions

are generally suggested by the manufacturers. However, the selection of suitable

proteolytic enzymes and the extent of hydrolysis need to be refined according to

the nature of application.

Fig. 14.2 Schematic flavor profile of lobster body hydrolysates with and without freeamino acid compensation. Compensation was done to match the free amino acid profileof hydrolysate with that of cook juice. Scored on 1±9 point scale (1 being very weak or

very poor and 9 very strong or excellent) (same data given in Table 14.3).


Protein hydrolysate as a flavor extract was prepared from red hake frame

mince as well as from the head-and-gutted (H&G) mince using Flavourzyme. A

general process flow is given in Fig. 14.3. A water to fish ratio of 1:2 can be

used for hydrolysate production at a natural pH of fish without significant loss of

yield. The addition of 1.5% NaCl and 0.4% sodium tripolyphosphate (STPP)

after 30 min enzymatic hydrolysis improved flavor quality of the hydrolysate by

masking bitterness and off-flavor. Hydrolysate produced from the mince on a

pilot plant production scale by 3 h hydrolysis at 50ëC with 2% (w/w on a sample

protein weight basis) or 0.3% (mince weight basis) Flavourzyme had a highly

acceptable quality, suggesting that a good quality fish flavor can be produced

from unutilized frame mince (Imm and Lee, 1999). The predominant amino

acids were Leu and Arg. The spray-dried or freeze-dried fish mince hydrolysate

can be used as flavor supplement for various seafood products such as seafood

sauce and chowder.

The extensive hydrolysis (degree of hydrolysis, DH > 40%) resulted in

increased bitterness intensity, thus necessitating the control of DH below 40%

(< 6 h hydrolysis at 50ëC) to obtain an acceptable hydrolysate flavor extract. The

comparisons for the sensory quality between frame and H&G mince, salted and

unsalted, and hydrolyzed and unhydrolyzed frame mince are given in Table

14.4. In salted hydrolysate preparation from H&G and frame mince, control

(cooking juice) showed a slightly higher overall liking score with no

significance differences between control and mince hydrolysate, and between

Fig. 14.3 A general process scheme of enzymatic flavor production from red hakemince. The optimum hydrolysis condititon: 2% Flavourzyme (protein weight basis or0.3% on a mince weight basis) at 50ëC for 3 h and 1:2 water to mince with addition of

1.5% salt and 0.4% STPP.


H&G and frame mince. The salt-added mince hydrolysate showed a

significantly higher overall acceptability than the unsalted mince hydrolysate.

The hydrolysate prepared from frame mince has a flavor quality comparable to

that of H&G mince hydrolysate, suggesting that a good quality fish flavor can be

produced from unutilized frame mince. The hydrolysate solution has a slightly

chalky mouthfeel which affects the overall acceptability. However, this will not

be a problem in commercial products because the chalky mouthfeel can be

easily masked by other added ingredients. Table 14.4 also shows that the

hydrolysate received significantly higher scores in overall liking than the

unhydrolyzed frame mince. This is probably due to the difference in the umami

score which reflects the presence of umami-giving free amino acids generated

by hydrolysis.

There was a slight increase in bitterness after hydrolysis without any adverse

impact on the overall liking. Barzana and Garcia-Garibay (1994) suggested that

the intensity of bitterness depends on the DH and protease specificity because

hydrophobic amino acids responsible for bitterness can be liberated by

endopeptidase. In order to reduce undesirable sensory attributes such as off-

flavor and bitterness, a salt mixture was added during hydrolysis. The addition

of salt mixture improved sensory quality of hydrolysates by effectively reducing

off-flavor and bitterness scores and thus increasing the overall acceptability.

This result was consistent with the finding of Gillete (1985) that addition of

sodium chloride enhanced fullness and balance of perception while decreased

bitterness and off-flavor note. The addition of 0.4% STPP reduced off-flavor and

oxidation during storage (Matlock et al., 1984).

Table 14.4 Comparison of sensory properties of hydrolysates prepared from H&G andframe mince of red hake with and without salt added

H&G mince Frame mince

Unsalted Salt mixture1 Salt mixture

Attribute Con2 Fla3 Con Fla Con Fla Unhyd4

Fish chowder flavor5 7.35ab 9.30b 10.36a 11.01a 10.29a 9.24a 6.78b

Off-flavor 5.00a 2.43a 2.36a 1.65a 2.10a 2.40a 3.03b

Bitterness 2.40a 2.39a 1.34a 1.44a 1.15a 2.13a 2.80a

Umami 5.30a 6.46b 7.45 7.34a 5.83a 6.96a 3.73b

Overall liking 4.75a 6.38b 7.00a 6.75a 6.75a 6.38a 4.25b

Values are means of scores provided by 8 panelists.1 A mixture of salt (1.5%) and STPP (0.4%) was added after 30 min hydrolysis.2 Freeze-dried cooking juice.3 Freeze-dried hydrolysate prepared with Flavourzyme for 6 h hydrolysis.4 Unhydrolyzed frame mince was prepared following the same procedure except enzyme addition.5 Liking as fish chowder flavor.a,b Means with different letters in a row within the same group are significantly different (p < 0:05).


14.5.3 Clam flavor production from sea clam processing by-product (clam

belly)

Sea clam (Spisula solidissima) processing by-product (CPB) is produced as

shown in Fig. 14.4. It consists of residual meat (body), belly and viscera

remaining after collection of clam juice and meat. The CPB contains 17.13%

solid of which 63.7% (or 10.9% on a total CPB weight) is protein. The cooking

juice prepared from the CPB still has a mild and sweet clam flavor. There are no

critical differences in off-flavor and bitterness of the cooking juice between CPB

and clam meat. However, the CPB juice has a slight astringency and less clam

flavor and umami taste than the clam meat juice.

The hydrolysate strictly from CPB lacks a good clam taste and has a green

color and a chalky mouthfeel with a slight bitterness. It is not considered

acceptable as a clam flavor. However, a mixture of three parts clam juice and

two parts hydrolysate was found to be a right combination in terms of flavor

quality and overall liking when it was presented in the chowder form for

evaluation. The optimum hydrolysis was achieved with 2% Flavourzyme-2%

Nutrase at 55ëC for 4 h which gave complete liquefaction (>15% DH) with 69%

yield and the highest umami score without bitterness (Imm and Lee, 2000).

As shown in the flow chart (Fig. 14.4), CPB was chopped to facilitate

enzymatic hydrolysis and then subjected to heat treatment in a steam cooker.

The internal temperature of the sample reached 85ëC in 35 min and final

temperature was about 86.5ëC after cooking for 45 min. This precooking is a

step to reduce some off-flavor and inactivate undesirable microorganisms. After

precooking, samples were cooled down to 55ëC and water (1/5 part of CPB) was

added to avoid high viscosity development. The pH of the sample before

Fig. 14.4 Preparation of clam flavor from clam processing by-product. The optimumhydrolysis condititon: 2% Flavourzyme-2% Nutrase at 55ëC for 4 h. The most desirableclam chowder flavor was obtained by blending 3 clam juice and 2 clam belly hydrolysate.

Clams shell on (100): soft flesh (37)-shell (63); soft flesh (22 meat: 14 belly).


hydrolysis was 6.43 and it was close to the optimum pH range of enzymes under

evaluation (Flavourzyme: 5.0±7.0, Neutrase: 5.5±7.5 and Alcalase: 6.5±8.5).

In order to find the optimum enzyme level and combinations, the DH was

determined at different enzyme combinations. For complete sample liquefaction

within 4 h hydrolysis, at least 2% (based on sample protein weight) of Flavourzyme

was required and the estimated point for complete liquefaction was around 15%

DH.

The mouthfeel of filtered hydrolysate varied with the particle size of

hydrolysate. The hydrolysate was filtered and separated according to the

particle size using different sizes of screen sieves. The noticeable chalkiness

was effectively controlled after filtering through 250�m sieve. In the prepara-

tion of enzyme combinations, 2% Flavourzyme (FZ) (on a sample protein

weight basis) was used as the principal enzyme source. Other commercial

enzymes, Alcalase (AC) and Nutrase (NT) (Novozyme North America), were

evaluated for their performance in assisting hydrolysis reaction in order to

increase the yield and reduce the processing time. Based on the yield results,

three hydrolysis conditions (FZ (4%), FZ (2%) + NT (2%), and FZ (2%) + AC

(1%)) were chosen and the sensory qualities of the resulting hydrolysates were

examined.

A preliminary sensory evaluation indicated that hydrolysate prepared using

2% FZ +2% NT had a better sensory quality than the other two combinations.

The 2% FZ+ 1% AC hydrolysate showed a slightly higher bitterness score while

the hydrolysate prepared with 4% FZ alone had a lower umami score. As a

major application of CPB hydrolysate, an actual clam chowder was prepared

using hydrolysate (2% FZ+ 2% NT) to evaluate its potential to replace clam

juice in clam chowder preparation (Table 14.5). When clam chowder was

prepared with hydrolysate, a significant difference was found in acceptability in

terms of clam chowder flavor and overall liking. The main reason for such a

difference was probably due to off-flavor remaining in the hydrolysate. The off-

flavor of hydrolysate added `fishy taste' to chowder and reduced acceptability.

Table 14.5 Comparison of sensory properties of hydrolysate, cooking juice andhydrolysate-cooking juice blend prepared from clam

Chowder Umami Saltiness Off- Overallflavor1 flavor liking

Hydrolysate2 4.2b 5.8 4.4 3.6a 4.3b

Clam juice3 6.1a 5.7 4.6 1.5b 6.6a

Hydro-juice4 6.8a 5.6 4.4 2.4b 6.5a

Values are the means of the sensory scores provided by 10 panelists.1 Acceptability as clam chowder flavor.2 Hydrolysate prepared from clam belly by Flavourzyme (2%) and Nutrase (2%) for 4 h.3 Commercial clam juice from Blount Seafood, Warren, RI.4 A mixture of clam juice and clam-belly hydrolysate at a ratio of 3:2 (v/v).a,b Means with different letter under each attribute are significantly different (p < 0:05).


This off-flavor might have originated from the organ because CPB mainly con-

sists of belly and viscera. The chowder prepared from the hydrolysate showed a

significantly higher off-flavor score than others. The chowder prepared with

clam juice and hydrolysate blend (3:2 v/v) also had a slightly higher off-flavor

than the chowder prepared with clam juice. However, the hydrolysate provided

the same level of umami taste and saltiness. When the clam chowder was

prepared with a hydrolysate-juice blend, all evaluated sensory characteristics

were not different from those of clam juice. The blend even showed a slightly

higher score in acceptability as clam chowder flavor. This result suggests that

the hydrolysate might have enough background clam flavor and blend well with

clam juice without the loss of desirable characteristics.

The proximate composition of CPB hydrolysate showed a markedly higher

lipid (9.0%) content than clam meat (3.9%) and slightly higher protein (78.5%

over 68.5%) on a solid weight basis. Predominant taste active free amino acids

(TAFAA) found in hydrolysates were Arg, Ala, Gly, Glu and Met. TAFAA

reflects the overall liking of hydrolysates when tested in the form of clam

chowder. Identified taste active free amino acids can be appropriately adjusted

to provide the most desirable flavor profile. When the cooking juice of clam

meat and CPB were prepared by heating at 85ëC for 30 min and free amino acids

were determined by the method of Sekiwa et al. (1997), clam meat cooking juice

contained more free amino acids than CPB cooking juice. Collection of cooking

juice prior to the removal of CPB might be the reason for smaller amounts of

free amino acids in CPB. The predominant free amino acids in clam meat

cooking juice were Arg, Ala, Gly, Glu and Met. The CPB cooking juice lacked

Met in the profile of free amino acids but contained a significant amount of Thr.

The overall proportion of free amino acids in clam meat cooking juice, CPB

cooking juice and the concentrated commercial clam juice were similar to each

other. Based on the above results, Arg, Ala, Gly, Glu and Met are characteristic

free amino acids imparting distinct clam flavor and these free amino acids are

considered as clam taste active free amino acids.

The flavor profile can be characterized by the proportion of taste active free

amino acids to the total free amino acids or by the distribution of free amino

acids representing sweet, bitter and umami taste. The proportion of calm taste

active components linearly increased as the concentration of Flavourzyme

increased. When Flavourzyme was used as the only enzyme source, there was no

noticeable change in the proportion of sweet or bitter taste free amino acids. The

umami taste gradually increased with increasing Flavourzyme concentration. In

the production of taste of soup stocks, the umami substance of glutamic acid and

50-nucleotide in clams play a significant role through the synergistic effect

between the two substances. Among the 50-nucleotide, IMP, GMP and AMP

have this synergistic function with glutamic acids (Fuke and Ueda, 1996). When

the 50-nucleotide compounds were determined by the method of McKeag and

Brown (1978), four nucleotides including UMP (uridine 50-monophosphate),

AMP, CMP (cytidine 50-monophosphate) and GMP were found in clam meat

whereas only UMP and CDP (cytidine 50-diphosphate) were detected in CPB.


CPB contained UMP less than one-third of that in clam meat but contained more

CDP than clam meat. The same kinds of nucleotide compounds were found in

the corresponding cooking juices.

The optimum hydrolysis was achieved with 2% Flavourzyme-2% Nutrase (on

a substrate protein weight basis) at 55ëC for 4 h, where Nutrase was required for

a higher yield (> 65%) without reducing the desirable flavor profile.

14.6 Flavor-imparting compounds and chemistry

14.6.1 Lobster

In both lobster body and meat homogenates, Gly, Arg, Ala and Pro were found

to be predominant amino acids, and constituted 86 and 82% of the total detected

free amino acids, respectively. Hydrophobic amino acids, such as Met, Val, Leu

and Ile, were found to be in low concentrations, and constituted only 5% and 7%

of the total detected free amino acids in lobster body and meat, respectively.

Glutamic acid (including glutamine) concentration was lower than the

predominant amino acids but higher than other amino acid concentrations in

the lobster body.

When nucleotides were measured in the spray-dried sample (95% solids), the

concentrations of AMP, GMP and IMP were found to be 0.73, 2.80 and 0.39 mg/

100 g, respectively. The 1:7 ratio of IMP to GMP was in agreement with our

earlier work on a UF/RO concentrated lobster extract (Jayarajah and Lee, 1999).

ATP and ADP, on the other hand, were not detected. Overall, the nucleotide

concentrations were very low or undetectable in the product, indicating that

nucleotides might not play a significant role in the lobster flavor quality.

The importance of free amino acids and short-chain peptides in lobster flavor

quality should be noted. In the muscle extract of the Japanese spiny and shovel-

nosed lobster, 25 different free amino acids were identified (Shirai et al., 1996).

Among them, Tau, Gly, Arg, Ala and Pro were predominant, while the

hydrophobic amino acids, such as Met, Val, Leu and Ile, were very low. An

omission test showed that Gly, Arg, Ala, and Pro were essential for the taste of

both species. While the hydrophobic amino acids (Met, Val, Leu and Ile) at low

concentrations were essential for the taste of shovel-nosed lobster. These four

hydrophobic amino acids contributed bitterness and thick mouthfeel to the

shovel-nosed lobster. The concentrations of Gly and Arg in lobster and prawn

vary with the season. Hujita et al. (1972a,b) recognized that the decrease in the

acceptability of the prawn (Penaeus japonicus) in fall was accompanied by the

decrease in glycine. They also noticed that the increase in the acceptability of

prawn in winter paralleled the increase in the glycine content. From these

results, they assumed that glycine was one of the most important contributors to

the acceptability of prawns and lobsters. Shimizu and Hujita (1954) and Hujita

et al. (1972a,b) found that lobster containing more free glycine was more

palatable. Moreover, they suggested that the other three sweet amino acids (Ala,

Pro and Ser) might contribute to the acceptability of the species to some extent,


because the sum of these four amino acids and the palatability were highly

correlated.

The American lobster has high concentrations of Gly, Arg, Ala and Pro, and a

low concentration of Ser. When the juice-removed lobster body homogenate was

subjected to enzymatic hydrolysis, the concentration of hydrophobic amino

acids increased quickly from their initial low concentrations. Thus the ratio of

hydrophilic amino acids to hydrophobic amino acid concentrations decreased,

and correspondingly, the acceptability of the hydrolysate decreased. By

increasing the hydrophilic to hydrophobic amino acid ratio, the acceptability

of the lobster body hydrolysate flavor significantly increased. It indicates that

the hydrophilic to hydrophobic amino acid ratio played an important role in the

flavor quality of the lobster body hydrolysate. However, it is difficult to preserve

the original lobster flavor in the hydrolysate because other factors, such as the

short-chain peptides, play an important role in taste quality. The formation of

volatile compounds during enzyme hydrolysis also plays an important role in the

acceptability of the hydrolysate. The dipeptides or tripeptides, which are soluble

in the 7.5% TCA solution are known to be responsible for the bitterness of

protein hydrolysate. Matoba and Hata (1972) showed that hydrophobic amino

acids give the strongest bitterness when positioned in the interior of the peptide.

It gave a slightly weaker bitterness when in the terminal position, and the lowest

bitterness intensity as free amino acid.

Enzymatic hydrolysis also had a significant impact on the volatile compound

profile. Pyrazine concentration increased significantly after enzymatic

hydrolysis of crayfish process by-products. The concentrations of dimethyl

disulfide, dimethyl trisulfide, and benzaldehyde also increased after enzymatic

hydrolysis (Baek and Cadwallader, 1996). Free amino acids and peptides act as

precursors of the volatile compounds which are formed through the Maillard

reaction. Each amino acid and peptide has its specific contribution to the volatile

compound profile (Hayashi et al., 1990; Izzo and Ho, 1992).

14.6.2 Red hake flavor

There is no critical difference in the compositions of free amino acids between

mince and frame mince. Leu and Arg are dominant free amino acids in mince

and frame mince control. Mackie (1982) reported that the amino acids com-

position is barely changed by digestion except some loss of the sulfur-containing

amino acids such as Cys and Met depending on the hydrolysis conditions.

However, the composition of free amino acids might be changed by enzyme

hydrolysis because enzymes can cleave peptide bonds specifically and liberate

amino acids. These free amino acids might be more meaningful than overall

amino acids composition in terms of sensory quality because free amino acids

contribute to the flavor with their own flavor characteristics. The proportion of,

Arg, His and Leu in the hydrolysates decreased while that of Ile, Glu, Lys, Met,

Pro, Ser, Thr, and Val increased approximately more than twofold by enzymatic

hydrolysis.


14.6.3 Cooking juice vs. hydrolysate in lobster and crab flavor production

Ochi (1980) stated that aqueous extraction is the best way to obtain the original

taste and aroma. The purpose of using enzymes was to maximally release the

original lobster flavor and increase the yield. In achieving this, one strategy is to

extract the cooking juice first and hydrolyze the juice-removed remaining

portion, mostly the water insoluble components. The cooking juice and the

hydrolysate from the juice-removed body homogenate are combined and

subjected to dehydration or concentration. In this way, the `freshness' of

cooking juice can be preserved by not subjecting it to a prolonged enzymatic

hydrolysis at an elevated temperature. The hydrolysate should not introduce off-

flavor and other negative properties, such as bitterness and chalkiness. Although

water insoluble components in the hydrolysate are not important for flavor

generation, they may play an important role in the mouthfeel of the hydrolysate.

14.6.4 Hydrolysis conditions

Many studies have been conducted on the optimization of hydrolysis conditions

for fish protein hydrolysate, such as the optimum temperature, pH, enzyme

concentration, hydrolysis time, and substrate concentration. The purpose of

these studies was to find the optimum conditions at which a high DH could be

achieved (Baek and Cadwallader, 1995; Hoyle and Merritt, 1994; Martin and

Porter, 1995; Shahidi et al., 1995; Vieira et al., 1995a,b). Generally, the higher

the enzyme concentration and the longer the hydrolysis time, the higher will be

the DH. A typical hydrolysis curve of changes in DH with hydrolysis time is

shown in Fig. 14.5. The optimum temperature and pH could usually be obtained

from the enzyme manufacturers. Based on the results of the factorial screening

(Yang and Lee, 2000), among enzymes tested, Flavozyme was preferred because

the hydrolysate had a better flavor quality, and was thicker but not chalky. The

Flavourzyme hydrolysate also had a lower TCA soluble peptide concentration,

and higher free amino acid concentration and hydrophilic to hydrophobic amino

acid ratio. The juice-removed lobster body homogenate had a neutral pH which

was within the optimum pH range for Flavourzyme. The optimum temperature

was suggested at 50±55ëC by the enzyme manufacturer (Novozyme North

America). The protein concentration in the juice-removed lobster body homog-

enate was 6.3%, in which the concentration of the total TCA soluble peptides

was very low. This means that the major substrate for Flavourzyme is TCA

insoluble meat proteins. Since it is below 10%, the usual protein concentration,

further dilution is not necessary. Thus, the two remaining factors to be con-

sidered are enzyme concentration and hydrolysis time. The enzyme usage level

is usually suggested by the enzyme supplier. However, due to the difference of

the substrate and the purpose of hydrolysis, the optimum enzyme concentration

has to be determined. Enzyme concentration and hydrolysis time rendered

significant effects on free amino acid concentration, hydrophilic-hydrophobic

amino acid ratio and TCA soluble peptide concentration in the lobster body

hydrolysate (Fig. 14.6).


14.6.5 Concentration and drying methods on flavor quality

When hydrolysis reached the optimum time, the enzyme was inactivated by

heating to 85ëC and holding for 5 min. The hydrolysate was filtered through a

150�m screen to separate the shell and other residues. Filtrate and cooking juice

were combined and dehydrated by freeze-drying, spray-drying, or concentrated

by rotary evaporation. Spray-drying was done using an Anhydro spray-dryer

(AVP Crepaco, Inc., Tonawanda, NY) at inlet air temperature of 220±230ëC,

outlet air temperature of 80±90ëC, feed temperature of 40±50ëC, and a flow rate

of 50±60ml/min. Freeze-drying was carried out using a Virtis freeze-dryer

(GPC-2, Virtis Co., Gardiner, NY) at a plate temperature of 30ëC. Rotary

evaporation was conducted on a Labconco rotary evaporator (Labconco, Inc.,

Kansas City, MI) at 45ëC in a water bath and under vacuum which allowed

gentle bubbling. Concentration was continued until the mixture of cooking juice

and hydrolysate became thick (around 30% solid).

Results of the sensory evaluation showed that the freeze-dried and rotary

evaporated products had a quality similiar to that of the freeze-dried cooking

juice in terms of sweetness, bitterness, umami, and lobster taste (Table 14.6).

The overall desirability of the freeze-dried hydrolysate-juice was slightly better

than that of the freeze-dried cooking juice and rotary evaporated hydrolysate-

Fig. 14.5 Changes in the DH of clam belly meat treated with Flavourzyme at differentconcentrations. The chopped belly meat was mixed with water (1/5 of meat weight) and

hydrolyzed with Flavourzyme at 55ëC for varying time periods.


juice. The spray-dried hydrolysate-juice had some burned off-flavor. This is

typical of spray-dried product due to the high inlet air temperature and the

occurrence of a Maillard reaction, both responsible for being less sweet and

more bitter. The products prepared by freeze-drying and rotary evaporation

received significantly higher sweetness, umami, lobster taste and overall

desirability scores with less bitterness and off-flavor scores than the product

Fig. 14.6 Changes in the total hydrophilic and hydrophobic free amino acidconcentrations with hydrolysis time in the juice-removed lobster body homogenate atdifferent Flavourzyme concentrations at 55ëC. Hydrophilic group (Gly, Arg, Ala andPro): (ú) 0.15% Flavourzyme (on a homogenate weight basis); (4) 0.5%; (�) 0.83%;hydrophobic group (Val, Met, Leu, Ile): (n) 0.15% Flavourzyme; (s) 0.5%; (l) 0.83%.

Table 14.6 Comparison of sensory properties of the freeze and spray dried, and rotor-evaporator concentrated lobster body hydrolysates and freeze-dried lobster body cookingjuice


Freeze-dried 7.2 1.4 6.9 7.5 1.0 8.3Spray-dried 5.2 3.0 5.1 5.1 2.8 4.8Rotary-evaporated 6.7 1.4 6.3 6.7 1.2 7.4Freeze-driedcooking juice 7.8 1.2 6.9 7.0 1.0 7.9

The hydrolysate was prepared from juice-extracted lobster body homogenate with 0.5% Flavourzymeat 55ëC for 5 h and combined with cooking juice before drying or concentrated. All samples wereprepared with water at 5% concentration before serving. The intensity or hedonic scale was 1 to 9 inwhich 1 was very weak or very poor and 9 was very strong or excellent. The number of panelists was8.


prepared by spray drying. Should the hydrolysate be spray-dried, the mix may

require thermoprotective and anti-Maillard reaction agents. Both freeze-dried

and spray-dried powders were hygroscopic and thus required stabilizing agents

which retard the development of hygrocopicity and allow free flowing during

and after an extended storage period.

14.6.6 Species-specific taste active compounds

Each species offers its own characteristic flavor which is based on species-

specific taste active compounds. Those compounds are identified from extrac-

tive components which are generally water soluble and low-molecular-weight in

nature. They can be divided into nitrogenous and non-nitrogenous compounds.

The former includes amino acids, nucleotides, and organic bases, while the latter

includes sugars and organic acids (Fuke, 1994).

According to Hujita et al. (1972b), glycine and glutamic acid are the most

important taste active components which determine the acceptability of lobster

and prawn. Alanine, proline and serine are also found be taste active. Konosu

(1979) and Hayashi et al. (1981) found that Glu, Gly, Arg, AMP and GMP are

the primary taste active components in snow crab with a taste synergism

between Gly and 50-ribonucleotides. The taste active components in clam were

Glu, Gly, Arg, Tau, AMP and succinic acid (Fuke and Konosu, 1989).

14.6.7 Formulation of seafood flavor extract

Unlike synthetic flavors, so called `natural seafood extracts' of a hydrolysate

origin give rather dull and round flavor notes. The intensity of taste-active

compounds is somewhat toned-down by the presence of other non-taste active

components such as taste neutral amino acids and flavor-binding proteins. To

make it distinctive and intense, the flavor needs to be heightened by addition of

flavor enhancer, primarily sodium chloride and umami-giving compounds such

as MSG (monosodium glutamate), ribonucleotides, or their combination.

Nowadays, because of consumer preference for natural ingredients, the natural

sources of umami substances rather than MSG are highly preferred. They are so

called `natural savory flavors' such as yeast or soy protein hydrolysate. In

addition, amino acid and nucleotide profiles as well as other flavor-imparting

compounds can be matched with those found in the cooking juice by appropriate

supplementation.

14.7 Future trends

The finished fraction of the current enzymatic hydrolysis contains non-flavor

imparting components, primarily high molecular weight (MW) proteins, in

addition to low MW flavor-giving components, mostly free amino acids, fatty

acids and nucleotides. The intensity and purity of flavor can be enhanced by


removal of non-flavor imparting fractions, especially high MW proteins that

tend to bind flavors and subsequently reduce the overall intensity. An additional

refining step to remove non-flavor imparting fractions needs to be developed in

an effort to produce a flavor that has a greater intensity and a better sensory

quality. The non-flavor imparting fractions can also be examined for quantity

and any potential commercial use. This refined flavor can be made available to

those who need high quality and intense flavor in addition to unrefined

hydrolysate flavor which will have its own market. This effort will require a new

set of process design using processing units of a larger production capacity and

subsequent modifications in processing conditions for process optimization,

especially hydrolysis and refining steps.

14.8 References

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15.1 Introduction

Calcium is known to be an essential element required for numerous functions in

our bodies including the strengthening of teeth and bones, nerve function and

many enzymatic reactions that require calcium as a cofactor. It is also necessary

for muscle contraction and regulation of the permeability of sodium ion across

cell membranes including those of nerve cells. The concentration of calcium in

the blood plasma remains almost constant and varies only slightly over time for

a given individual (Allen, 1982; Anderson and Garner, 1996).

In various industries such as food, electronics, leather, and others, calcium

originates from dolomite, bone meal, and oyster shell and is utilized as an

important ingredient. For example, calcium is used to produce acryl resin, make

emulsion coagulant in rubber, produce additives in paper making, and make

early strengthening agent (concrete strengthening agent and coating material

coagulating agent, and others) in the construction industry. Especially in food

and agricultural industries, calcium is utilized as a food stuff antiseptic to

prevent putrefaction of fruits and vegetables and help the process of cheese

making.

Although most people are aware of calcium as an important element in their

bodies, it is still severely deficient in most diets. Calcium deficiency in the

United States has been considered a major cause of osteoporosis, affecting

approximately 26 million people annually (Melton, 1995). In 1994, the National

Institute of Health (NIH) Consensus Panel revised the recommendations for

calcium intake (NIH Consensus Development Conference, 1994). As shown in

Table 15.1, the optimal calcium intake has been recommended to be 800mg/day

during childhood below five years of age, 800±1200mg/day for children from

15

Fish and bone as a calcium sourceS.-K. Kim and W.-K. Jung, Pukyong National University,Republic of Korea

age six to ten, 1200±1500mg/day for adolescents or young adults from age 12±

24 and pregnant or lactating women, 1000mg/day from age 25 to the time of

estrogen deprivation or age 65, and 1500mg/day for elderly people.

Generally, the most common and trusted source of calcium (Table 15.2) is

milk or other dairy products (Anderson and Garner, 1996). However, some

people, especially Asian people, prefer not to consume milk due to lactose

indigestion and intolerance which makes them allergic to milk. As an alter-

native, these people prefer to take calcium-fortified fruit-juice, calcium-rich

foods and calcium salt supplements, such as calcium fumarate, citrate, lactate,

carbonate, di- and tri-basic phosphate, and gluconate. These salts are available in

ingredient forms, each with its own calcium content, solubility, taste and cost

issues. The solubility and bioavailability of calcium-containing ingredients are

especially important. Although the low pH condition in stomach renders all

calcium into its ionic form, precipitation as insoluble calcium phosphate,

depending on the amount of phosphate present, can occur in the intestine, where

the pH range is 6 to 7. The human body cannot absorb the calcium in precipi-

tated calcium phosphate.

In order to improve solubility and bioavailability of calcium, various

proprietary blends of calcium salts have been developed with milk protein, food

acids and sugar, polysaccharides and calcium/amino acid chelate complexes like

Ca-casein phosphopeptides (CPPs), as specific end-use products, depending on

their final pH (Allen, 1982).

Table 15.1 Recommended calcium intake for various population groups

Age (years) Calcium needs

Children 800±1200mg/dAdolescents 1200±1500mg/dAdults 1000mg/dElderly 1500mg/d<65 on hormonal replacement therapy 1000mg/d

Source: NIH Consensus Development.

Table 15.2 High-bioavailable calcium sources in foods

Food source Serving size Calcium (mg)

Milk and yogurt 8 oz or 1 cup 300±450Cheese 3 ounces 300±450Bones in canned sardines and salmon 3 ounces 181±325Calcium fortified foods 8 ounces 200±300(i.e. orange juice, soy milk, tofu)Dark green, leafy vegetables 1/2 cup cooked, 1 cup raw 50±100Nuts and seeds 1 ounce 25±75

Source: http//:ag.arizona.edu/pubs/ health/az1296.pdf

Fish and bone as a calcium source 329

Annually, more than 50% of the total fishery products (over 120 million tons

per year) are discarded as inedible by-products such as bone, skin, fins, internal

organs and head. Thus many studies have been performed to utilize a large

amount of protein, oil, mineral, carbohydrate and nucleic acid originated from

fishery by-products, and improve their functional properties (Nair and

Gopakumar, 1982; Rodriguez-Estrada et al., 1994; Nagai and Suzuki, 2000;

Kim et al., 2001; Shahidi and Kamil, 2001; Kim et al., 2003). However, the

studies on the utilization of organic components or minerals in the fish bone are

scarce (Larsen, 2000, 2003; Kim et al., 2003).

Recently, as a part of a research to utilize large amounts of fishery by-

products from fish processing, many studies have been carried out to improve

functional properties of fishery by-products such as oyster shell, hoki frame, and

crab shell by enzymatic modification (Kim et al., 1997a,b,c, 1998a,b, 2001,

2003, 2005; Jeon et al., 2000, 2001a,b, 2002; Jung et al., 2002, 2003, 2005a,b,c,

2006a,b). This chapter focuses on biochemical property of fish bone and

utilization of fish bone as a calcium supplement or fortifier.

15.2 Biochemical properties of fish bone

Fish bone consists of both organic and inorganic (mineral) parts. According to the

report of Jung et al. (2005a), the organic portion (30.54% on dry basis) of hoki

(Johnius belengerii) bone was composed of 28.04% protein, 1.94% lipid and

0.56% carbohydrate. Collagen represented at 86.21% of total protein, and non-

collageneous protein content was 13.79%. As reported by Garner et al. (1996),

90% of organic components in the bone matrix are composed of type I collagen,

and the remaining 10% consisted of non-collageneous proteins such as

osteocalcin, osteopontin, osteonectin, fibronectin, thrombospondin, proteoglycan

I/II and growth factors (IGF-1, PDGF, TGF-�, etc.). These molecules are

produced by osteoblast-like cells and their functions are related to bone formation

and cell attachment. Carp (Cyprinus carpio) osteocalcin, known as bone

formation factor, was detected and characterized by Nishimoto et al. (2003).

The inorganic mineral portion (69.46% on dry basis) was mainly composed

of 59.69% of calcium (Ca) and 35.81% of phosphorus (P) with the mole ratio of

Ca/P of 1.67. The inorganic portion of vertebrate bone is primarily composed of

hydroxyapatite (HA) crystals deposited within an organic matrix of cross-linked

collagen fibrils (Anderson and Garner, 1996). The HA crystals make up

approximately 60±65% of bone and the HA has an extremely complicated

crystaline structure [(Ca2+)10-x(H3O+)2x(PO4

3-)6(OHÿ)2]. In vertebrate animals,

the crystals are usually organized with x value range from 0±2 and 1.67 mole

ratio of Ca/P.

As reported by Hamada et al. (1995), the skeletal Ca/P mole ratio in 15

species of commercially processed marine teleosts, in Asia, varied within the

range of 1.63±1.20 (Table 15.3), and these bones are organized as a combina-

tional structure with hydroxyapatite and beta type Ca3(PO4)2.


15.3 Utilization of fish bone calcium and organic compound

15.3.1 Degradation of fish bone

Skeletal frames discarded from industrial processing of J. belengerii were

digested by a heterogeneous enzyme extracted from the intestine of a carni-

vorous fish (also discarded from industrial processing), bluefin tuna (Thunnus

thynnus), in order to utilize the bone to produce nutraceuticals with a high Ca

bioavailability (Jung et al., 2005a). Fish bone with well-organized tissue

structures containing hard minerals and organic fibers should be degraded. In a

previous study (Jung et al., 2005a), fish skeletal frames discarded from industrial

processing had been digested by a heterogeneous enzyme extracted from the

intestine of a carnivorous fish (also discarded from industrial processing).

Further, T. thynnus intestinal enzymes (TICE) could effectively biodegrade the

hoki bone matrices composed of collagen, non-collageneous proteins,

carbohydrates and minerals using enzymatic degradation system (Fig. 15.1).

In addition, specific proteolytic activities of TICE were examined with various

substrates; type I collagen as a natural substrate, N-benzoyl-L-tyrosine ethyl

ester (BTEE) and N-acetyl-L-tyrosine ethyl ester (ATEE) as a synthetic ester

type substrate of �-chymotrypsin, N-benzoyl-L-arginine ethyl ester (BAEE) as a

synthetic ester type substrate of trypsin and N-benzoyl-DL-arginine-

pnitroanilide (BAPNA) as a synthetic nitroanilide type substrate of trypsin.

Table 15.3 Ca and P composition in various fish bones

Sample species Ca (% w/w)a P (% w/w) Ca/P (molar ratio)

Hokia 36.2 16.8 1.67Sea breamb 35.4 17.5 1.54Norse mackerelb 35.6 17.3 1.60Carpb 34.9 16.9 1.63Sharkb 34.9 16.9 1.60Sardineb 35.8 17.3 1.63Mackerelb 33.9 17.0 1.59Tilefishb 35.1 18.4 1.52Croakerb 35.1 17.8 1.56Trigger fishb 34.4 17.8 1.54Lizard fishb 35.7 17.8 1.60Spanish mackerelb 34.0 18.6 1.47Flying fishb 34.4 17.8 1.55Conger eelb 35.7 17.7 1.60Flat fishb 35.9 18.1 1.57Anchovyb 24.4 16.3 1.20Fowlb 36.1 17.4 1.60Cattleb 36.5 17.2 1.64Swineb 36.9 17.0 1.68Human 37.8 17.1 1.69

a Minerals were calculated according to the ratio of a mineral/total ash.b The data were cited in Hamada et al. (1995).


The TICE could hydrolyze four kinds of synthetic substrates. The activity of

TICE was the highest for BAEE, approximately 2.5U/mg; TICE exhibited

potent activity (1.6U/mg) against BTEE. Collagenolytic activity of TICE was

determined as 16.5U/mg type I collagen. The results indicated that TICE

contained have considered tryptic and collangenic enzymes.

In a previous study (Kim et al., 1997c), the specific activities of tuna pyloric

caeca crude proteinase (TPCCP) were determined using casein as a natural

substrate and the same kinds of synthetic substrates. TPCCP showed a specific

activity of 0.54U/mg casein, and its activity was also the highest for BAEE (2.8

U/mg). Some other proteolytic activities have also been elucidated from internal

organs of other carnivorous fish species (mackerel, carp, cod, salmon and trout)

as heterogeneous or homogeneous proteinases (Gudmundsson and Hafsteinsson,

1997; Shahidi and Kamil, 2001). These can be classified into two large families,

serine and aspartic proteinases. Among well known proteinases, pepsin and

chymosin are aspartic proteinases, and trypsin, chymotrypsin, collagenase and

elastase are serine proteinases. Ramakrishna et al. (1987) reported that generally

dogfish enzyme could hydrolyze collagen molecules more efficiently than

bovine enzyme regardless of the solubility of collagen. Bezerra et al. (2000)

reported that the highest proteolytic activity was found in the stomach, and

alkaline activity was greatest in the pyloric caeca of tambaqui (Clolssoma

macropomum).

In the other methods (Young and Lorimer, 1960; Sato et al., 1989; Miura and

Nakano, 1998), acetic acid and citric acid were often treated to recover Ca and

protein-like gelatin and collagen from fish bone or bovine bone. Citric acid is

widely used for food-grade gelatin from fish because it does not impart

objectionable color or odor to the gelatin (Gudmundsson and Hafsteinsson,

1997).

Fig. 15.1 Enzymatic degradation system for preparation of soluble calcium from fishskeletal frames.


Because of an acid lability of cross-linking in the collagen matrix (Montero et

al., 1995), a treatment with acidic solution should be enough to affect solubiliza-

tion. In detail, acidic treatment can break non-covalent bonds to disorganize the

protein structure, thus producing adequate swelling and cleavage of intra- and

inter molecular bonds, leading to subsequent collagen solubilization. In par-

ticular, acidic treatment at high temperature can easily convert insoluble collagen

to gelatin (Stainsby, 1987), as a result Ca can be extracted from its organic

matrix.

15.3.2 Calcium solubilization using fish bone peptide

Casein phosphopeptides (CPP) derived from the intestinal digestion of casein

have been shown to enhance bone calcification in rats (Lee et al., 1980; Tsuchita

et al., 1993). Calcium fortifiers like CPP, egg yolk phosphopeptide (phosvitin),

and some organic ingredients (citrate, malate, acetate, etc.) have the capacity to

chelate Ca ion and to prevent precipitation of Ca-phosphate salts at neutral

intestinal pH (Berrocal et al., 1989), thereby increasing the amount of soluble Ca

available for absorption across the mucosa (Yuan and Kitts, 1991, 1994).

In the previous study (Jung et al., 2005a), fish bone hydrolysate liberated by

TICE consisted of 13.36% collagen and 10.25% non-collageneous protein.

Phosphoprotein was determined to be 16.65% of non-collageneous protein, and

the content of soluble calcium liberated by the TICE 6.55%. Proteins in the

hydrolysates were mainly composed of Gly, Thr, Glx, Ala, Asx, Ser, Hyp, and

Arg. As reported by Jiang and Mine (2000), Ca-binding phosphoproteins such as

osteocalcin, phosvitin and casein phosphoprotein mainly consist of Ser, Thr, Ala

and Tyr residues phosphorylated or bound to Ca (Houben et al., 1999). Pro, Gly,

Pro and Hyp residues are known as typical amino acids of collagen (Edwards

and O'Brien, 1980). Calcium-binding phosphoproteins derived from non-

collageneous materials in the bone have a high affinity to Ca2+ on the surface of

hydroxyapatite (Hoang et al., 2003).

As shown in Fig. 15.2, the fish bone phosphopeptides (FBP I < 1 kDa of MW;

1 < FBP II < 5 kDa; FBP III > 5 kDa) could inhibit the formation of insoluble

calcium phosphate, as measured by the calcium contents of the supernatant after

the formation of Ca-FBP complex. Calcium binding activity of the FBP II was

similar to that of casein oligophosphopeptide (CPP). The solubility of Ca was

dependent on the concentration of FBP, and 41.06mg/l of Ca was obtained at a

concentration of 250mg/l. The pH of the reaction system was maintained at 7.8,

because low pH could increase the solubility of the insoluble calcium salt.

Various concentrations up to 500mg/l were mixed with 5mMCaCl2 and

20mM sodium phosphate buffer (pH 7.8). The mixture was stirred at 22ëC for

30 min, and the pH was maintained at 7.8 in the buffer system. When the pH

changed, it was adjusted with 6MHCl or NaOH and monitored by a pH meter.

After removal of insoluble calcium phosphate salts by filtration with a 0.45mm

membrane, Ca contents of the supernatant fraction were determined by flame

atomic absorption spectrometry. The experiments were performed in triplicate.


Values are means, with standard deviations represented by vertical bars. (l),

Control; (�), FBP I; (t), FBP II; (5), FBP III; (n), casein phosphopeptide.

As reported by Jiang and Mine (2000), the solubility of 36.3mg/l of Ca was

obtained at 200mg/l of the oligophosphopeptide from egg yolk phosvitin with

35% phosphate retention, and the solubility was higher than that of commercial

CPP II (Meiji Seika Co., Ltd., Tokyo, Japan). According to the data of amino

acid composition of the FBP, the relative contents of Gly, Pro, Hyp, and known

typical collageneous amino acids, were significantly lower than those of hoki

bone hydrolysates. However, the contents of Thr, Ser, Glx, and Ala, which are

phosphorylated or Ca2+-binding, showed remarkable increments as compared to

the bone hydrolysates. Nishimoto et al. (2003) isolated and characterized an

osteocalcin from carp Cyprinus carpio, and carp osteocalcin consisted of a high

proportion of Ala, Tyr, Thr, Gln, and Asp. The calcium binding oligophospho-

peptide prepared from hen egg yolk phosvitin by Jiang and Mine (2000) mainly

consisted of Ser, Asx, Glx and Arg. They reported that phosphoseryl groups in

the oligophosphopeptide played an essential role in Ca2+-phosphopeptide

interaction. As reported by Hoang et al. (2003), specific residues of osteocalcin

implicated in HA binding are located on the same surface of helix chain,

coordinate five Ca2+ in an elaborate network of ionic bonds. These five Ca2+ are

sandwiched between two crystallographically related osteocalcin molecules

from bone tissue and show both monodentate and malonate modes of chelation

with extensive bridging (Fig. 15.3).

Fig. 15.2 In vitro assay for calcium solubility.


15.4 In vivo availability of soluble calcium complex from fishbone

In vivo effects of FBP II on Ca bioavailability were further studied in the

ovariectomized rats (Jung et al., 2006b). Menopause is a time when oestrogen

deficiency leads to accelerated bone resorption and negative bone balance. The

present study was undertaken to evaluate the beneficial effects of FBP as a Ca

fortifier in osteoporosis induced by ovariectomy and a concurrent low-Ca diet.

During the experimental period corresponding to the menopause with osteo-

porosis disease, the loss of bone mineral (Ca) was decreased by FBP II

supplementation in the ovariectomized rats. After the low-Ca diet, the FBP II

Fig. 15.3 Hypothetical pattern of calcium-peptide binding after enzymatic digestion.Crystallographic dimer interface. Light and dark distinguish the two molecules. Spheres

and the broken lines represent Ca2+ ions and ionic bonds, respectively.

Table 15.4 Effects of calcium fortifier intakes in the ovariectomized rats

Experimental groups Control CPP FBP II(n=8 per group)

Mean SD Mean SD Mean SD

Body weight gain (g/d) 14.4 1.5 14.2 2.5 13.9 1.9Food intake (g/d) 12.9 0.9 13.0 1.3 12.5 1.5Ca intake (mg/d) 54.8 5.9 54.2 4.8 54.4 5.5Fecal Ca (mg/d) 53.3a 3.4 44.8b 4.2 46.5ab 3.6Urinary Ca (mg/d) 0.9 0.4 1.4 0.5 1.5 0.5Ca retention* (mg/d) 0.2a 0.5 8.0b 1.2 6.4b 1.7Femoral total Ca (mg) 143a 7 153b 9 155b 10Femur length (mm) 33.9 0.5 34.8 0.4 34.3 0.6Femur wet weight (g) 1.12a 0.06 1.32b 0.05 1.22ab 0.06Bone mineral density (BMD) 0.161a 0.018 0.229b 0.029 0.213b 0.025of the distal femur (g/cm2)Breaking force (kg) 3.96a 0.57 8.97b 1.03 8.48b 0.97

a,b Mean values within a row with unlike superscript letters were significantly different (P < 0.05).* Calcium retention (balance) was calculated as: Ca intake ± fecal Ca ± urinary Ca.


diet, including both normal levels of Ca and vitamin D, significantly decreased

Ca loss in faeces and increased Ca retention as compared with the control (Table

15.4). The levels of femoral total Ca, bone mineral density, and breaking

strength were also significantly increased by FBP II diet to a level similar to

those of the CPP diet group (no difference; P < 0.05). It illustrates that increased

Ca retention by FBP II intake led to the prevention of mineral loss in the

osteoporosis-modelling rats. As reported by Larsen et al. (2000, 2003), the

intake of small fish with bones can increase Ca bioavailability, and the small fish

may be an important source of Ca, especially in population groups with low

intakes of milk and dairy products. In the present study, the results proved the

beneficial effects of fish-meal in preventing Ca deficiency due to increased Ca

bioavailability by FBP intake. Furthermore, it is possible to provide a novel

nutraceutical with a high bioavailability for Ca to oriental people with lactose

indigestion and intolerance and Ca-fortified supplements, such as fruit juice or

Ca-rich foods, as alternatives to dairy products.


This research was supported by a grant (p-2004-01) from Marine Bioprocess

Research Center of the Marine Bio 21 Center funded by the Ministry of

Maritime Affairs and Fisheries, Republic of Korea.

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Biochem. Biophys. 88, 373±381.

YUAN, Y. V. and KITTS, D. D. (1991). Conformation of calcium absorption and femoral

utilization in spontaneously hypertensive rats fed casein phosphopeptide

supplemented diets. Nutr. Res. 11, 1257±1272.

YUAN, Y. V. and KITTS, D. D. (1994). Calcium absorption and bone utilization in spon-

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protein. Br. J. Nutr. 71, 583±603.


16.1 Introduction

Chitin is a major component of the exoskeleton of invertebrates, crustaceans,

insects and the cell wall of fungi and yeast and acts as a supportive and

protective component (Knorr, 1984; Lower, 1984; Tan et al., 1996). Chitin is the

second most abundant natural polymer on Earth after cellulose (Brzeski, 1987;

Ornum, 1992). At least 10 gigatonnes (1� 1013 kg) of chitin are produced and

hydrolyzed each year in the biosphere (Muzzarelli, 1999). Chitin, poly-(164)-N-

acetyl-D-glucosamine, is a cellulose-like biopolymer found in a wide range of

products in nature (Shahidi et al., 1999). Chitin is biosynthesized from

polymerization of uridine diphosphate-N-acetyl-D-glucosamine by chitin

synthase (EC 2.4.1.16) (Hirano, 1996).

Chitosan, a copolymer of mainly D-glucosamine and a small proportion of N-

acetyl-D-glucosamine with �-(164) linkage, is obtained by alkaline or enzymatic

deacetylation of chitin and is also an abundant polymeric product in nature.

Chitosan was first discovered in the nineteenth century when chitin was heated to

boiling in a concentrated KOH solution (Dunn et al., 1997). Chitosan is found in

different morphological forms such as a primary, unorganized structure, crystalline

and semicrystalline forms. For different reasons, especially problems of

environmental toxicity, these two biopolymers are considered to be interesting

substances (Sorlier et al., 2001). Owing to their unique structures, they possess high

biological and mechanical properties as they are biorenewable, biodegradable, and

biofunctional (Hirano et al., 2000). Both chemical and enzymatic methods are

known for preparation of chitin, chitosan and their oligomers, with different

degrees of deacetylation, polymerization and molecular weight. Chitin, chitosan

16

Chitin and chitosan from marineby-productsF. Shahidi, Memorial University of Newfoundland, Canada

and their oligomers can be produced chemically using concentrated HCl followed

by column chromatographic fractionation (Jeon et al., 2000). Three methods are

known for modification of the process of isolation of chitin and chitosan oligomers

(Jeon et al., 2000). These are acetolysis, fluorohydrolysis and sonolysis.

Meanwhile, chitin and chitosan oligomers can be prepared through microbiological

and fungal treatments (enzymatic preparation). Chitin and chitosan may be

degraded by certain enzymes such as chitinases and chitosanases, respectively, and

the process is environmentally friendly. Chitin, chitosan and their oligomers may

be employed in medical uses as wound-healing agents, as dietary and

hypocholesterolemic agents, anti-tumor and anti-ulcer compounds and as coating

of artificial parts of the body such as leg, tooth and arm, among others. They may

also be used in food preservation such as for seafoods (Shahidi et al., 1999) and

fruits (El Ghaouth et al., 1992a,b) as well as for acidity adjustment (Scheruhn et al.,

1999) and as antibacterial and antifungal agents (Shahidi et al., 1999).

This chapter provides a cursory account of the chemistry and uses of chitin,

chitosan and their oligomers.

16.2 Chemical characteristics

16.2.1 Structure and properties of chitin and chitosan

Chitin is a high-molecular-weight polymer of 1000±3000 units of N-acetyl-D-

glucosamine (NAG) linked together by �-D (164) bonds (Lower, 1984). The

chemical structure of chitin is the same as that of cellulose with the hydroxyl

group at position C-2 replaced by an acetamido group (Fig. 16.1). Chitin can be

deacetylated to produce chitosan, which is soluble in dilute acidic solutions and

is highly viscous when dissolved; this makes it distinctly different from chitin

(Jeon et al., 2000; Shahidi et al., 1999).

Chitin and chitosan are different in their solubility characteristics. There are

few solvents for chitin, whereas almost all aqueous acids dissolve chitosan. Most

solvents, namely dimethylformamide, lithium chloride, hexafluoroacetone and

hexafluoroisopropanol, among others used for dissolution of chitin are toxic and

hence cannot be used in food processing applications. Nonetheless, when chitin

is ground to a fine mesh, it could be used to increase viscosity of liquids.

Solvents for chitosan, such as acetic acid, glutamic acid and glutamine acids, are

generally safe to consume, allowing the formation of solutions that are appro-

priate for gel production. Thus, chitosan is better matched to the viscosity of

foods (Winterowd and Sandford, 1995). The solubility characteristics of

chitosan are generally dictated by the extent of N-acetylation, the distribution

of acetyl groups, the pH and the ionic strength (Anthonsen et al., 1993). The

amino group in chitosan has a pKa value of 6.2±7.0, which makes chitosan a

polyelectrolyte at low pH values (Claesson and Ninham, 1992). Recently, it was

reported that a highly deacetylated chitin with a number of acetylated chitin

joining each other in a block and then a number of units with free amino groups

Chitin and chitosan from marine by-products 341

in a block could produce products which are soluble in water (Kristbergsson et

al., 2003). However, no details are yet available in the non-proprietary literature

in this regard.

Solubility issues associated with chitin and chitosan may limit their use in

physiological and functional foods. The intestines of most animals lack the

ability to produce chitinase and chitosanase that are able to hydrolyze chitin and

chitosan, respectively. Therefore, they will be excreted unchanged in the feces.

On the other hand, chitin and chitosan oligomers are considered to have more

physiological functions because they are water-soluble and their solutions are

less viscous, so they are readily absorbed in the human intestine (Jeon et al.,

2000).

Fig. 16.1 The chemical structures of chitin, chitosan and cellulose.


Chitosan has many useful applications in different fields as summarized in

Table 16.1. These are mainly due to the presence of amino groups at the C-2

positions, and also because of the primary and secondary hydroxyl groups at the

C-3, and C-6 positions, respectively (Kurita, 1986). Chitosan is the simplest and

the least expensive derivative of chitin (Ornum, 1992). Presence of positively

charged amino groups repeatedly placed along the chitosan polymer chain

allows the molecule to bind to negatively charged surfaces via ionic or hydrogen

bonding (Muzzarelli, 1973; Rha, 1984; Shahidi, 1995). The term chitosan is

favored when the nitrogen content of the molecule is higher than 7% by weight

(Muzzarelli, 1985) and the degree of deacetylation is more than 70% (Li et al.,

1992).

Chitin and chitosan are weak bases and hence go through the usual

neutralization reactions of basic compounds. The non-bonding pair of

electrons on the primary amino group of the glucosamine unit accepts a

proton, and thus becomes positively charged (Winterowd and Sandford, 1995).

In addition, chitosan serves as a strong nucleophile because of the presence of

non-bonding pair of electrons on its primary amino groups. Chitosan reacts

readily with most aldehydes to produce imines (Kurita et al., 1988). It also

reacts with acyl chlorides to form the corresponding acylated derivatives

(Hirano et al., 1976).

Chitin and chitosan are capable of forming complexes with many of the

transition metals (Muzzarelli, 1973). The heavy metal complexes are supposed

to form as a result of donation of non-bonding pair of electrons on the nitrogen

and/or on the oxygen of the hydroxyl groups to a heavy metal ion. Cupric ion

appears to form one of the strongest metal complexes with chitosan in the solid

state (Domard, 1987; Kentaro et al., 1986; McKay et al., 1986). Ferrous ion has

the ability of binding to chitosan (Koshijima et al., 1973). Under experimental

conditions (100mg of powdered chitosan mixed with a solution of ferrous

nitrate (25mg) in 50ml water, at 30ëC, and reaction time of 74 h), about 28% of

the ferrous ions were complexed with chitosan. The rate of formation and

stability of these complexes are generally affected by the presence of counter-

ions, competing heavy metal ions, temperature, and pH of the solution, as well

as particle size, crystallinity, and the degree of N-acetylation of chitin and

chitosan (Winterowd and Sanford, 1995).

Chitin and chitosan are labile to acid- or alkaline-assisted degradation. Under

acidic or basic conditions, acetic acid can be freed as N-acetyl groups at the C-2

positions of N-acetyl glucosamine units are released, leaving behind primary

amine groups (Muzzarelli, 1977). In addition, presence of the primary amino

groups in chitosan presents further potential for modification of the molecule

such as N-acylation and N-alkylation, among others. Acidic conditions also

cause partial depolymerization and degradation of the �-glycosidic bonds

(Madhaven and Ramachandran, 1974). Depolymerization under basic conditions

occurs, but to a lesser extent, and chitosan can be hydrolyzed using nitrous acid

(Allan and Peyron, 1989).


Table 16.1 Applications of chitin, chitosan and their derivatives

Area of application Examples

Antimicrobial agent BactericidalFungicidalMeasure of mold contamination in agricultural commodities

Edible film Controlled moisture transfer between food and the surroundingenvironmentControlled release of antimicrobial substancesControlled release of antioxidantsControlled release of nutrients, flavors and drugsReduction of oxygen partial pressureControlled rate of respirationTemperature controlControlled enzymatic browning in fruitsReverse osmosis membranes

Food additive Clarification and deacidification of fruit juicesNatural flavor extenderTexture adjusting agentEmulsifying agentFood mimeticThickening and stabilizing agentColor stabilization

Nutrition Dietary fiberHypocholesterolemic agentLivestock and fish feed additiveReduction of lipid absorptionProduction of single cell proteinAntigastritis agentInfant food ingredient

Water treatment Recovery of metal ions, pesticides, phenols and PCBsRemoval of dyes, radioisotopes

Agriculture Seed and fruit-coveringFertilizerFungicide

Cosmetics Skin and hair products

Biomedical and Artificial skinpharmaceutical Surgical structuresmaterials Contact lens

Treating major burnsBlood dialysis membranesArtificial blood vesicles

Others Enzyme immobilizationEncapsulation of nutraceuticalsChromatographyAnalytical reagentSynthetic fiberChitosan-coated paperManufacturing material for fiberFilm and sponges


Natural chitin has a molecular weight exceeding 1,000 kDa while commer-

cially available chitosan has a MW of 100±1200 kDa (Lower, 1984; Li et al.,

1992). Numerous forces during commercial production may influence the

molecular weight of chitosan. Factors such as high temperature (above 280ëC

thermal degradation of chitosan occurs and the polymer chains quickly break

down), dissolved oxygen concentration and shear stress may cause these

changes to occur (Muzzarelli, 1977; Li et al., 1992).

16.2.2 Production of chitin and chitosan

Two hydrolytic methods are known for preparation of chitin and chitosan. These

are acid hydrolysis (chemical treatment) and enzymatic hydrolysis. Shahidi and

Synowieki (1991) and Shahidi et al. (1999) reported isolation and characteriza-

tion of chitin from shrimp and crab processing by-products. Different parts of

crab shells contained varying amounts of chitin with the legs having the

maximum amount. On a dry weight basis, 17.0 to 32.2% chitin was present in

shrimp and crab section by-products (Shahidi and Synowiecki, 1991).

The normal procedure for preparation of chitin from crustacean shells

includes the use of sodium or potassium hydroxide for deproteination, hydro-

chloric acid for demineralization and agents to remove the remaining proteins,

calcium, and color, respectively (Fig. 16.2). The chitin that is produced can

then be deacetylated with concentrated base (40±50%) at high temperatures

(100±130EC) to produce chitosan (Jaworska and Kowieczna, 2001; Tsai et al.,

2002).

Deacetylation proceeds rapidly during the first hour of treatment with 50%

NaOH at 100ëC leading to 68% deacetylation (Oh et al., 2001). This is followed

by a slower step and by the end of 5 h, about 78% deacetylation is achieved.

Increasing time does not lead to any further deacetylation of chitin, but it lowers

the molecular weight of the product.

Chitosans produced from both chemical and enzymatic methods are different

with respect to their degree of deacetylation (DD), distribution of acetyl groups,

chain length, and conformational structure of chitin and chitosan molecules.

These factors, together, will affect the characteristics of the products. Optimum

conditions for chitosan pre-treatment (deacetylation by 45% alkali solution for

1 h) were studied by investigating the coagulation efficiencies of chitosan

prepared under different conditions (Huang et al., 2000). The procedure

involved crushing of crab shells to a powder and isolation of chitin. Sub-

sequently, chitin was deacetylated using NaOH at 100ëC (Kamil et al., 2002)

followed by multiple rinsing of the product with deionized water to reach pH 7,

and finally drying at 80ëC for 48 h. The resulting chitosan was dissolved in

different concentrations of acetic or hydrochloric acid, stirring at room

temperature, until it was completely dissolved. As the concentration of acid

increased, the viscosity of dissolved chitosan coagulants decreased due to

binding of positively charged chitosan to the negatively charged acid anion in

the solution. The conformation of chitosan polymers changes and becomes more


compact in the acidic solution and thus lowers the viscosity of the solution; the

best solution was obtained at pH 2.0.

16.2.3 Deacetylation and MW of chitosan and its activity

Chitin with varying degrees of acetylation (DA), ranging from fully acetylated

to totally deacetylated, may be procured. The degree of acetylation affects the

physical properties of chitin; thus increasing the degree of acetylation leading to

a decrease in the degree of solubility in different solvents. Oh et al. (2001)

reported that the DD of chitosan is affected by the concentration of alkali,

temperature, reaction time, prior treatment of chitin, particle size, and chitin

concentration.

Heux et al. (2000) reported that after partial deacetylation (to less than 50%),

the product of chitin becomes soluble in acidified water. Therefore, chitosan is

characterized by its degree of acetylation (DA), which is the average mole

fraction/percentage of N-acetyl-D-glucosamine units within the macromolecular

chain (Desbrieres, 2002); other methods may also be employed for this purpose

Fig. 16.2 Flowsheet for preparation of chitin, chitosan, their oligomers and monomersfrom shellfish processing by-products.


(Huang et al., 2000). Many different techniques are used to evaluate the average

degree of acetylation of chitosan, such as infrared, solid state NMR, ultraviolet

spectrometry and potentiometric titration, 1H liquid-state NMR, and elemental

analysis (Heux et al., 2000), as well as 13C solid-state NMR and elemental

analysis; these techniques do not require solubilization of the polymer.

Techniques used for evaluation of DA of chitin and chitosan over the whole

range of DA include 13C and 15N cross-polarization/magic angle spin (CP-MAS)

solid-state nuclear magnetic resonance (NMR) and 1H liquid-state NMR. These

methods afford results in good agreement, but the limitation of solid-state NMR

is that it requires a detection threshold not higher than 5%. Meanwhile the15N

CP-MAS technique was found to be a powerful technique to assess the acetyl

content in the case of complex association of chitin and other polysacchrides

(Heux et al., 2000).

The degree of acetylation and the molecular weight of chitin/chitosan may be

determined using circular dichroism and viscometric methods, respectively

(Zhang and Neau, 2001). The degree of deacetylation has no effect on the acid-

binding properties of chitosan (Scheruhn et al., 1999). Chitosans which have a

relatively high degree of deacetylation enhance fibroblast proliferation, but

those with lower levels of deacetylation exhibit less activity. However, the

molecular weight and polymer chain length were of little consequence (Howling

et al., 2001).

The molecular weight of chitosan has an important effect on its activity.

Chitosan preparations with a molecular weight of 5±50 kDa reduced serum

cholesterol levels in rats (Ikeda et al., 1993). Meanwhile, chitosans with a

molecular weight of 8 kDa were more effective as hypocholesterolemic agents in

rats than chitosans with a MW of 2 or 220 kDa (Enomoto et al., 1992). Chitosan

with a MW of 12 kDa (DDA, 87%) was most effective against L. fructivorans,

and chitosan with MW of 32.5 kDa (DDA, 80%) was most effective against L.

plantarum (Oh et al., 2001). The molecular weight of chitosan had no effect on

S. liquifaciens. Thus, a relationship between the type of microorganism and

antimicrobial activity of different MW chitosans is evident. Chitosans with

average molecular weights of over 10 kDa had a positive effect on enhancing

fecal excretion of neutral steroids. In addition, as the viscosity or the degree of

deacetylaion of chitosan preparation increased, the effects on the apparent fat

digestibility were more clear (Ylitalo et al., 2002). Thus an increase in the DD,

and hence the number of NH2 groups, resulted in stronger antimicrobial activity

of chitosan (Tsai et al., 2002). This result agrees with the findings of Chang et

al. (1989), Darmadji and Izumimoto (1994), Simpson et al. (1997), and Wang

(1992).

16.2.4 Depolymerization and N-acetylation

Thermal depolymerization of chitosan chloride in solid state showed increasing

of intrinsic viscosity with increasing degree of acetylation, which is an important

parameter for thermal degradation (Holme et al., 2001). The presence of oxygen


had no effect on the rate of chitosan degradation, but pH did affect the degrada-

tion of chitosan. Furthermore, acid hydrolysis was the primary mechanism

involved in thermal depolymerization of chitosan chloride in the solid state

(Holme et al., 2001). Chitosan, similar to other polysaccharides, is influenced by

several degradation mechanisms, including oxidative-reductive free radical

depolymerization, and acid-, alkaline- and enzymatically-catalyzed hydrolysis

(Holme et al., 2001). In these, cleavage of glycosidic bonds is involved, hence it

is important to control the depolymerization process in order to maintain other

properties such as viscosity, solubility, and biological activity. Decomposition

(release of material) of chitosan begins at 200ëC, but Holme et al. (2001)

reported that chitosan chlorides were thermally degraded at 60, 80, 105, and

120ëC, the degradation rate of chitosan increasing with increasing temperature

and degree of acetylation during acid hydrolysis (Holme et al., 2001).

N-acylation of chitosan fibers upon treatment with a series of carboxylic acid

anhydrides in methanol at room temperature was examined (Hirano et al., 2000).

The N-acylation had little effect on mechanical properties of the resultant

filaments such as tenacity and elongation values. Treatment of chitin fiber and

chitin-cellulose mixed fiber with 40% NaOH at 95±100ëC for 4 h in suspension,

afforded a chitosan fiber and a novel cellulose-chitosan mixed fiber, respec-

tively. Novel N-acylchitosan fibers produced were N-acetyl-, N-propionyl-, N-

butyryl-, N-hexanoyl-, and N-octanoyl chitosans. These fibers were insoluble in

water, aqueous basic and acidic solutions.

16.2.5 Comb-shaped chitosan

Chitosan has a considerable advantage over chitin for modification purposes

because it possesses free amino groups. N-substituted chitosan derivatives may

be obtained using reducing sugars, aldehydes or ketones via reductive alkyla-

tion, which is a typical example of reactions of chitosan. Reductive alkylation of

chitosan leads to production of comb-shaped chitosan derivatives with mono-

aldehydes from tri- and tetra (ethylene glycol) monosubstituted derivatives. The

introduction of such branches clearly increased the affinity of molecules for both

water and organic solvents without loosening the attractive characteristics of

chitosan, such as metal ion adsorption capacity (Kurita et al., 1999). In order to

prepare comb-shaped chitosan derivatives, chitosan may be completely

deacetylated via treatment with monoaldehyde derived from tri(ethylene glycol)

under homogeneous conditions in an acetic acid-methanol solution. Sodium

cyanoborohydride was added to the solutions which afforded a weak gel. The

resultant mixtures were then dialyzed against deionized water to afford clear

solutions which were concentrated and freeze-dried in order to afford slightly

yellowish solids (Kurita et al., 1999).

16.2.6 N-alkylation of chitin for improved solubility

Lack of solubility of chitin in usual solvents, except in fluorinated solvents, N,N-

dimethylacetamide/LiCl, and methanol/CaCl2 leads to preservation of chitin in


nature. Deacetylated chitin (50% random), and chitin derivatives having tosyl,

iodo, trimethylsilyl and glycosyl groups are soluble in water or organic solvents

(Kurita et al., 2002). The N-alkylation of chitin improves solvent affinity and

lowers crystallinity of chitins. Thus, many experiments have been conducted to

synthesize polymers with N,N-dimethylacetamide moieties in their backbone by

ring-opening polymerization of 2-oxazolines.

Introduction of methyl, ethyl, and pentyl groups into chitin at the nitrogen of

C-2 acetamido moiety via an adjusted 5-step modification process was reported

by Kurita et al. (2002) (Fig. 16.3). Chitosan was completely deacetylated and

treated with formaldehyde (methanal), acetaldehyde (ethanal) or valenaldehyde

(pentanal). The Schiff bases of chitosan were then reduced to N-alkylated

chitosan using sodium cyanoborohydride (NaCNBH3).

The N-alkyl chitosans were subsequently changed into corresponding N-alkyl

chitins via acetylation using acetic anhydride followed by transesterification to

eliminate partially formed O-acetyl groups. This synthetic pathway is direct and

effective to provide well-defined novel chitin derivatives. The resulting N-

methyl-, N-ethyl-, and N-pentylchitins were amorphous and displayed a high

affinity for solvents (Kurita et al., 2002).

16.2.7 Trapping-retention of heavy metals

Chitinous materials are known to interact with metal ions, including radio-

active products. However, chitosan derivatives are better known for their

trapping-retention of heavy metal. Cardenas et al. (2001) described a method

for preparing chitosan mercaptan derivatives with mercaptoacetic acid and

Fig. 16.3 Schiff base formation with aldehydes, reduction, and N-acetylation of chitin.Symbols are: Ac, Acetyl; Ac2O, acetic anhydride.


1-chloro-2,3-epoxypropane and evaluated their retention capacities using

different concentrations of copper and mercury. These derivatives are shown

in Fig. 16.4 and include N-hydroxy-3-mercaptopropylchitosan (chitosan 1), and

N-(2-hydroxy-3-methylaminopropylchitosan, (chitosan 2). All chitosan

derivatives tested were thermally stable; N-hydroxy-3-mercaptopropylchitosan

showed the highest thermal stability at 314ëC compared with chitosan at 290ëC.

The copper ion adsorption was less than that of mercury ions at either pH2.5 or

pH4.5, suggesting a lower selectivity for Cu (Cardenas et al., 2001).

Hydrolysis of chitin and chitosan may occur upon the action of chitinases,

chitosanases, lysozymes, and cellulases (Fig. 16.5) (Shahidi et al., 1999).

Tsigos et al. (2000) reported the necessity for pre-treatment (alkali treatment)

of crystalline chitin before adding the enzyme to increase the rate of

deacetylation in order to produce new polymers with new physical and

chemical characteristics. The compounds are easily soluble if produced with

different distribution of N-deacetylated residues. A synthetic procedure for

chitin with N-acetyl-D-glucosamine and chitosan derivatives with D-

glucosamine branches has been reported (Kurita et al., 2000). These resulting

non-natural branched chitin and chitosan have extra amino sugars in branches

that render them much improved properties in comparison with linear ones,

such as the affinity for solvents and hygroscopicity. These characteristics

would be of great interest in different applications, such as moisturizers for

cosmetics and antimicrobial substances for fiber and textile treatment (Kurita

et al., 2000).

16.2.8 Enzymatic hydrolysis and preparation of chitin and chitosan

oligomers

Several reports on chemical hydrolysis (including acid hydrolysis) for prepara-

tion of chitin oligomers have appeared (Bosso et al., 1986; Defaye et al., 1989;

Inaba et al., 1984; Hirano and Nagano, 1989; Kendra et al., 1989; Kurita et al.,

1993; Rupley, 1964; Sakai et al., 1990; Takahashi, 1995). A series of commer-

Fig. 16.4 Production of mercaptan derivatives of chitosan via conversion with 1-chloro-2,3-epoxypropane.


cially available chitin oligomers, up to hexamer, has been prepared by partial

hydrolysis of chitin with concentrated HCl, followed by fractionation using

column chromatography (Rupley, 1964). The procedures for isolation of chitins

and oligomers include acid hydrolysis, neutralization, demineralization, frac-

tionation by charcoal-celite column, fractionation by HPLC (high performance

liquid chromotography) and lyophilization. These methods suffer from several

disadvantages, such as being time consuming, laborious, and environmentally

unfriendly (Tsigos et al., 2000). In addition, these methods may afford a low

yield of oligomers with a high degree of polymerization (Takahashi et al., 1995).

To overcome drawbacks associated with the conventional methods, procedures

such as acetolysis (Bosso et al., 1986; Defaye et al., 1989; Inaba et al., 1984;

Hirano and Nagano, 1989; Kendra et al., 1989; Kurita et al., 1993; Rupley,

1964; Sakai et al., 1990; Takahashi, 1995), fluorosis (Bosso et al., 1986),

fluorohydrolysis (Defaye et al., 1989), and sonolysis (Takahashi et al., 1995)

(Fig. 16.6) have been considered.

Acetolysis is a procedure for preparation of oligomers from chitin using

acetic anhydride and sulfuric acid (Fig. 16.6). Beta-chitin from squid was used

as a starting material for simple acetolysis, leading to the formation of

Fig. 16.5 Preparation of products from chitin.


N-acetylchitooligosaccharide peracetate in high yields with reasonable repro-

ducibility (Kurita et al., 1993).

Fluorohydrolysis is another method used for preparation of chitin oligomers

in which anhydrous HF is employed (Fig. 16.6). Defaye et al. (1989) reported

that fluorohydrolysis of chitin in anhydrous hydrogen fluoride (HF) yields chitin

oligomers ranging from 2 to 9 residues and chitin oligomer isomer (�-(166)-linked acetamino-2deoxy-D-glycosyloligosaccharides) nearly quantitatively.

In addition, sonolysis may be used for preparation of oligomers from chitin

using hydrochloric acid hydrolysis under ultrasound irradiation (Fig. 16.6)

(Takahashi et al., 1995). The above combined method that includes a mild acid

hydrolysis and sonolysis was able to hydrolyze polymers independent of

temperature of the bulk solution and degradation of chitin by HCl under

ultrasound irradiation (Takahashi et al., 1995). This method saves time and does

not require more than 2 h. However, caution should be exercised to avoid

deacetylation of the acetamido group.

Chitosan oligomers were first prepared by Horowitz et al. (1957). Acid

hydrolysis of chitosan with concentrated HCl led to the production of chitosan

oligomers with a low degree of polymerization (DP), but in a quantitative

manner. Several studies have described the production of chitosan oligomers

with a DP of less than 6 residues (Sakai et al., 1990; Takahashi et al., 1995;

Tsukada and Inoue, 1981). On the other hand, Domard and Cartier (1989)

Fig. 16.6 Mechanism for acid hydrolysis of chitin.


reported that a wide distribution of glucosamine oligomers could be easily

produced and separated up to DP of 15 in the pure form. Defaye et al. (1994)

prepared chitosan oligomers by fluorolysis in anhydrous hydrogen fluoride.

They obtained oligomers with DP of 2±11. The majority of acidic hydrolysis

methods have reported production of chitosan oligomers with a low DP, mainly

from monomer to tetramer in quantitative amounts. The yields of relatively

higher DP (pentamer to heptamer) oligomers were low. However, physiological

function is rendered best by high DP oligomers.

Chitin and chitosan oligomers can also be prepared by enzymatic methods.

Enzymatic methods offer many benefits over chemical hydrolysis as they produce

desirable oligomers with a high DP under milder conditions (Jeon et al., 2000).

Jaworska and Konieczna (2001) investigated the effect of supplemental

components (Fe+2, Co+2, Mn+2, trypsin, and chitin) on the in vivo activity of two

enzymes (chitin synthase and chitin deacetylase) to produce chitosan from

fungus Absidia orchodis. Manganese ions (Mn+2) and ferrous ions (Fe+2) gave

rise to the highest increase in the amount of biomass rather than chitosan content

in cell walls of the fungus. The effects of trypsin and chitin on biomass and

chitosan content in cell walls were not significant, while Co+2 totally inhibited

the growth of fungi. Ferrous ions decreased the activity of chitin deacetylase.

Chitosan from fungi cultivated with ferrous ions had a higher DD (26±30%) than

chitosan from unsupplemented medium (15%). The same trend was observed for

Mn+2. The amount of chitosan from fungi cultivated in the presence of Mn+2 was

higher (about 30%) than that produced in an uncultivated medium (15%).

The effects of degree of deaceylation (DD) and preparation procedures for

chitosan were evaluated for their antimicrobial activity (Tsai et al., 2002). Chitin

was chemically (CH-chitin) and microbiologically (MO-chitin) prepared from

shrimp shells. The resulting chitins were subsequently deacetylated chemically

to produce chitosan with DD ranging from low (47±53%) to medium (74±76%)

to high (95±98%). The antimicrobial activities of both chemically and micro-

biologically prepared chitin/chitosan were the same, and in both cases the

activity increased with increasing DD. Moreover, the size and conformational

characteristics of chitin and chitosan were crucial for their antimicrobial

function. In general, chitosan has a stronger effect against bacteria than fungi.

Chitosan with a high DD (98%) efficiently inhibited various bacteria (Tsai et al.,

2002). Therefore, chitosan displays potential for increasing the shelf life of

refrigerated fish fillets (Shahidi et al., 1999). Furthermore, Uchida et al. (1989)

showed that enzymatic hydrolysis produced a high amount of high DP oligomers

from chitin and chitosan when compared to acid hydrolysis.

16.3 Applications of chitin, chitosan and their oligomers

Different applications of chitins, chitosan and their oligomers have been

summarized in Table 16.1. As indicated, there is a myriad of areas in which such

products could be used (see below).


16.3.1 Medical applications

Chitin and chitosan have both material and biological properties that might be

beneficial to enhance wound-repair. As well, both of them have great influence

on different stages of wound-healing in experimental animal models (Howling et

al., 2001). Howling et al. (2001) found that chitosan polymers can interact with

and modulate the migration behavior of neutrophils and macrophages modifying

subsequent repair processes such as fibroplastia and reepithelialization. Chitin

and chitosan have both stimulatory and inhibitory effects on proliferation of

human dermal fibroblasts and keratinocytes (Howling et al., 2001). They also

have enhancing effects on the survival function of osteoblasts and chondrocytes

(Lahiji et al., 2000). The procedure for promoting wound-healing by chitosan

was tested as follows: chitosan was coated onto plastic coverslips that had been

filled into 24-well plates. Human osteoblasts and articular chondrocytes were

seeded on either uncoated or chitosan-coated coverslips. The incubation

temperature of the culture was 37ëC and under 5% CO2 for 7 days. By using

a fluorescent molecular probe, cell viability was judged. Reverse transcriptase±

polymerase chain reaction and immunocytochemistry were used for pheno-

typing expression of osteoblasts and chondrocytes. The results showed that the

chondrocytes and osteoblasts appeared spherical and refractile of the chitosan-

coated coverslips, while 90% of the cells on the plastic coverslips were

elongated and spindle shaped after this period of incubation (Lahiji et al., 2000).

It was reported that the wound recovering material composed of polyelectrolytic

complexes of chitosan and sulfonated chitosan that speeded up wound healing

and afforded a good-looking skin surface (Lahiji et al., 2000).

Chitosan has the ability to promote wound-healing; this is due to the tendency

to form polyelectrolyte complexes with polyanion heparin, which possesses

anticoagulant and angiogenic properties (Lahiji et al., 2000). By forming a

complex with heparin and acting to lengthen the half-life of growth factors,

chitosan supports tissue growth and helps wound-healing.

Other studies have examined the effect of chitin and chitosan samples with

different deacetylation levels and polymer chain length on the proliferation of

human dermal fibroblasts in vitro (Howling et al., 2001). It was found that

chitosans with a high degree of deacetylation strongly motivated fibroblast

proliferation; meanwhile, samples with lower degrees of deacetylation showed

less activity.

Cho et al. (1999) used water-soluble chitin (WSC) prepared at room tem-

perature through depolymerization by ultrasonication after alkaline treatment of

chitin. The degree of deacetylation and molecular weight were controlled. Chitin

with DD of 8.60%, chitosan with DD of 83.9% and WSC were embedded to the

wounded backs of rats after full thickness skin cuts. It was noticed that the WSC

had the highest efficiency in recovering strength of the wounded skin due to the

hydrophilicity and high biodegradability of WSC that maximized its activity as a

wound-healing accelerator. In addition, the arrangement of the collagen fibres in

the wound was the same as that of the normal skin. Hirano and Zhang (2000)

described the preparation of a novel blend fiber. This fiber is a mixture of


cellulose with each of hyaluronate (HA), heparin (Hep), chondroitin 4-sulfate,

chondroitin 6-sulfate and a chitin-chondroitin 6-sulfate blend using an aqueous

10% sulfuric acid solution containing 40±43% ammonium sulfate as a

coagulating solution. These blend fibers could be used as covering materials

for the wound-healing tissues of animals and plants.

Recently, bandages made of chitosan were investigated in the military field in

the new war in Iraq (Brown, 2003). Z-Medica, a small company supplied these

products to the US ground troops in Iraq and Afghanistan (Becker, 2003). These

bandages were used immediately after injury to control bleeding at this critical

time and were found to save numerous lives (Becker, 2003). Arterial bleeding

was stopped in about a minute when these bandages were applied with pressure

to a wound (Brown, 2003). The use of such bandages was approved by the Food

and Drug Administration (FDA) in November 2002 (Mientka, 2003). They

called it `shrimp' bandage that contains chitosan. This bandage can stop

capillary bleeding and stanch severe arterial hemorrhaging (Mientka 2003).

Mientka (2003) reported that chitosan bandages had the ability to stop bleeding

at a rate of 600 mL per minute. Moreover, there was no sign of alergenicity for

use of these bandages in soldiers who were allergic to shrimp (Mientka, 2003).

Dietary applications

Chitosan may be considered as a dietary supplement for reducing body weight in

humans. Industrial production of chitosan tablets (Muzzarelli et al., 2000) and

chitosan dietary fibers (Hughes, 2002) has occurred. In addition, Schiller et al.

(2001) reported that a rapidly-soluble chitosan (LipoSan Ultra that has a higher

density and solubility than chitosan itself) facilitated weight loss and reduced

body fat. This effect was due to the fact that this chitosan was able to prevent

dietary fat absorption in overweight and mildly obese individuals that consumed

a high-fat diet.

Chitosans have also been used to prevent body weight increase in animals

(Hughes, 2002). Meanwhile, negative results were recorded regarding chitosan

effectiveness in this field (Hughes, 2002). During a high-fat diet and chitosan

supplementation, no increase in fecal fat content was noticed, meaning that

chitosan had no effect on fat absorption (Hughes, 2002). Use of chitosan in the

diet has been questioned by some researchers for individuals suffering from

allergic reaction to crustaceans (Ylitalo et al., 2002).

Although the reactivity of chitosan toward lipids is not clear, it is claimed that

chitosan, due to its cationic nature, binds to appropriate bile and fatty acids and

brings about their excretion (Muzzarelli et al., 2000; Ylitalo et al., 2002). This

claimed efficacy of chitosan in reducing the body weight, hypercholesterolemia,

and hypertension stimulated production of chitosan tablets. The capacity of

chitin, chitosan, N-lauryl chitosan and N-dimethylaminopropyl chitosan on

sequestering steroids was investigated (Muzerelli et al., 2000). They reported

that chitin might be more effective in holding olive oil and enriching the retained

oil fraction with steroids sequestering than chitosan. As well, chitin derivatives

were able to distinguish between different lipids. These results put into question


the need for high cationity for sequestering cholesterol. The use of chitosan

monomer, glucosamine sulfate, for joint building is also commonplace.

Antihypercholesterolemic agent

Chitosan has been reported to render a significant hypocholesterolemic activity

in different experimental animals (Hirano et al., 1990; Sugano et al., 1978,

1980; Ylitalo et al., 2002). Sugano et al. (1988) noted that chitosan oligomers

did not exhibit this effect. The studies were carried out on rat groups fed on a

diet rich in cholesterol to find the effect of chitosan hydrolyzates with different

molecular weights and viscosity on the hypocholesterolemic activity. The lower

the molecular weight of chitosan, the better was its cholesterol-lowering

potential.

The mechanism of antihypercholesterolemic activity of chitosan has been

described by Ylitalo et al. (2002). In the stomach, the acidic condition

protonates the amino groups. Fats, fatty acids (oleic, linoleic, palmatic, stearic

and linolenic acids) and other lipids as well as bile acids, due to their negative

charge (X-COOÿ), attach themselves strongly to the positively charged amino

groups (ÿNH3+) of chitosan. This binding might inhibit their absorption and

recycling from the intestine to the liver. However, this interruption of entero-

hepatic circulation of cholic acid and other bile acids can lead to an increase in

the biosynthesis of cholic acid from cholesterol in the liver. The cholesterol

content of liver cells is thus decreased and this may lead to subsequent activation

of LDL-receptor expression, and could further increase LDL uptake via LDL-

receptors in the liver (Ylitalo et al., 2002). We have previously, reported that

production of dietary cookies, potato chips and noodles enriched with chitosan is

commonplace in certain countries. The products enriched with chitosan are

expected to render hypocholesterolemic effects. As well, vinegar products

containing chitosan are produced and sold in Japan because of their cholesterol-

lowering ability (Shahidi et al., 1999).

Anti-tumor activity

Suzuki (1996) reported that chitin and chitosan oligomers have the ability to act

as inhibitors of growth tumor cells via their immuno-enhancing effects. Suzuki

et al. (1985) found that chitin oligomers from (GlcNAc)4 to (GlcNAc)7 have

strong attracting responses to peritoneal excudate cells in BALB/c mice.

However, chitooligosaccharides from (GlcN)2 to (GlcN)6 did not exhibit such an

effect.

With regard to hexamers, both (GlcNAc)6 and (GlcN)6, were reported to

process growth inhibitory effects against allogenic and syngeneic mouse systems

(Suzuki et al., 1986a). These results indicate that the effect was not by direct

cytocidal action on tumor cells, but was in fact host-mediated.

Anti-ulcer agent

Ito et al. (2000) reported that chitosans with different molecular weights had

ulcer healing actions. The effects of low molecular weight (LMW) chitosan,


high molecular weight chitosan (HMW), and chitin on ethanol-induced gastric

mucosal injury and on the healing of acetic acid-induced gastric ulcers in rats

were compared. It was found that orally administrated LMW chitosan could

prevent ethanol-induced gastric mucosal injury. Repeated oral administration of

LMW chitosan, in a dose-dependent manner, accelerated the gastric ulcer

healing. The effects of HMW chitosan and chitin on gastric ulcer healing were

less than those of LMW chitosan.

A coating agent for prosthetic articles (artificial parts of the body)

Muzzarelli et al. (2000) described a method for coating prosthetic articles with

chitosan-oxychitin. Plates of Ti (titanium) and its alloys were plasma-sprayed

with hydroxyapatite and glass layers, and subsequently a chitosan coat was

deposited on the plasma-sprayed layers using chitosan acetate. These layers

were treated with 6-oxychitin to form a polyelectrolytic complex. This complex

was optionally contacted with 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide

at 4ëC for 2 h to form amide links between the two polysaccharides, or

acetylation with acetic anhydride in methanol to obtain a chitin film. In all cases,

the modified coats were insoluble, uniformly flat and smooth. Prosthetic

materials coated with chitosan-oxychitin were capable of provoking coloniza-

tion by cells, osteogenesis and osteointegration.

There were two main reasons behind the selection of chitosan-oxychitin

coated orthopedic plates. Firstly, chitosan enhances the integration of the

implant and secondly, chitosan stimulates bone regeneration.

16.3.2 Food applications of chitin, chitosan and their oligomers

New applications of chitin and its oligomers led to over 50 patents in the

1930s and the early 1940s. However, commercialization of these products was

hindered by inadequate manufacturing services and competition from synthetic

polymers (Averbach, 1981). However, after the 1970s industrial utilization of

chitin and its oligomers has increased (Kaye, 1985). Furthermore, improve-

ment in research and small-scale production of chitin and chitosan has

extended the number and the varieties of potential applications of chitinous

materials. In addition, environmental problems and cost of disposal of shellfish

processing disards have increased the urgency for development of

environmentally safe alternatives for numerous plastic or polymeric products

(Ashford et al., 1976; Berkeley, 1979; Shahidi and Synowiecki, 1991). Some

food applications of chitin, chitosan and their olgomers are summarized in

Table 16.1 on page 344.

Chitin, chitosan and their derivatives offer a wide range of applications

(Table 16.1) including bioconversion for the production of value-added food

products, preservation of foods from microbial spoilage, formation of

biodegradable films, recovery of waste material from food processing discards,

purification of water as well as clarification and deacidification of fruit juices

(Shahidi et al., 1999).


Antimicrobial activity

Chitin, chitosan and their derivatives have antimicrobial activity against

bacteria, yeast and fungi (Yalpani et al., 1992). The exact mechanism of

antimicrobial action of chitin and chitosan and their derivatives remains illusive,

but different mechanisms have been proposed (Shahidi et al., 1999). Chitosan

has the ability to produce phytoalexins, cell wall phenols and callose (Tsai et al.,

2002). Chitosan is considered to be a soluble chelating agent and activator due to

the presence of a positive charge on the C-2 of its glucosamine monomer at pH

values below 6. This characteristic gives it a higher antimicrobial activity than

chitin (Chen et al., 1998). A leakage of proteineous and intercellular com-

ponents occurs due to the interaction between the positively charged chitosan

molecules and the negatively charged microbial cell membranes (Chen et al.,

1998; Papineau et al., 1991; Sudharshan et al., 1992; Young et al., 1982). This is

affected by the molecular weight of chitosan (Tsai et al., 2002). Being a

chelating agent, chitosan has the ability to selectively bind trace metals, which

prevents production of toxins and microbial growth (Cuero et al., 1991).

Chitosan is also an activator for several defence processes in the host tissue (El

Ghaouth et al., 1992c), having the ability to bind water and inhibit various

enzymes (Young et al., 1982).

Tsai et al. (2002) studied the effects of degree of deacetylation (DD) and

preparation methods for chitin and chitosan on their antimicrobial activity. It

was found that chemically-(CH-chitin) and microbiologically-prepared chitin

(MO-chitin) could undergo further chemical deacetylation to produce chitosan

with different DDs. However, MO-chitin that was deacetylated by various

proteases had no antimicrobial activity (Tsai et al., 2002). However, for

chitosan, as the DD increased, its antimicrobial effect on bacteria increased,

even to a greater extent than that on fungi.

Genetically, chitosan can enter the nuclei of a microorganism and bind with

DNA. This binding inhibits the mRNA and protein synthesis (Hadwiger et al.,

1985; Sudharashan et al., 1992).

The effect of concentration of chitosan for complete inactivation of certain

types of bacteria has been reported (Shahidi et al., 1999). Wang (1992) observed

that a much higher concentration of chitosan (1±1.5%) was required for

complete inactivation of Staphylococcus aureus after two days of incubation at

pH 5.5 or 6.5 in the medium. Furthermore, Chang et al. (1989) found that

chitosan concentrations of � 0.005 were sufficient to elicit complete inactivation

of S. aureus. This was in accordance with the findings of Darmadji and

Izumimoto (1994) on the effect of chitosan in meat preservation. Different

concentrations of chitosan and their effect on the growth of different cultures of

bacteria on raw shrimp was examined (Simpson et al., 1997). Bacillus cereus

required chitosan concentrations of 0.02% for anti-bacterial effect, while

Escherichia coli and Proteus vugaris exhibited minimal growth at 0.005% and

growth was inhibited at � 0.0075%. The effect of different concentrations of

chitosan on Escherichia coli growth has also been studied. Wang (1992)


reported complete inhibition after 2 days incubation with 0.5 or 1% chitosan at

pH 5.5. It was also reported that if chitosan concentration increased by about 1%

in the broth, it could afford complete inactivation. However, Darmadji and

Izumimoto (1994) reported that growth inhibition of E. coli required a 0.1%

chitosan concentration. Simpson et al. (1997) found that only 0.0075% chitosan

was required to inhibit the growth of the same species. Existing differences in

the degree of acetylation of chitosans employed might explain the observed

variations (Shahidi et al., 1999).

Iida et al. (1987) and Nishimura et al. (1984) have reported that if chitin is

partially deacetylated, especially at 70%, it has the ability to stimulate non-

specific host resistance against E. coli and Sendai virus infection in mice.

Meanwhile, chitin and chitosan have the ability to increase the number of mouse

peritoneal exudate cells that generate reactive oxygen intermediates and then

display candidacidal activities (Suzuki et al., 1984). Suzuki et al. (1986b)

reported that chitin hexamer (GlcNAc)6 had a strong candidacidal activity.

Chitosan was found to decrease the in vitro proliferation of many fungi with

the exception of Zygomycetes (Allan and Hadwiger, 1979). Chitosan acts as an

antifungal agent via the formation of gas permeable coats, interference with

fungal growth, and stimulation of many defence processes, including accumula-

tion of chitinases, production of proteinase inhibitors, and lignifications and

stimulation of callous synthesis (Bai et al., 1988; El Ghaouth et al., 1992c).

Antifungal effect of chitosan on in vitro growth of common post-harvest

fungal pathogens in strawberry fruits was reported by El Ghaouth et al. (1992b).

Chitosan with a 7.2% NH2 significantly decreased the eradial proliferation of

Botrytis cinerea and Rhizopus stolonife, with a greater impact at higher

concentrations. Fang et al. (1994) reported the preservative influence of chitosan

on low-sugar candied Kumquat (fruit). Chitosan (at 0.1±5mg/ml) inhibited the

growth of Aspergillus niger, whereas chitosan at less than 2mg/ml was

ineffective in inhibiting mold proliferation and aflatoxin synthesis by

Aspergillus parasiticus. Cuero et al. (1991) conducted a similar study and

observed that N-carboxymethylchitosan decreased aflatoxin formation in A.

flavus and A. Parasitius by more than 90% while fungal growth was decreased

to less than half. Furthermore, Savage and Savage (1994) reported that apples

coated with chitosan reduced the rate of molds occurring on them over a period

of 12 weeks. Cheah and Page (1997) found that chitosan coating of carrot with a

2 or 4% chitosan solution considerably reduced their Sclerotinia rotting by 28 to

88%.

El-Katatny et al. (2001) reported the characterization of a chitinase and endo-

�-1,3-glucanase from Trichoderma harzianum strain Rifai T24. These two

enzymes are the key enzymes in the lyses of cell walls during their myco-

parasitic effect against plant diseases caused by fungi, including S. rolfsii. The

chitinase from T. harzianum was purified in two steps using ammonium sulfate

precipitation followed by hydrolytic interaction chromatography. SDS-PAGE

showed that the enzyme exhibited a single band at 43 kDa. The �-1,3-glucanase


was purified and found to have a molecular mass of 74 kDa. The optimum pH of

both enzymes was 4.5. The optimum temperature of the T24 chitinase was 40ëC,

whereas the optimum temperature of �-1, 3-glucanase was 50±60ëC. Both the T.

harzianum T24 chitinase and �-1,3-glucanase were strongly inhibited by Hg+2,

suggesting that sulfhydryl groups are involved in the catalytic reaction (El-

Katatny et al., 2001). A mixture of these enzymes, which showed thermo-

stability and low effective dose (ED50) values against S. rolfsii, may be

considered a potential tool for controlling of plants' pathogens.

Chitin may be used to determine the total content of mycelium based on

chitin (Bishop et al., 1982; Donald and Mirocha, 1977). Bishop et al. (1982)

used chitin to evaluate the presence of mold in tomato products, ketchup, paste

and puree. They noticed variations in chitin content among different fungal

species depending upon the cultural age and growth conditions; values ranged

from 5.7 to 43�g of glucosamine per mg dry weight.

Preservation of foods

Chitosan can be used for food preservation in order to inhibit the growth of

spoilage microorganisms (Oh et al., 2001). By treating crude chitin with various

NaOH concentrations (45, 50, 55, and 60% w/v), four kinds of chitosans were

prepared (chitosan-45, -50, -55 and -60, respectively) (Ylitalo et al., 2002). Four

species of food spoilage microorganisms were treated with chitosans in order to

examine their effects on microbial activity (Oh et al., 2001). These were

Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquifaciens and

Zygosacchaomyces bailii. Chitosan had a biocidal effect; the number of cells

grown was clearly reduced. It has been found that after an extended phase, some

strains recovered and started to grow. As the concentration of chitosan

increased, the activities of these strains increased. It was noticed that chitosan-50

was most effective against L. fructivorans; meanwhile, the inhibition of L.

plantarum growth was mostly by chitosan-55 and no difference was found

among the chitosans tested against S. liquefaciens and Z. bailii. Thus, for

mayonnaise, during its storage at 25ëC, the addition of chitosan decreased the

viable cell counts of L. fructivorans and Z. bailii.

Seafood and meat preservations

A 1% solution of chitosan with a high degree of deacetylation increased shelf

life of fish from 5 to 9 days. Kamil et al. (2002) showed that chitosans prepared

from snow crab shells had different viscosities, closely correlated to the time of

deacetylation. Different viscosity chitosans (14, 57, 360 cP chitosans) were

prepared and used to examine the impact of chitosan covering on fish quality

during refrigerated storage. This study showed the potential of chitosan as a

protective coating for herring and cod in decreasing or preventing moisture loss,

lipid oxidation, and microbial growth. Cod samples coated with 57 and 360 cP

chitosans demonstrated a considerably (p < 0:05) lower relative moisture loss in

comparison with those of uncoated samples and those coated with 14 cP chitosan


throughout the storage period. Furthermore, crab chitosan showed a medium to

high viscosity-dependent protective effect in both fish model systems. In

general, 360 cP chitosan exhibited a better preservative effect in comparison

with 57 and 14 cP chitosans in both systems at 4� 1ëC. The chitosan showed

antioxidant activity in cooked comminuted fish model system, as revealed in

their peroxide value and content of 2-thiobarbituric acid-reactive substances

(TBARS) which were reduced in a concentration-dependent manner. However,

the antioxidant efficiency of relatively high viscosity chitosan in both model

systems was lower than that of the low viscosity chitosan at the same

concentration. The mechanism of action appears to be a result of chelation of

metal ions found in fish muscle proteins, gas exchange adjustment (particularly

oxygen) between fish meat and the surrounding environment, and bactericidal

effect of chitosan itself. Thus, chitosan as an edible coating would enhance the

quality of seafoods during storage (Jeon et al., 2002).

Weist and Karel (1992) studied the effect of using chitosan powders in a

fluorescence sensor for monitoring lipid oxidation in muscle foods. The

efficiency of chitosan powders was explained to be due to the ability of the

primary amino groups of chitosan to form a stable fluorosphere with volatile

aldehydes such as malonaldehyde which is derived from the breakdown of fats

(Weist and Karel, 1992). On the other hand, chitosan was used to improve the

preservation of vacuum-packaged processed meats stored under refrigerated

conditions (Quattar et al., 2000). These authors used chitosan matrix to produce

antimicrobial films by adding acetic or propionic acid (with or without addition

of lauric acid or cinnamaldehyde) to this matrix (Quattar et al., 2000). These

films were applied on to bologna, regular cooked ham, or pastrami. The amounts

of antimicrobial agents found in the chitosan matrix were measured several times

during storage. It was found that within the first 48 hours of application,

propionic acid was nearly completely released from the chitosan matrix, whereas

2±22% of acetic acid remained in chitosan even after 168 hours of storage. With

regard to the presence of lauric acid, but not cinnamaldehyde, it was found that

the release of acetic acid was reduced significantly and more limited on to

bologna than on to ham or pastrami (Quattar et al., 2000). In another study, Li et

al. (1996) found that addition of 3000 ppm of N-carboxymethylchitosan to

cooked pork was sufficient to prevent the oxidative rancidity of the product.

Fruit preservative

Chitin, chitosan and their derivatives have been used as food wraps, due to their

film-forming properties. The chitosan film controls moisture movement between

food and the surrounding environment, thus decreasing the rate of metabolism,

respiration, and rendering high impermeability to certain substances such as fats

and oils, in addition to temperature. These would lead to a delay in ripening of

fruits (Shahidi et al., 1999).

The coating of fruits with chitosan delays the rate of ripening and the

occurrence of decay in tomato (El-Ghaouth et al., 1992a), bell pepper and


cucumber (El-Ghaouth et al., 1991a), and strawberries (El-Ghaouth et al.,

1991b). The control of disease in fruits by chitosan could account for the

antifungal activity of chitosan and its capacity to provoke defence enzymes and

phytoalexins in the plant tissue or a combination of both (El Ghaouth et al.,

1992c). Chitosan (7.2% NH2) inhibited the growth of post harvest pathogens,

namely B. cinerea, A. alternata, C. gloesporioides, and R. stolonifer (El

Ghaouth et al., 1992c). Among the fungi examined, R. stolonifer was least

affected by chitosan (El Ghaouth et al., 1992c). While chitin did not influence

the growth of any of the fungi tested, the growth delay of fungi provoked by

chitosan increased with increasing of the degree of deacetylation (El Ghaouth et

al., 1992c). The inhibitory effects of chitosan correlated with its cationic nature

and the size of the polymers. Moreover, the importance of cationic groups and

the length of the polymer chain was demonstrated by the low fungicidal activity

displayed by N,O-carboxymethyl chitosan compared to that of chitosan, and by

the improved activity of chitosan with increasing levels of deacetylation. The

antifungal effects may, in part, account for the capacity of chitosan to enhance

membrane permeability and result in cellular leakage.

Three mechanisms may be involved in the action of chitosan as an antifungal

agent in the preservation of post harvest crops. Firstly, the treatment of potato

with chitosan, challenged with Erwinia carotovora (the soft rot pathogen of

potato), showed a declined count of bacteria and tissue maceration, thus

resulting in an increase in cell viability. Secondly, potatoes treated with chitosan

showed an inhibition in bacterial reproduction as well as secretion of pectic

enzymes (produced by pathogenic bacteria capable of attacking the plant tissue).

The third mechanism of chitosan action is by controlling the pH. The pathogenic

bacteria that cause decay of crops such as potatoes after harvesting secrete

macerating enzymes (negatively charged proteins), leading to an outflow of

protons and cations from the cell wall of the plant and hence a pH increase in the

cell and cell wall.

Acidity adjustment

Chitosan could be used for deacidification of fruit juices because chitosan salts

carry strong positive charge that could interact with proteins and hence act as

dehazing agents in fruit juice (Shahidi et al., 1999). Scheruhn et al. (1999)

reported that treating of coffee drinks with chitosan increased the pH and

decreased the acid content of the coffee drinks due to the acid binding properties

of chitosan in the coffee. This treatment depended on the concentration of

chitosan and the acid content of the drinks; in addition to the raw material of

chitosan and its processing (Scheruhn et al., 1999).

Antioxidant activity

Muscle food products containing a high content of unsaturated lipids are highly

labile to off-flavor and rancidity development. Warmed-over flavor is developed

in cooked poultry and uncured meat upon storage, resulting in the loss of


attractive meaty flavor (Shahidi et al., 1999). Darmadji and Izumimoto (1994)

noticed that 1% chitosan added to meat resulted in a decline of 70% in the 2-

thiobarbituric acid (TBA) values after three days of storage at 4ëC. St. Angelo

and Vercellotti (1989) reported that N-carboxmethylchitosan was effective in

preventing the formation of warm-over flavor (WOF) over a broad range of

temperature. Moreover, ground beef treated with 5000 ppm of N-carboxy-

methylchitosan exhibited 93 % inhibition of TBA values and 99% reduction in

hexanal content. Furthermore, Shahidi et al. (1999) reported that N,O-carboxy-

methylchitosan (NOCC) and its lactate, acetate and pyrrolidine carboxylate salts

were effective in controlling the oxidation and off-flavor development in cooked

meat stored for nine days at refrigerated temperature. The mechanism by which

this inhibition occurred was thought to be related to the chelation of free iron,

which was released from hemoproteins during heat processing. These results

were further confirmed by Li et al. (1996) who found that addition of 3000 ppm

N-carboxmethylchitosan to cooked pork was sufficient for inhibiting the

development of oxidative rancidity in the product.

16.3.3 Agricultural applications and retention of nutrients

Smither-Copperl (2001) found that chitin exhibits several functions, including

retention of nutrients, in the soil. Chitin contributes to the cycling of nutrients

such as nitrogen. When chitin decomposes, it produces ammonia, which takes

part in the nitrogen cycle. Furthermore, chitin is a main constituent in

geochemical recycling of both carbon and nitrogen. Fungi, arthropods, and

nematodes are the major contributor of chitin in the soil. Among these the fungi

provide the largest amount of chitin in the soil (6±12% of the chitin biomass

which is in the range 500±5000 kg/ha).

In another study, Kokalis-Burelle (2001) reported that chitin contributes

significantly to soil enrichment. It was found that chitin could control plant

pathogens, pathogenic nematodes and provoke the development of host plant

resistance against these pathogens. Chitin led to an increase in microorganism

population; this sharp increase could shift and prompt their action as anti-plant

pathogens in two ways. Firstly, the microorganism may act as parasite for plant

pathogens. Secondly, they can kill or inhibit these pathogens through production

of toxins or metabolites or enzymes. Furthermore, the increase in microorganism

numbers increases the number of non-parasitic nematodes, which results in a

decline in the number of pathogenic nematodes.

16.3.4 Industrial applications and water purification

Treatment of industrial wastewater is necessary before their use or disposal

because of the environmental and health difficulties associated with heavy

metals and pesticides and their deposit through the food chain (Shahidi et al.,

1999). Traditional methods for the elimination of heavy metals from industrial


wastewater may be inefficient or costly, particularly when metals are present at

low concentrations (Deans and Dixon, 1992; Volesky, 1987). Recovering of

metal ions from discards can be achieved using a chelation ion exchange

process. Biopolymers, such as chitin and chitosan, have the ability to lower the

concentration of transition metal ions to parts per billion levels. These

biopolymers should be ecologically safe, commercially available and bear a

number of different functional groups, such as hydroxyl and amino groups in

their backbones (Deans and Dixons, 1992). Chitosan can be used for treatment

of wastewater because it has a good sorption capacity (Jeuniaux, 1986). In

Japan, chitin and chitosan have been used for water purification due to their

ability to complex metal ions via their amino groups (Simpson et al., 1994).

Chitosan powder and dried films of it have free amino groups above the pKa of

their NH2 groups. Therefore, chitosan powder and dried films have potential use

in complexing metal ions (Tirmizi et al., 1996). The United States Environ-

mental Protection Agency (USEPA) has approved the use of commercially

available chitosan for wastewater treatment up to a maximum level of 10mg/L

(Knorr, 1984). Muzzarelli et al. (1989) have demonstrated the effectiveness of

cross-linked N-carboxymethylchitosan in removing lead and cadmium from

drinking water. Micera et al. (1986) have shown that chitosan has a high binding

capacity for metals such as copper and vanadium. Deans and Dixons (1992)

observed that unfunctionalized chitosan was efficient in eliminating Cu2+, but

not Pb2+. Thome and Daele (1986) examined the ability of chitosan to remove

polychlorinated biphenyls (PCB) from polluted stream water. The authors

showed that chitosan was highly effective, compared to activated charcoal, for

purification of (PCB) of polluted water. Use of chitosan for purification of

potable water is also in practice.

16.4 Safety and regulatory status

Chitosan has many industrial, agricultural, food, pharmaceutical, and cosmetic

applications. Consequently, safety and toxicological studies have been per-

formed on chitosan in order to address issues related to its regulatory status. Rao

and Sharma (1997) reported no toxicity for 2% chitosan solution in acetic acid,

when applied on punctured bleeding capillaries in mice, rabbits and guinea pigs.

These researchers further observed that eye irritation tests in rabbits and skin

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Similar results were obtained by Mou et al. (2003) who reported no obvious

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material in tissue engineering. Chitosan received the `Generally Recognized As

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reported by Shepherd et al. (1997). The use of chitosan for purification of

potable water was approved by the US Environmental Protection Agency (EPA),


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food ingredients. Based on their definition of functional foods, chitin and

chitosans possess most of the required attributes related to enhancement of

immunity, prevention of illness, delaying of aging, recovery for illness and

control of biorhythm (Subasinghe, 1999). Thus, the use of chitosan in foods such

as potato chips has been in commercial practice for some time. Therefore,

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17.1 Introduction

World fish and shellfish production has increased from 40 million metric tonnes

(MMT) in 1962 to 133 MMT in 2002, with a concomitant increase in processed

fish and production of fish by-products (Vannuccini, 2004). Frozen and fresh

fish were the main fishery products accounting for over 60% of total production

followed by canning/curing (7% and 4%, respectively) and non-food uses

(26%). While the wild-caught fisheries production appears to have leveled off

by the mid 1990s, aquaculture has shown considerable growth and now repre-

sents about 30% of the total production (Johnson, 2004). Currently, only about

50 to 60% of total catch is used for direct human consumption, and the annual

discards from the world fisheries have been estimated to be approximately 25±

30 MMT of fish and shellfish caught each year (Sovik and Rustad, 2005). While

much of these discards can be used to address increasing demands for fish meal

there is also potential for the use of certain by-products for the extraction of

unique compounds such as marine enzymes.

Enzyme technology has been used extensively in the food processing industry

including fish processing. Enzymes are used as processing aids and can be

produced from by-product material from both new and traditional fish pro-

cessing operations (Vecchi and Coppes, 1996). There are several excellent

reviews on the utilization of enzymes from aquatic organisms (Shahidi and

Kamil, 2001; Gildberg et al., 2000; Haard, 1998). Marine enzymes have drawn

the attention of numerous researchers as they can possess unique specificity and

characteristics. Enzyme recovery from seafood by-products could also help the

17

Marine enzymes from seafoodby-productsM. T. Morrissey and T. Okada, Oregon State University SeafoodLaboratory, USA

environmental and ethical concerns surrounding discards and improve the

bottom line for seafood companies wishing to exploit new technologies and

markets. In this chapter, the current literature on the extraction and purification

of various marine enzymes is reviewed and their potential use and limitations in

their utilization are discussed.

Extractable enzymes from seafood and seafood by-products include digestive

and cellular proteinases, extracellular gastric proteinases, chitinases, lipases,

phospholipases, transglutaminases, polyphenoloxidase and others. An excellent

reference source on this topic is the text Seafood Enzymes by Haard and

Simpson (2000). A partial list of enzymes which have been purified from fish

and processing by-products include lipase from tilapia stomach (Taniguchi et

al., 2001), amino peptidase, carboxypeptidases from tilapia intestine (Taniguchi

and Takano, 2002, 2001), glycogenolytic and alpha glucosidase from Pacific

mackerel, Japanese eel, black sea bream and frog flounder (Nakagawa et al.,

1996), carbohydrases from butterfish, silver drummer, and marble fish, (Skea et

al. 2005), transglutaminase from walleye pollock liver (Kumazawa et al., 1996)

and oyster gill (Kumazawa et al., 1997), trypsin-like enzyme from shrimp

(Honjo et al., 1990), protease from arrowtooth flounder (Wilson and Choudhury,

2004), myosin heavy chain-degrading proteinase from squid muscle (Ehara et

al., 1994), cathepsin S from carp hepatopancreas (Pangkey et al., 2000), �-glucosidase, �-glucosidase and amylase from digestive cecum of scallop (Nakai

et al., 2005), amylases from hard clam viscera (Tsao et al., 2004), acid and

alkaline phosphatase from intestine, liver, kidney of mackerel, sea robin, tongue

sole, and others (Kuda et al., 2004), and cellulase and xylanase from crayfish

and marine prawns (Crawford et al., 2005).

Marine-based enzymes often have unique physical, chemical and catalytic

properties compared to corresponding enzymes from terrestrial animals and

plant sources (Shahidi and Kamil, 2001). Fish muscle, for example, contains

approximately ten times more catheptic enzyme activities than mammalian

muscles (Haard et al., 1994), and marine enzymes are generally more suscept-

ible to hydrostatic pressure inactivation than their mammalian counterparts

(Ashie and Simpson, 1996). Since the habitat of many marine animals tends to

be in cold temperatures, their enzymes often have cold-adapted properties. They

are known to be more catalytically active at low temperatures and possess lower

thermal stability making them suitable for enzymatic applications in food

processing as they can often be inactivated at lower temperatures (Gerday et al.,

2000). Several enzymes have been shown to be salt tolerant as well, which can

be an advantage in certain food applications (Caviccholi et al., 2002; Haard,

1998). Compared to their mesophilic analogues, cold-adapted enzymes tend to

have a lower number of hydrogen bonds, less densely packed structures,

increased surface hydrophilicity, a higher number of methionine residues, a

different fold of the autolysis loop as well as the carboxyterminal region

(Gudmundsdottir and Palsdottir, 2005).

Enzyme extraction and utilization from seafood by-products include enzymes

from both solid and liquid waste streams that are often discarded or traditionally

Marine enzymes from seafood by-products 375

processed into fish feed or fertilizer. Seafood by-products include viscera, skins,

bones, heads, and frames from fish as well as shells and exoskeletons from

shellfish in both traditional and modern processing operations. Fish and shellfish

processing discards can consist of two thirds of incoming raw materials (Lee,

2000) depending on the process and species. Whether it is economically feasible

to commercially extract enzymes from these by-products depends on the volume

and quality of raw material, extraction technology and potential markets. The

following sections will describe several different categories of marine enzymes

and some of their unique properties. Extraction and purification methods will

then be discussed followed by some examples of commercial operations as well

as a discussion of limitations in the use of by-products for marine enzymes.

17.2 Marine enzymes

17.2.1 Proteolytic enzymes

Among the extractable enzymes from seafood and seafood by-products, diges-

tive proteolytic enzymes have received a considerable amount of interest from

researchers over the last two decades due to the availability of raw materials

such as viscera and their high rate of enzymatic activity. Proteases hydrolyze the

peptide bonds in proteins and polypeptides and are characterized as endo-

(proteinases) and exo- (peptidases) proteases (Shahidi and Kamil, 2001). The

increased production of value-added products such as fresh and frozen fillets and

others has created large volumes of discards such as viscera that could be used as

a raw material source. Proteolytic enzymes can also be found in muscle cells, the

extracellular matrix or other organs and the hepatopancreas in shellfish. They

can be extracted from seafood digestive tract and frame muscle as well as waste

water from fish, shellfish and surimi processing. Digestive proteolytic enzymes

include pepsin, trypsin, chymotryosin, elastase, gastricin and others. Pepsin can

be categorized as aspartic proteinase and formed by an autocatalytic reaction

from pepsinogen, which is normally located within the fish stomach and has

peak activity at acidic conditions (pH 2.0 for most of substrates). Trypsin can be

categorized as serine proteinase and it has a dual role in that it cleaves ingested

proteins and activates the precursor forms of several other digestive proteinases

including chymotrypsin. Trypsin is normally located in pyloric cecum and

shows its activity at neutral and alkaline conditions (Naz, 2002). Muscle

proteinases include catepsin A, B, C, D, H, and L, calpains and muscle col-

lagenases, and they are mainly located in lysosomes, the sacroplasm, and

extracellular matrix of the connective tissue (An and Visessanguan, 2000).

Proteolytic enzymes have been purified and characterized from many species

and seafood by-products. These include Atlantic cod (Amiza and Owusu

Apenten, 2002; Jonsdottir et al., 2004), skipjack tuna, yellowfin tuna, tongol

tuna (Suppashith et al., 2004), anchovy (Heu et al., 1995), white croaker skeletal

muscle (Makinodan et al., 1987), salmon (Yamashita and Konagaya, 1995),

decapoda muscle (Ehara et al., 1992), Pacific whiting (An et al., 1994), orange


roughy stomach (Xu et al., 1996), Atlantic menhaden muscle (Choi et al.,

1999a,b), Asian bony tongue (Natalia et al., 2003), mackerel white muscle (Aoki

et al., 1995), gastric fluid of crab (Saborowski et al., 2004), Antarctic krill

(Yoshitomi, 2005; Sjodahl et al., 2002) jumbo squid (Cardenas-Lopez and

Haard, 2005), and surimi waste water (Mireles-DeWitt and Morrissey, 2002a;

Benjakul et al., 1996, 1997).

Proteolytic activity of Atlantic cod by-products from different fishing

locations was studied by Sovik and Rustad (2005). They found that the

proteolytic activity in viscera and frame muscle was the highest at pH 3 and 35ëC,

whereas the proteolytic enzyme from liver showed its maximum activity at pH 3

and 50ëC. The proteolytic activity in viscera was 20 times higher than liver and

250 times higher than that of frame muscle. A significant difference in enzymatic

activity was also reported for cod viscera harvested from different locations.

There were seasonality differences in cod from the Icelandic and Barents Sea in

April±June as well as differences from cod in these locations and cod from the

South coast of Ireland during October±December. These researchers also studied

seasonal changes in trypsin and chymotrypsin activity in viscera from cod, saithe,

haddock, tusk and ling viscera from different locations (Sovik and Rustad, 2004)

and found that enzyme activities differed among species. Krill proteases have

been researched thoroughly and are well defined. Sjodahl et al. (2002) have

identified three trypsin-like proteinases, and four carboxypeptidases. The trypsin-

like proteinases were as much as 60 times as active as bovine trypsin. Yoshitomi

(2005) found that crude krill digestive proteases were much more active in the

Antarctic summer season (December to February) than the winter season and was

correlated with phytoplankton abundance. Benjakul et al. (1996) recovered a

major proteinase from Pacific whiting surimi wash water which was cathepsin L

with molecular weight of 39.5 kDa. These researchers also characterized

biochemical properties and stability of the proteinase recovered from Pacific

whiting surimi wash water by ohmic heating, ultrafiltration, and freeze-drying

(Benjakul et al., 1997) and found that they were stable at pH range of 3.0±9.0,

having the highest activity at pH 4.0.

17.2.2 Collagenolytic enzymes

Collagenolytic enzymes are capable of degrading the polypeptide backbone of

native collagen under conditions that do not denature the protein (Kim et al.,

2002). They can be divided into serine collagenases and metallocollagenases

due to different physiological functions that they possess. Serine collagenases

show their greatest activity in protein digestion, blood-clotting, fibrinolysis,

complement activation and fertilization whereas metallocollagenases are

involved in remodeling the extracellular matrix (Park et al., 2002).

Collagenolytic enzymes have been extracted from shrimp (Brauer et al.,

2003; Van Wormhoudt et al., 1992; Oh et al., 2000), cod (Kristjansson et al.,

1995), filefish (Kim and Kim, 1991), yellow-tail pyloric ceca (Yoshinaka et al.,

1972), crab (Roy et al., 1996; Grant et al., 1981, 1983; Klimova et al., 1990),


winter flounder (Teruel and Simpson, 1995), and mackerel (Park et al., 2002).

Related studies regarding its effect on autolysis of fish muscle tissues during

storage period include cod (Hernandez-Herrero et al., 2003), and salmon

(Hultmann and Rustad, 2004). These enzymes were most active in the pH range

of 6.5±8.0 and are inactivated at pH < 6.0 (Haard and Simpson, 1994). Col-

lagenase from internal organs of mackerel was purified and characterized, and

its molecular weight was estimated to be 14.8 kDa, and the optimum pH and

temperature were approximately pH 7.5 and 55ëC, respectively (Park et al.,

2002).

17.2.3 Lipases

Lipase is an enzyme produced primarily in the pancreas and hydrolyzes fatty

acids from the glycerol backbone at the hydrophobic/hydrophilic interface of the

lipid substrate. It is necessary for the absorption and digestion of lipid including

mono-, di-, and triacylglycerols. Lipase has been purified and characterized

from various aquatic organisms including cod (Gjellesvik et al., 1992), salmon

(Gjellesvik et al., 1994), stomach, pyloric ceca, liver, and intestine of rohu, oil

sardine, mullet, Indian mackerel (Nayak et al., 2003), red sea bream hepato-

pancreas (Iijima et al., 1998) and others. The optimum pH of lipase ranges from

6.0 to 9.0, and lipase is normally stable between 30 and 45ëC. These lipases

could be suitable for various kinds of food applications because of their high

activity at mild pH and temperature conditions.

Phospholipases have also been extracted from pollock muscle (Audley et al.,

1978), Atlantic cod muscle (Chawla and Ablett, 1987), gill membranes of red

sea bream (Uchiyama and Nozaki, 2005), and hepatopancreas of red sea bream

(Iijima et al., 1990, 1997). Recently, phospholipase A2 isozyme from starfish

(Asterina pectinifera) was characterized (Kishimura and Hayashi, 2004)

demonstrating that phospholipase A2 isozyme II had an optimum temperature

of 50ëC and pH of 9.0. It did not show fatty acid specificity for hydrolysis of

phosphatidylcholine; however, it had ten times the activity than that of

commercially available porcine pancreatic phospholipase A2 and has been

suggested as a potential source for phospholipase A2 for industrial production.

Lipase from tilapia stomach and intestine were extracted and characterized by

Taniguchi et al. (2001). The study showed that this lipase had a molecular

weight of 54 kDa with optimum pH of 6.5, and optimum temperature of 40ëC.

The lipases were stable at the pH range of 5.0 to 7.0 at 40ëC for 30 min while

lipase from tilapia intestine showed its molecular weight of 46 kDa, optimum

temperature of 35ëC, and was stable in the pH range of 6.5 to 8.5. The lipases

were identified as non-specific in terms of position on the glycerol backbone but

preferentially hydrolyzed triacylglycerol rather than di- and monoacylglycerol.

The highest lipase activity was found when soybean oil was used for substrate

showing 100% hydrolysis.

A bile salt-activated lipase from hepatopancreas of red sea bream was

extracted and characterized (Iijima et al., 1998). Molecular weight of this lipase


was determined as approximately 64 kDa and had a pH optimum of 7.0 to 9.0.

This lipase was homologous to mammalian bile salt-activated lipase which

preferentially hydrolyzed ethyl esters of polyunsaturated fatty acids.

17.2.4 Chitinolytic enzymes

Chitinolytic enzymes are widely distributed in crustaceans and play an important

role in the degradation of chitin. Two chitinolytic enzymes are involved in

crustaceans, which are endo-type (chitinase) and exo-type (�-N-acetylhexo-saminidase), which are related to molting in several crustaceans (Kono et al.,

1995). These enzymes have been purified mainly from processing by-products

such as squid liver (Matsumiya, 2004), shrimp waste silage (Matsumoto et al.,

2004), and shrimp by-products (Olsen et al., 1990). Chitinase was purified from

the stomach of red sea bream (Pagrus major) and kinetic analysis (Karasuda et

al., 2004) showed high enzyme activity at pH 2.5 and 9.0 toward glycolchitin,

and at pH 2.5 and 5.0 toward N-acethylchitopentasaccharide. These multiple

pH-peak activities are unique for chitinases.

Prawn shell waste was used as a raw material for chitinase production by the

marine fungus Beauveria bassiana BTMF S10 by solid state fermentation

(Suresh and Chandrasekaran, 1998). Likewise, shrimp waste silage was used for

production of �-N-acetylhexosaminidase by Verticillium lecanii in submerged

and solid state fermentations taking advantage of the abundance and com-

position of crustacean wastes (Matsumoto et al., 2004). They concluded that

shrimp silage was an efficient inducer of the extracellular enzyme, compared

with media supplemented with sucrose where enzymic activity was not detected.

17.2.5 Transglutaminases

Transglutaminase is an aminoacyltransferase that catalyzes an acyl transfer

reaction between -carboxyamide groups of glutamine residues in polypeptides

and proteins. When the �-amino group of lysine acts as an acyl acceptor, �-( -glutamyl) lysine crosslinks are formed in proteins which change protein texture

and structure (Kumazawa, 2002). Protein properties, gelation capability, thermal

stability, and water-holding capacity are uniquely affected by this crosslinkage

(Kuraishi et al., 2001). A study showed that seafood products contained the

highest level of �-( -glutamyl) lysine crosslinks (43�moles/100 g protein) fol-

lowed by meat products (38�moles/100 g protein) and soy product (15�moles/

100 g protein) (Kuraishi et al., 2001). Transglutaminases have been purified and

characterized from walleye pollock liver (Kumazawa et al., 1996), Japanese

oyster (Kumazawa, 1997), red sea breammuscle (Yasueda et al., 1994; Noguchi et

al., 2001), botan shrimp squid, carp, rainbow trout, atka mackerel (Nozawa et al.,

1997), squid gill (Nozawa et al., 2001), scallop striated adductor muscle (Nozawa

and Seki, 2001), and tilapia (Worratao and Yongsawatdigul, 2003, 2005).

Two different kinds of transglutaminases were extracted from the Japanese

oyster (Kumazawa, 1997) and had molecular weights of 84 and 90 kDa with


optimum pH of 8.0 for both enzymes. The optimum temperature differed and

showed 40 and 25ëC, respectively. Squid gill was used for transglutaminase

extraction due to its high activity compared with other tissues (Nozawa et al.,

2001). Its molecular weight was estimated to be 94 kDa and its activity showed

an optimum pH and temperature at 8.0 and 20ëC, respectively with 10mM

CaCl2. Tissue transglutaminase from scallop striated adductor muscle was

purified (Nozawa and Seki, 2001), and its molecular weight was estimated to be

95 kDa. Optimum pH and temperature were pH 8.0 and 35ëC, respectively. This

enzyme was Ca2+ dependent and inactivated by �-chloromercuribenzoic acid, N-

ethylmaleimide, Cu2+, and Zn2+ showing that it belongs to the thiol group of

enzymes.

17.2.6 Polyphenoloxidases

Polyphenoloxidase which is also known as tyrosinase, polyphenolase, phenol-

ase, catechol oxidase, cresolase, and catecholase is widely distributed in nature

(Chen et al., 1991). Polyphenoloxidase oxidizes diphenols to quinones, which

undergo autoxidation and polymerization to form dark pigments in fruits, vege-

tables, and crustacean species (Bartolo and Birk, 1998). Of these, crustacean

species can be a source for polyphenoloxidase from seafood by-products of the

shellfish processing industry. Polyphenoloxidase can be found mainly in shell-

fish and by-products including shrimp (Nakagawa et al., 1992), prawns

(Montero et al., 2001), lobster (Opoku-Gyamfua et al., 1992) and cuttlefish

(Zhou et al., 2004).

A polyphenoloxidase fraction was isolated and characterized from lobster

(Opoku-Gyamfua et al., 1992) using the skin layer between the muscle and the

exoskeleton (Homarus americanus) and compared with those of commercial

tyrosinase. The lobster polyphenoloxidase fraction was activated by trypsin, and

it showed a more heat labile nature as compared with commercial tyrosinase.

The enzyme was most stable at pH 7.5, while tyrosinase exhibited a much

broader pH stability ranging between 6.5 and 10.0.

The thermotolerance of polyphenoloxidase activity could depend on its source

and the environmental factors under which the species are grown. One of the

significant environmental factors is water temperature although polyphenol-

oxidase from shrimp are usually stable between 30 and 50ëC. Polyphenoloxidase

activity from Florida spiny lobster (Panulirus argus) and Western Australian

lobster (Panuliruscygnus) was studied (Chen et al., 1991). Both enzymes showed

similar characteristics that catalyzed oxidation of catechol and dl-�-3,4-dihydroxyphenylalanine and showed optimum pH stability at pH 7.0. However,

they differed with respect to activation energy and thermal stability. Western

Australian lobster polyphenoloxidase showed decreased activity when pre-

incubated at temperatures greater than 30ëC whereas that of Florida spiny lobster

showed greater stability at 35ëC. Chen et al. (1991) suggested that this is because

Florida spiny lobsters live in warm water areas while Western Australian lobsters

live in cold water areas. Authors stated that these differences in environmental


conditions of their natural habitats may account for the difference in optimal

thremostability between the enzymes.

17.3 Producing enzymes from seafood processing by-products

Enzyme extraction/solubilization, concentration, fractionation, and purification

are the main steps of enzyme production. To produce enzymes at the industrial

level from seafood by-products, there are several requirements including: 1) a

large quantity of raw material is needed because the target enzymes are usually

in very low concentration; 2) facilities are required to efficiently remove sig-

nificant amounts of water to concentrate/purify enzyme; and 3) the removal of

particulate matter (including cell and/or cell debris) from the final product is

required. The following applications are examples achieving these objectives

using a combination of several techniques.

17.3.1 Enzyme extraction/solubilization

Extraction of enzymes largely depends on the localization of the target enzymes.

For instance, if the starting material contains intracellular enzymes, cell

membrane should be disrupted and homogenized in a buffer. The membrane

disruption can be done by various methods; alkalization, addition of Ethylene-

diaminetetraacetic acid (EDTA), detergents or osmotic shock, or by physical

methods, including sonication, alternating freezing and thawing phases, solid or

liquid shear, or grinding or agitation with abrasives. Then, insoluble impurities

and cellular waste should be removed by filtration or centrifugation (Bartho-

meuf, 1989). On the other hand, if the enzyme is located in the extracellular

fluid, cells should be removed by centrifugation to eliminate contaminants.

17.3.2 Enzyme concentration

As mentioned earlier, the target enzyme is normally of very low concentration in

a large volume of raw material; therefore, the concentration of enzymes after

extraction is necessary. To maintain enzyme activity, harsh conditions such as

high temperature and strong mechanical stress should be avoided. The enzyme

concentration process includes removal of water and other molecules or use of

precipitation methods. The following methods are examples that can be applied

with relatively mild condition throughout the process.

Membrane technology

The food industry started incorporating membrane technology with reverse

osmosis technology for water purification as well as ultrafiltration technology

for concentration of products (Cuperus and Nijhuis, 1993). Since then,

membrane processes have become major tools in food processing and for

extraction of bioactive ingredients.


A membrane is a thin sheet of artificial or natural polymeric material with

pores distributed throughout the material. Under certain pressure, the membrane

rejects large particles, and allows species smaller than the pores to pass through

(Morrissey et al., 2005). Membrane technology includes microfiltration, ultra-

filtration, nanofiltration, reverse osmosis and electrodialysis. Ultrafiltration,

where particles smaller than 0.2�m pass through the membrane, is the most

widely used process for protein and enzyme recovery.

Ultrafiltration has been used extensively in many industries including the

pharmaceutical as well as the food and beverage industry. A good example of

membrane technology and use in the dairy industry is to recover and separate

out different whey proteins (Dsouza and Mawson, 2005). Ultrafiltration is a

pressure-driven process and works by passing low-molecular-weight products

through the membrane while retaining high-molecular-weight compounds such

as protein. Cellulose acetate is one of the traditional materials for ultrafiltration

membrane. Compared to others such as polyacrylnitrile and polyethersulphone,

it is extremely hydrophilic. Having fewer fouling problems, it tends to have

weak resistance against heat and chemicals. With the development of chemical

and heat stable membrane such as polysulphone membrane, the feasibility of

this technology has greatly increased.

Protease recovery from surimi wash water containing various cathepsins was

studied using ultrafiltration technology with various pretreatments (Mireles-

Dewitt and Morrissey, 2002b). Preliminary tests showed that fish proteins will

rapidly clog membrane pores actually changing the dynamics of the filtration

(Huang and Morrissey, 1998). Acidification of wash water (pH 4.0) and mild

heat treatment (60ëC) followed by centrifugation were necessary as pre-

treatments before ultrafiltration was applied. This helped to remove high-

molecular-weight proteins that might interfere with ultrafiltration without

reducing protease activity. The supernatant was then subjected to both high-

molecular-weight ultramicrofiltration and low-molecular-weight ultrafiltration.

The result showed that concentration of protease using 50 kDa ultrafiltration

polysulphone membranes was successful in recovering approximately 80% of

original protease activity (Mireles-Dewitt and Morrissey, 2002b).

Enzyme concentration by precipitation

An alternative and relatively traditional way to concentrate enzymes is to use

salts for enzyme precipitation. This is due to its ability to change electrostatic

forces which affect enzyme solubility. Enzymes are usually soluble in water/

buffer solutions because hydrophobic residues tend to locate the interior of the

globular proteins (enzymes). At low salt concentration (0.5±1M), protein solu-

bility increases (salting in) as ions from salt decrease the electrostatic attraction

between opposite charges of neighboring molecules. At high salt concentration

(>1M), protein solubility decreases resulting in protein precipitation. The

protein can also be precipitated by adjusting pH to pI of proteins which is

usually between 5.2 and 5.5. However, use of salt, especially ammonium

sulphate (NH4)2SO4, is the most common way to precipitate and concentrate


enzymes. Ammonium sulphate possesses a relatively strong `salting out' effect

without causing significant levels of protein denaturation. It has been intensively

used in research areas including seafood and seafood by-products as a first step

to accomplish separation of crude enzymes. The advantages of using these

tecniques include: 1) high solubility, 2) high commercial availability with

inexpensive costs, 3) lack of toxicity, and 4) its stabilizing effect on precipitates.

A disadvantage of using mineral salts is that the method requires another step

such as dialysis or gel filtration (Barthomeuf, 1989). In industries where

enzymes are purified on a large scale, the corrosion of stainless steel by ammo-

nium sulphate is a disadvantage and possibly causes additional environmental

concerns. Sodium sulphate may be more suitable from this point of view, but it

is not as effective (Naz, 2002).

Freeze-drying or lyophilization

Freeze-drying, also known as lyophilization, has been widely used in the food

industry for various protein products including enzymes. Freeze-drying removes

water from a frozen sample by sublimation and desorption in a three-step

process, which includes freezing, primary drying and secondary drying. Freeze-

drying technology is used in combination with other concentration/purification

methods including ultrafiltration in seafood enzyme recovery research. Dry

powder forms of enzymes are more stable than enzymes in aqueous solution,

since water can facilitate enzyme denaturation. However, it is also known that

conformational changes of enzymes by freeze-drying can result in lower enzyme

activity. Alternative forms of enzyme preparations have been developed to

increase enzyme stabilization, including immobilization with sol-gel methods,

cross-linked enzyme crystals (Altus Biologics, Inc., Cambridge, MA, USA),

soluble enzymes with polymers such as ethylene glycol, and surfactant-modified

enzymes such as sorbitan monostearate-modified lipase (Roy and Gupta, 2004).

17.3.3 Fractionation/purification

Concentration techniques will remove considerable extraneous material but only

partially purify the enzyme. The degree of purity requires further downstream

processing that often includes chromatography and electrophoresis. Since

electrophoresis is mainly an analytical procedure, only chromatography will be

discussed here.

Chromatography

Although there are limitations for industrial scale use of chromatography, it is

frequently used in the laboratory to fractionate components including enzymes

with high degrees of purity. Chromatography technology can be categorized into

various methods based on protein size, charge, hydrophobicity and molecular

recognition. These include size exclusion chromatography, ion exchange

chromatography, and affinity chromatography. Enzymes can be fractionated

depending on their hydrodynamic volume by size exclusion chromatography


whereas affinity chromatography is based on specific recognition between two

relevant biomolecules (An and Visessanguan, 2000). Affinity chromatography is

commonly used for the separation of one enzyme from others. Fractionation of

target enzyme by ion exchange chromatography is based on the electrostatistic

interactions between the enzyme and charged groups on the exchangers.

Cellulosic ion exchange chromatography has been one of the most common

methods for protein separation (Naz, 2002). Separation of enzymes can be

controlled by changing pH of the elution buffer.

Gastricsin-like proteinase was purified from Atlantic cod viscera after

lyophilization with a single step purification scheme on ion-exchange of

Amberlite CG-50 with efficient recovery (Amiza and Owusu Apenten, 2002).

Crude cod pepsinogen was dissolved in 10ml of 0.2M sodium citrate buffer (pH

2.1) and introduced to the column. The pH of elution buffer was changed to 3.8,

4.2, and 4.6 to fractionate proteinases followed by pooling the target peaks,

dialyzing, and freeze-drying to produce purified proteinaes. Pepsin A was eluted

at a lower pH (pH 4.0) while gastricsin appeared at higher pH (pH 4.4). The

study demonstrated that final proteinase recovery was high which could be

related to the salt activation by citrate buffer or removal of inhibitors. Chen et al.

(1997) investigated the purification methods for shrimp polyphenoloxidase from

frozen powder of white shrimp (Penaeus setiferus) and pink shrimp (P

duorarum) and found that the use of butanol treatment followed by phenyl

sepharose CL-4B chromatography was better than ammonium sulphate

fractionation and then phenyl sepharose chromatography. They also found that

different species exhibited different activity through the purification process

which demonstrated that activity of white shrimp polyphenoloxidase was more

susceptible than that of pink shrimp during the process.

Taniguchi and coworkers (2001) extracted lipase from the stomach and

intestines of tilapia. They extracted crude lipase by chromatofocusing and

applying the extract on a polyexchanger column previously equilibrated with

25mM imidazole-acetic acid buffer. The lipase was eluted with polybuffer 96-

acetic acid. Iijima et al. (1998) characterized a bile salt-activated lipase from

hepatopancreas of red sea bream. A delipidated powder processed from red sea

bream hepatopancreas was used as a raw material. Lipase was extracted by

fractional precipitation with ammonium sulphate and sequential chromatography.

17.4 Marine by-product enzyme utilization

Despite extensive research in marine enzyme technology, there are only a few

applications in the food processing sector. Limitations of marine by-product

enzyme utilization are often due to the cost of enzyme recovery and competition

with more mainstream enzyme sources. However, there are some commercial

operations that use marine by-product enzymes such as the enzyme recovery

process from cod viscera decribed in Fig. 17.1 (Gildberg, 2004). This figure

shows a Norwegian multi-purpose processing plant manufacturing several


products including a protein hydrolysate and a crude pepsin extract as a fish

processing aid for fish caviar and descaling operations. The crude extract will

vary in pepsin concentration from 2 to 10%, depending on the quality and source

of raw material and operating conditions.

Crude extracts can undergo further seperation by chromatography or other

methods to produce a purified enzyme. However, these are often expensive

procedures and most commercial uses require only the crude extract form except

in the medical/biotechnology field. Several of these uses, from traditional

seafood products to advanced biochemical/medical applications, are described

below.

17.4.1 Fermented fish products

Fish fermentation products, such as fish sauces, are expanding in the market-

place and there have been increased efforts to better define their manufacturing

parameters and control the fermentation process (Lopetcharat et al., 2001). More

success has been found using enzymes to hasten the process and quality of fish

sauce production. Researchers (Tungkawachara et al., 2003; Chaveesuk et al.,

1993) have shown the efficiency of using specific enzymes for the production of

fish sauce and producing an acceptable product. Maatjes is a fermented product

using the natural viscera enzymes in herring and several efforts have been made

to describe the enzymatic reaction and mimic the end result (Olsen and Skara,

1997; Nielsen and Borrensen, 1997; Nunes et al., 1997).

17.4.2 Protein hydrolysates

There is an increasing amount of interest in protein hydrolysate production as

this product has shown unique biological properties such as stimulating the

immune system as well as having peptide fractions that stimulate growth. A

good general review on hydrolysates can be found by Kristinsson and Rasco

Fig. 17.1 Flowchart of by-product processing and crude extract production using codviscera (Gildberg 2004).


(2000). Recent research in the area of production hydrolysates from by-products

include threadfin bream (Normah et al., 2005), cod by-products (Slizyte et al.,

2004, 2005), gold carp (Sumaya-Martinez et al., 2005), salmon heads (Gbogouri

et al., 2004), mackerel (Wu et al., 2003), and herring (Sathivel et al., 2003).

Although enzymes from plant and microbial sources are commonly used for

the production of fish protein hydrolysates, research with marine enzymes has

shown comparable production rates (Benjakul et al., 1997). Shahidi and his

coworkers have produced fish protein hydrolysates using mixtures of extracted

and endogenous enzymes in both capelin and seal meat (Shahidi et al., 1995;

Shahidi and Synowiecki, 1997).

17.4.3 Deskinning

The enzymatic removal of fish skin from certain species has proven advan-

tageous as an alternative method for mechanical deskinning (Tschersich and

Choudhury, 1998; Kim et al., 1993) since mechanical methods tend to be harsh

processes resulting in lower fillet recovery. The use of proteases for removal of

squid skin is a standard process, although non-marine proteases are often used.

Several researchers have shown that marine enzymes can be used and often

leave a product that has several advantages over papain or ficin proteases (Wray,

1988).

17.4.4 Fish roe (caviar) production

Caviar is the salt-cured and preserved eggs of aquatic animals that have been

separated from the supporting connective tissue. The most widely recognized

and valued caviar is made from sturgeon harvested from the Caspian Sea

(Bledsoe et al., 2003). There are enzyme-based processes for removing the

connective tissue that surrounds the eggs, which reduces human handling and

increases caviar recovery. Commercially available fish enzymes such as

RozymTM (Biotec-Mackzymal AS, Tromso, Norway) and DigestaseTM (Alaska

Russia Salmon Caviar Co., Anchorage, AK, USA) have been used for salmon

caviar. Enzymes from fish viscera such as pepsin can be also used for skein

removal. Pepsins split the linkages that adhere the egg cells to the roe sack

without affecting the eggs. Such application with marine derived pepsin has

advantages over the enzymes from mammalian origin including higher optimum

pH and higher activity at lower temperatures (Raa, 1996). Research has shown

the efficacy of proteolytic enzymes for higher yields in fish caviar for salmon

and lumpfish (Raa, 1997).

17.4.5 Gel formation of food

Transglutaminase can be used in certain food products such as surimi, seafood

and meat products that require improved gel formation and gel strength resulting

in better texture. The advantages of transglutaminase addition in the surimi


products include: 1) to increase gel strength of surimi resulting in higher quality,

and 2) to lower production costs by increasing water content in surimi

(Kumazawa, 2002). Currently, transglutaminase has also been used for meat

products, noodles, soy protein products such as tofu, dairy and baked products.

Commercially available transglutaminase, such as Activa TG-K has high

potential for food applications which to date are only from microbial sources

(Kuraishi et al., 2001).

17.4.6 Meat tenderization

Currently, proteases from plant sources, such as papain and bromelain, are the

principle enzymes used as meat tenderizers (Haard et al., 1994). However, these

enzymes attack both connective and myofibrillar proteins which often lead to

over-tenderization whereas marine collagenase could only hydrolyze connective

tissue protein possibly resulting in optimum tenderization of meat products.

Aoki et al. (2004) extracted collagenase from northern shrimp by products and

suggested potential use for the meat industry in reducing toughness of the meat

products cased by connective tissues claiming that it works better than currently

available enzymes from plant sources.

17.4.7 Biotechnology/medical

Although many of the marine enzyme technologies are in the initial phases,

there have been notable successes in biochemistry/biotechnology field. The

isolation and purification of shrimp alkaline phosphatase (SAP) from cold water

shrimp (Pandalus borealis) has led to the use of this compound in gene splicing

with plasmid and bacteriophage vectors in most biotechnology laboratories

(Olsen et al., 1991; Sambrook and Russell, 2001). SAP completely dephos-

phorylates DNA and has the advantage over other alkaline phosphatases as it can

be inactivated at lower temperatures (65ëC for 15 min) and thus not denature

DNA materials. The enzyme is recovered and purified from the shrimp

processing waste water and is a highly sought after chemical. This success has

created other potential opportunities with by-product enzymes and Biotec, ASA

in Norway also lists cod uracil-DNA glycosylase and other enzymes for use in

biotechnology and food processing fields.

Atlantic cod viscera is an abundant fishery by-product in Iceland and the

purification and characterization of trypsin followed by chymotrypsin from the

viscera has led to its utilization (Asgeirsson et al., 1989; Asgeirsson and

Bjarnason, 1991). After ten years of research and collaboration between Uni-

versity of Virginia and University of Iceland, researchers developed a new

product using both trypsin and chymotrypsin from cod viscera. The product,

called Penzim, is used as a gel and lotion in the treatment of skin ailments

including psoriasis and other skin conditions. Recently, researchers have

reported the successful cloning and expression of trypsin I from cod in E. coli

(Jonsdottir et al., 2004).


There has also been active research in investigating krill proteolytic enzymes

as an active ingredient in wound debridement or the removal of necrotic tissue in

wounds (Mekkes et al., 1997, 1998). Research suggests that it is more active

than several plant proteases and can help accelerate wound healing.

17.5 Future trends

Despite tremendous scientific strides in identification and purification of marine

enzymes and technological advances in the recovery of specific compounds from

fish by-products, the question remains concerning the economic feasibility of

enzyme recovery from fish and shellfish by-products. Many of the specific

enzymatic activities, e.g. proteases for fish hydrolysates, have been preempted by

other enzymes from plant and microbial sources that often prove to be less

expensive. In addition to scale-up limitations, there is also strong market

competition for the by-product raw material. Many fish processing operations

already use solid by-products for established-market products such as fish meal,

fertilizers, silage and more recently hydrolysates. As aquaculture expands over the

next decade, demand for the raw material for producing fish meal and other

products for fish feed formulations will also increase (Kilpatrick, 2003). These

products have relatively stable global markets in which there are known risks,

capital investment needs and available technology which facilitates entry into the

marketplace. Other markets for fish by-products have also developed over the last

decade. Viscera by-products, e.g. stomachs from the Alaska pollock industry and

other organs, are now being marketed into Asian niche markets (Morrissey et al.,

2005). Extraction of specific enzymes from fish by-products requires considerable

investment in technologies that often have high costs, varying efficiencies and

require skilled technicians which can be problematic in remote areas.

Some of the best examples of utilization of marine enzymes from seafood by-

products come from the biotechnology/medical field. Companies such as Biotec

Pharmacon ASA in Tromso, Norway, which produces shrimp alkaline phosphatase

for biotechnology laboratories and Zymetec in Iceland currently marketing a

marine trypsin as a skin healing lotion, are examples of companies that have

successfully taken extractive by-product research into the marketplace. Perhaps the

future of marine enzymes utilization rests more in biotechnology/medical uses than

in food processing; however, even this is a two-edged sword. Rapid advances in

biotechnology have also revolutionized the field of enzyme production providing

researchers and companies with the potential to produce specific enzymes more

economically. Haard (1998) addressed the uniqueness of aquatic enzymes in their

diverse environments in his review of `specialty enzymes'. It is unlikely that many

of these marine enzymes could be produced in large enough quantities due to

limitations in obtaining sufficient raw material. However, their unique properties

may have industrial or medical applications that warrant production through

biotechnological techniques. The advent of biotechnology and the production of

specific compounds through gene transfer and use of microbial organisms for


production are making inroads in the commercial production of enzymes. As this

technology continues to develop over the next decade, marine enzymes with unique

characteristics could be cloned and produced more economically through

biotechnology than from by-product recovery operations. Even in the case of

shrimp alkaline phosphatase and Penzim, there is active research to transfer these

genes to bacteria which would be able to produce commercial quantities of this

valuable enzyme. In many cases, the scientific information about biochemical

properties of the marine enzymes themselves, might prove to be the most valuable

component of the by-product itself.

Although the science of seafood enzyme research remains an exciting one

due, in part, to the uniqueness of its resources and the opportunities it may

provide in the biotechnology/medicine fields, the question remains whether it

will reach its true potential beyond the laboratory setting and become a viable

force in the global marketplace.

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18.1 Introduction

Lipid oxidation, which involves the generation of reactive oxygen species (ROS)

such as superoxide anion and hydroxyl radicals, is one of the major reasons for

food quality deterioration during processing and storage with concurrent decrease

in nutritional value, safety and appearance of products. In living organisms,

oxidation is associated with aging, membrane damage, heart disease, stroke,

emphysema and cancer through free radical-mediated modification of DNA,

proteins, lipids and small cellular molecules (Marx, 1987). Antioxidants in foods

can retard lipid oxidation and thus extend the shelf-life of products. Furthermore,

intake of antioxidants can protect the body against oxidative stress and lead to

disease risk reduction. Synthetic antioxidants such as butylated hydroxyanisole

(BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and

propyl gallate (PG) are commonly used in food products. However, these

synthetic antioxidants are suspected to cause potential health hazards, and their

use in foods has been discouraged. Therefore, the search for effective and safe

antioxidants from natural sources is of great interest to researchers, producers and

consumers alike.

Higher plants and their constituents provide for a rich source of natural

antioxidants such as tocopherols and polyphenols. Spices and herbs and hulls

from seeds as well as their extracts are known to exert antioxidant activity, albeit

to different degrees. More recently, marine organisms have attracted special

interest for their potential use in drugs and value-added food production, thus

their antioxidant properties have been investigated. Antioxidants from marine

sources may be used as substitutes for plant antioxidants such as those from

rosemary and sage.

18

Antioxidants from marine by-productsF. Shahidi and Y. Zhong, Memorial University of Newfoundland,Canada

Most of the research on marine antioxidants has focused on potency of crude

extracts with effective antioxidant compounds remaining unisolated and

unidentified. Only few studies have been carried out aiming at purification

and characterization of antioxidant compounds from marine resources. The

previously characterized marine antioxidants are mainly substances that are

structurally related to plant-derived antioxidants (Takamatsu et al., 2003). These

include pigments such as chlorophylls and carotenoids as well as tocopherol

derivatives, related isoprenoids and certain phenolic compounds and UV-

absorbing mycosporine-like amino acids (Takamatsu et al., 2003). Naturally

occurring antioxidants can be found in a variety of marine organisms including

marine algae, invertebrates, fish, shellfish and marine bacteria. For instance,

polyphenols and carotenoids are present in marine algae and micro-algae; some

seafoods such as oyster and eel are well known to contain high levels of

tocopherols; antioxidants produced by marine bacteria isolates were also

reported. In addition, protein hydrolysates from marine animals or their

processing by-products as well as chitinous material exhibit antioxidant activity.

This chapter provides a cursory overview of antioxidants from marine sources

and their by-products.

18.2 Antioxidants from marine algae

Marine algae are well known as a rich source of polyunsaturated fatty acids

(PUFA), especially omega 3 PUFA. However, in spite of their high content of

highly unsaturated fatty acids (HUFA) which are very susceptible to oxidation,

their quality is not changed during storage (Sakata, 1997). It is believed that

marine algae are protected against oxidative deterioration by certain antioxidant

systems. While marine algae are primarily used for production of single-cell oil

rich in docosahexaenoic acid (DHA, 22:6n-3) and other omega 3 PUFA (Kyle,

2001; Zeller et al., 2001), the leftover material after processing may contain a

variety of antioxidative substances, including phenolics, and can potentially be

utilized as a source of natural antioxidants. A number of studies have been

conducted to verify and evaluate the antioxidant activity of marine algae. Mori

et al. (2003) found that methanol extract of a marine brown alga Sargassum

micracanthum inhibited oxidation in rat liver homogenates (Table 18.1). A red

alga Grateloupia filicina was reported to contain compounds with high

antioxidant efficacy equal to or better than that of commercial antioxidants

such as BHA, BHT and �-tocopherol, thus its use as an antioxidant in food

formulations was suggested (Athukorala et al., 2003a,b, 2005). Mediterranean

marine algae of genus Cystoseira were found to possess antioxidant activity

comparable to that of �-tocopherol (Table 18.2) (Foti et al., 1994; Ruberto et al.,2001). Furthermore, water, methanol and ethanol extracts of an edible seaweed

Hizikia fusiformis showed significant ROS scavenging activity, indicating that

this alga might be a valuable source of both water- and fat-soluble antioxidants

(Siriwardhana et al., 2003). In addition, Park et al. (2004b) demonstrated that


enzymatic hydrolysates of an edible seaweed Sargassum horneri exhibited

strong radical scavenging activity on hydroxyl and alkyl radicals. Enzymatic

extracts from various brown algae were reported to exert a positive effect in

reducing oxidative damage to DNA (Heo et al., 2005a,b; Park et al., 2005).

Antioxidant activity of marine algae may arise from pigments such as

chlorophylls and carotenoids, vitamins and vitamin precursors including �-tocopherol, �-carotene, niacin, thiamin and ascorbic acid, phenols such as

polyphenolics and hydroquinones, phospholipids particularly phosphatidyl-

choline, terpenoids, peptides, and other antioxidant substances. These com-

pounds directly or indirectly contribute to inhibition or suppression of free

radical generation.

Although chlorophyll-related compounds are photosensitizers under the light,

they are potent antioxidants in the dark. Chlorophyll a, chlorophyllonolacetone

a, chlorophyllonic acid a methyl ester and pyropheophorbide a produced by

microalgae showed higher antioxidant activity at certain concentrations in

linoleic acid than �-tocopherol and exerted about the same level of potency as

BHT (Sakata, 1997). Carotenoids, another important group of pigments in

nature, can also act as effective antioxidants. While most other well-known

Table 18.1 Effects of methanol extract of Sargassum micracanthum (SM) andtocopherol acetate on CCl4 induced liver injury in rats1

Groups Dose (mg/kg) Inhibition of formation ofmalondialdehyde in rat liver (%)

SM extract 120 4.4SM extract 400 11.1SM extract 1200 14.7

1 Percent inhibition of malondialdehyde formation for tocopheryl acetate at 400mg/kg dose was17.5.Adapted from Mori et al. (2003).

Table 18.2 Effects of Cystoseira extracts on linoleic acid micellar suspension measuredas conjugated dienes

Algae species Relative antioxidant activity

�-tocopherol 100Cystoseira amentacea var. stricta 83Cystoseira amentacea var.amentacea 57Cystoseira algeriensis 54Cystoseira elegans 62Cystoseira elegans x C. algeriensis 31Cystoseira jabukae 37Cystoseira barbata 58Cystoseira crinita 43

Adapted from Ruberto et al. (2001).

Antioxidants from marine by-products 399

carotenoids such as �-carotene, lutein and lycopene are derived from plant

sources, astaxanthin originates from marine sources, primarily from microalgae.

Astaxanthin is both water- and fat-soluble, and is considered 80 times more

potent than vitamin E and 10 times more potent than �-carotene as an anti-

oxidant (Express press release, 2005). It is thought to have great potential in

improving the health of the eyes and the skin (Express press release, 2005).

Astaxanthin from microalgae is commercially available.

Phenolics, in many cases, are claimed to be the major active constituents that

account for the antioxidant activity of marine algae. Duan et al. (2006) have

demonstrated that antioxidant potency of crude extract from a red alga

(Polysiphonia urceolata) correlated well with its total phenolic content. Strong

correlation also existed between the polyphenol content and DPPH radical

scavenging activity of a seaweed (Hizikia fusiformis) extract (Siriwardhana et

al., 2003). Phenolic antioxidants act as free radical scavengers, reducing agents

and metal chelators, and thus effectively inhibit lipid oxidation. Phenolic com-

pounds, especially polyphenols, are widely distributed throughout the plant

kingdom. Some important polyphenols in higher plants, such as catechin,

epicatechin, epigallocatechin, catechin gallate, epicatechin gallate and epigallo-

catechin gallate, are also present in marine algae such as Halimada algae

(Yoshie et al., 2002).

The content and profile of phenolic substances in marine algae varies with the

species (Table 18.3). In marine brown algae, a group of phloroglucine polymers

called phlorotannins comprises the major phenolic compounds (Chkhikvishvili

and Ramazanov, 2000). Brown algae contain various phlorotannins such as

fucolls, phlorethols, fucophlorethols, fuhalols and halogenated and sulphited

phlorotannins (Chkhikvishvili and Ramazanov, 2000); structures of these

phlorotannins are similar to those of condensed tannins (Fig. 18.1). Phloro-

Table 18.3 Content of phenolic compounds in selected brown algae

Alga Phenolic compounds (% of dry weight)

Cystoseira compressa 4.83Cystoseira foeniculaceae 2.16Dictyota sp.1 0.03Dictyota sp.2 0.001Dictyota ciliolate 0.08Dictyopteris membranacea 0.09Focus spiralis 2.17Halopteris scoparia 0.16Lobophora variegate 1.20Padina pavonica 0.69Sargassum desfontrainessi 1.68Sargassum furcatum 2.97Stypopodium zonale 1.22Zonaria tonznefortii 1.06

Adapted from: Chkhikvishvili and Ramazanov (2000).


tannins are known to possess a number of biological activity properties,

including antiplasmin inhibition (Nakayama et al., 1989), detoxification of

heavy metals (Eide et al., 1980), antibacterial effects (Nagayama et al., 2002),

UV protection (Swanson and Druehl, 2002) and chemoprevention against

vascular risk factors (Kang et al., 2003). It has also been reported that

phlorotannins extended the induction period in oxidation of methyl �-linolenate(Nakamura et al., 1996) and ROS generation (Kang et al., 2004). These findings

suggest that phlorotannins, the natural antioxidant compounds found in edible

brown algae, can protect food products against oxidative degradation as well as

preventing and/or treating free radical-related diseases.

In addition to phlorotannins, bromophenols also play an important role as

antioxidants in marine algae, especially in red algae. Fugimoto et al. (1985)

isolated four bromophenols from a red alga Polysiphonia ulceolata. Takamatsu

et al. (2003) showed that bromophenols (Fig. 18.2) isolated from several marine

algae not only exhibited activity in antioxidant assays, but also were taken up by

living cells and maintained their activity.

Tetraprenyltoluquinols, known phenolic compounds formed by coupling of a

hydroquinone ring and a diterpenoidic chain, are characteristically synthesized and

accumulated in algal species Cystoseira (Ruberto et al., 2001). Species Cystoseira

are the most widely-spread marine flora along the Mediterranean coasts. The

extracts of Cystoseira showed significant antioxidant activities comparable to that

of �-tocopherol in a micellar model system (Ruberto et al., 2001). The antioxidant

activities were found to be proportional to the tetraprenyltoluquinol contents in the

extracts, and were ascribed to the presence of these compounds (Ruberto et al.,

2001). Tetraprenyltoluquinols are tocopherol-like compounds with their

diterpenoid chain being very simple, linear and little functionalized in some cases,

and complex, largely cyclized and functionalized in others (Fig. 18.3).

Fig. 18.1 Chemical structures of monomeric units of phlorotannins.


Tocopherols, the most important natural antioxidants, are also tetraprenyltolu-

quinols. Considering the high content of tetraprenyltoluquinols and their potential

antioxidant efficacy, genus Cystoseira can provide for an alternative source of

natural antioxidant for food and cosmetic industries (Ruberto et al., 2001).

Fig. 18.2 Chemical structures of selected bromophenols.

Fig. 18.3 Chemical structures of chemical tetraprenyltoluquinols.


Polysaccharides are another group that account for the antioxidant activity of

marine algae. Polysaccharides are present in the cell walls of marine algae,

generally in the form of alginates, fucans, lamininarans, cellulose and sulphated

galactans such as agar and carrageenans (Ruperez et al., 2002). Cell walls of

marine algae characteristically contain sulphated polysaccharides, which are not

found in land plants and which are believed to possess specific functions

(Ruperez et al., 2002). Zhang et al. (2003) found that water extracted poly-

saccharides from Porphyra haitanesis exhibited antioxidant activity. Anti-

oxidant effects of polysaccharides from Fucus vesiculosus (Ruperez et al., 2002)

and Laminaria japonica (Xue et al., 2001) have also been reported. Sulphated

polysaccharides are by-products in the preparation of alginates from edible

brown seaweeds and could be used as a good source of natural antioxidants with

potential application in the food industry (Ruperez et al., 2002). The hydrolysis

products of polysaccharides, mainly oligosaccharides also showed antioxidant

activity, which is thought to be associated with their radical scavenging and

metal chelation capacity. Agar oligosaccharides produced by marine bacterial

agarase were found to be effective in inhibiting lipid oxidation and scavenging

superoxide anion and hydroxyl radicals (Wang et al., 2004). The antioxidant

activity of oligosaccharides was structure-dependent; the activity increased with

increasing molecular mass and sulphate content (Wang et al., 2004).

Some unsaturated fatty acids have been reported to play an effective role in

antioxidant activity. The lipophilic extracts from 16 species of seaweeds showed

potential antioxidant activities proportional to the content of unsaturated fatty

acids (Huang and Wang, 2004). In addition to the groups of substances

mentioned above, other compounds also make contribution to the antioxidant

efficacy of marine algae. Among them are indoles and dimethylsulphonio-

propionate (DMSP). Indole compounds isolated from marine algae have proven

to exert inhibitory effect on lipid oxidation (Takahashi et al., 1998). Investiga-

tions on DMSP have recently revealed that this compound from marine algal

species could serve as an effective antioxidant (Athukorala et al., 2005). Besides,

some unknown compounds present in marine algae may also act as active

constituents in inhibiting lipid oxidation. Various extraction methods have been

used to release these identified and unidentified antioxidant substances from

marine algae. Solvent extraction methods employ different solvent systems

depending on the solubility of the desired bioactive materials in certain solvents.

More recently, enzyme-assisted extraction has been proposed to prepare potential

natural water-soluble antioxidants from marine algae. Enzymes such as carbo-

hydrases and proteinases are used to macerate the tissues of the algae, break down

the cell walls and complex interior storage materials of algae, such as

laminarians, to release interior compounds (Heo et al., 2005c). In the meantime,

the breakdown/releasing of high-molecular-weight polysaccharides and proteins

themselves may contribute to the antioxidant activity of the extracts (Heo et al.,

2005c). Strong dose-dependent radical scavenging capacities were found in the

proteolytic hydrolysates of a brown marine alga (Ecklonia cava) (Heo et al.,

2005a).


18.3 Antioxidants from marine animals and their by-products

Marine animals including fish, shellfish and mammals have received consider-

able attention for their application in food and pharmaceutical industries. The

processing of marine animals for food production yields a large amount of by-

products, which have been recognized to have special values as natural

materials. Issues have been addressed on the utilization of marine discards and

by-products. Heu et al. (2003) investigated the components and nutritional

quality of shrimp processing by-products. Onodenalore (1998) showed that

enzymatic extract of shrimp heads displayed antioxidant activity in a meat

model system. Crude and purified extracts from shrimp shell waste have been

found to inhibit lipid oxidation and improve colour stability of red rockfish (Li

et al., 1998). A study on hag fish and eel skin extracts revealed that they were

rich with heat-stable antioxidants with strong radical scavenging activities

(Ekanayake et al., 2004, 2005). Compounds responsible for the antioxidant

properties of marine animals and/or their processing by-products have been

isolated and characterized. Protein hydrolysates and chitosan are among those

most frequently studied.

Marine animals and their processing by-products are rich in protein.

Hydrolysis of protein leads to the production of protein hydrolysates, which

have been shown to exert inhibitory effects on lipid oxidation. Protein hydroly-

sates from many animal and plant sources, individual peptides and amino acids

have been tested as antioxidants in a number of studies. Large quantities of yeast

and soybean protein hydrolysates were shown to inhibit the oxidation of

tocopherol-free corn oil (Benshov and Henick, 1972, 1975). Some amino acids

showed strong antioxidant activity in linoleic acid and methyl linoleate model

systems (Marcuse, 1962). A polar fraction from krill extract containing a

mixture of numerous amino acids possessed strong antioxidant activity (Seher

and LoÈschner, 1985). A combination of tryptophan and lysine was effective in

inhibiting the oxidation of butterfat (Merzametov and Gadzhieva, 1976).

Furthermore, antioxidant properties of proline, methionine, histidine and

thronine in fish and vegetable oil or oil emulsion model systems have been

reported (Revankar, 1974; Sims and Fioriti, 1977; Riison et al., 1980). Taurine,

hypotaurine, carnosine and anserine were found to exert antioxidant effects in

vivo (Aruoma et al., 1988). On the other hand, some amino acids such as

cysteine may act as prooxidants (Marcuse, 1962; Kanner, 1979). Amino acids

which show marked antioxidant activity at low concentrations may become

prooxidants at high concentrations (Marcuse, 1962).

Protein hydrolysates from marine animal sources and their antioxidant

activity have been investigated. Shahidi et al. (1995) reported that capelin

protein hydrolysates at a level of 0.5±3.0% inhibited the formation of thio-

barbituric acid reactive substances (TBARS) by 17.7±60.4% (Table 18.4). By

combining membrane filtration, separation and chromatography techniques, the

antioxidant fraction could be partly purified (Guerard et al., 2005). Peptides

fractions from protein hydrolysates showed different antioxidant effectiveness.


According to Amarowicz and Shahidi (1997), among the four peptide fractions

isolated from capelin protein hydrolysates, one fraction possessed a notable

antioxidant activity and another two had a weak efficacy while the fourth

fraction exerted prooxidant effect in a �-carotene-linoleate model system. He et

al. (2006) demonstrated that protein hydrolysates prepared from shrimp Acetes

chinensis by a crude protease inhibited hydroxyl radical generation by about

42%. The inhibition by their ultrafiltrate which contained 41% of oligopeptides

with a molecular mass of lower than 3 kDa, however, was nearly 68% (He et al.,

2006). Kim et al. (2001) isolated two peptides composed of 13 and 16 amino

acid residues, respectively from Alaska Pollack skin, both of which contained a

glycine residue at the C-terminus and the repeating motif Gly-Pro-Hyp (Table

18.5). The peptide with sequence of Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-

Gly-Pro-Hyp-Gly was more effective in inhibiting the formation of TBARS in

linoleic acid compared to another peptide fraction whose sequence was Gly-Glu-

Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly. The difference

in antioxidant activity between the two peptide isolates was thought to be

Table 18.4 Inhibition of TBARS formation by capelin protein hydrolysates (CPH) incooked meats stored at 4ëC

CPH (%) % inhibition (days)

0 1 3 5 Mean

0.5 29.4 15.9 7.3 18.3 17.71.0 36.0 16.5 14.6 19.9 21.82.0 62.3 62.3 34.6 41.2 44.43.0 76.4 76.4 48.2 58.1 60.4

Adapted from Shahidi et al. (1995).

Table 18.5 Antioxidative peptides from gelatin hydrolysate of Alaska pollack skin incomparison with that of soy 75 protein

Peptide Amino acid sequence

Alaska pollack skinP1 Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-GlyP2 Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly

Soy 75 proteinP1 Val-Asn-Pro-His-Asp-His-Glu-AsnP2 Leu-Val-Asn-Pro-His-Asp-His-Glu-AsnP3 Leu-Leu-Pro-His-HisP4 Leu-Leu-Pro-His-His-Ala-Asp-Ala-Asp-TyrP5 Val-Ile-Pro-Ala-Gly-Tyr-ProP6 Leu-Glu-Ser-Gly-Asp-Ala-Leu-Arg-Val-Pro-Ser-Gly-Thr-Tyr-Tyr

Adapted from Shahidi (2003).


attributed to the additional three amino acid residues (Gly-Glu-Hyp) at N-

terminus of the latter peptide (Kim et al., 2001). Antioxidant properties of

peptides isolated from protein hydrolysates of fish by-products such as skin and

frame has also been reported (Jun et al., 2004; Mendis et al., 2005; Je et al.,

2005).

Proteins can be recovered from marine organisms and their processing by-

products by base extraction. An alkali-assisted extraction process was employed

to obtain protein hydrolysates from meat and bone residues of heap seal (Shahidi

and Synowiecki, 1996). Besides, enzyme technology has been applied in con-

verting marine by-products and under-utilized species into protein hydrolysates.

Use of protein hydrolysates obtained from marine animals, especially from their

processing waste, as a source of natural antioxidants has been discussed

(Guerard et al., 2005). In addition to their antioxidant effectiveness, protein

hydrolysates were found to be useful in improving water-binding capacity of

meat products as phosphate alternatives (Shahidi and Synowiecki, 1997).

Shellfish processing by-products are also a rich source of chitin (poly-N-

acetyl-D-glucosamine). Deactylation of chitin affords chitosan. Depending on

the duration of the deacetylation process, the chitosan produced may assume

different viscosities and molecular weights. Chitosan is a linear polysaccharide

composed mainly of �-(1-4)-linked D-glucosamine and is one of the most

common polymers found in nature. The most practical source for chitosan is

processing discards of shellfish such as shrimp, crab, lobster and crayfish.

Chitosan possesses multiple functional properties and has been used in the food,

pharmaceutical, cosmetic, paint and textile industries. These include the use of

chitosan in medical area as a wound-healing agent, a coating agent for prosthetic

articles, a dietary supplement for reducing body weight, an antihypercholesterol-

emic agent, and an antitumor and antiulcer agent (Shahidi and Abuzaytoun,

2005). The food applications of chitosan include its role as an antimicrobial

agent, fruit preservation agent, acidity adjusting and antioxidant agent (Shahidi

and Abuzaytoun, 2005). The potential antioxidant activity of chitosan has been

investigated. Chitosans of different viscosity were found effective in controlling

lipid oxidation in cooked comminuted fish, and the inhibition of oxidation was

concentration- and viscosity-dependent (Shahidi et al., 2002; Kamil et al., 2002;

Jeon et al., 2002). The use of chitosan as an edible invisible film for quality

preservation of fish fillet has been proposed. The content of propanal, an

indicator of oxidation of omega 3 fatty acids, was decreased when chitosan was

used as an edible invisible film in herring (Table 18.6). Deactylated chitosan

showed strong free radical scavenging activity, which positively correlated with

the degree of deactylation (Park et al., 2004a). Kanatt et al. (2004) reported that

antioxidant activity of chitosan was increased six-fold by gamma irradiation at

25 kGy dose. In addition, chitosan act as an elicitor that induces phytochemicals,

mainly phenolic compounds, in plants and hence enhancing the antioxidant

activity of the plant, as observed in sweet basil (Kim et al., 2005). Chitosan

derivatives may also be produced in order to obtain more effective products for

certain applications. For instance, N, O-carboxymethylchitosan (NOCC) and its


lactate, acetate and pyrrolidine carboxylate salts were able to inhibit lipid

oxidation and off-flavor development in cooked meat stored for nine days in a

refrigerator (Shahidi et al., 1999).

Chitosan is water-insoluble and highly viscous in dilute acidic solutions,

which may restrict its use in physiological functional foods. The oligomers of

chitosan, however, are not only water-soluble with low viscosity values, but may

also be absorbed in the human intestine, suggesting that they may have much

physiological functionality in the in vivo systems (Jeon et al., 2000). Chitosan

oligomers with strong physiological activities can be prepared by chemical and

enzymatic hydrolyses (Jeon et al., 2000).

18.4 Antioxidants from other marine sources

Marine invertebrates and bacteria have also been explored for their potential

applications in biomedicine, food processing as well as in cosmetics and related

products. Marine invertebrates, especially tropical marine invertebrates which

are chronically exposed to high levels of solar UV radiation, suffer from

oxidative stress. Furthermore, unicellular algae residing in symbiosis within

their tissues continuously release photosynthetic oxygen that far exceeds the

respiratory demand of the invertebrate (Dunlap et al., 2003). For example, coral

tissues are hyperoxic (>250% air saturation) during daylight exposure (Dunlap

et al., 2003). This, in combination with the high levels of light intensity, can

cause photooxidative toxicity to the invertebrate via photodynamic production

of cytotoxic ROS (Dunlap et al., 2003). However, marine invertebrates are

protected against deleterious ROS, possibly by endogenous antioxidants in their

tissues or metabolites and/or the ÙV-extremophilic' bacteria inhabiting their

tissues. This sheds light on discovery of structurally novel and biologically

active antioxidants rich in biodiversity. Novel sunscreening agents derived from

tropical marine organisms continue to be developed (Dunlap et al., 1999).

Investigations on marine invertebrate metabolites have revealed that

metabolites consisting of benzenoid (phenol or quinoid) and terpenoid parts

are among the most active antioxidant substances in marine invertebrates

Table 18.6 Content of propanal (mg/kg dried fish) in headspace of chitosan-coatedherring samples stored at 4ëC

Chitosan Storage period (days)

0 2 4 6 8 10

Uncoated 12.6 23.7 29.9 34.3 44.1 46.314 cps 13.8 18.3 24.6 30.9 33.0 39.757 cps 12.6 15.5 19.7 24.9 22.8 24.2360 cps 14.2 15.7 17.6 20.2 18.3 22.7

Adapted from Shahidi (2003).


(Utkina et al., 2004). A number of terpenoid phenols and sesquiterpenequinones

were isolated from marine sponges. These compounds exhibit various degrees of

activity in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and in

inhibiting Fe2+/ascorbate-induced lipid oxidation in a rat brain homogenate

(Table 18.7) (Utkina et al., 2004). Nevertheless, some of these compounds such

as curcuphenol and curcudiol, although active in chemical assays, had no

significant activity inside living cells, as reported by Takamatsu et al. (2003). It

is suggested that these compounds did not enter the cells due to their poor

cellular uptake or low solubility, or perhaps their antioxidant capacity was

suppressed in the cellular environment.

Marine bacteria have been found to produce compounds with antioxidant

activity, such as the sunscreen pigment scytonemin from Scytonema spp.

(Takamatsu et al., 2003), astaxanthin and 4-ketozeaxanthin from Agrobacterium

auranticam (Yokoyama et al., 1994), as well as 3,4-dimethoxyphenol and indole

from Ruditapes phillipinarum bacterial isolate (Sakata, 1997). Sakata (1997)

obtained 112 bacterial isolates from 16 fish and shellfish species that possessed

antioxidant activity.

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19.1 Introduction

The term `pigments from aquatic species' is used here to encompass all those

compounds that are naturally present in aquatic animals, plants and algae, and

impart a plethora of colors to these species. These colors may be bright yellow,

red or orange as found in the flesh of salmon and trout, or in the exoskeletons of

raw and/or cooked shrimp, crab, krill and lobster; they may be brown as found in

the marine algae collectively known as the bryophytes; or they may range from

brown to black ± as occurs in the eyes, skins and other tissues of some of these

species. There are yet others that are either intensely red in color as found in the

red algae that inhabit greater depths of the ocean where hydrostatic pressure is

high; or they may be intensely green as in the green algae or seaweeds. At first

glance, it may be tempting for one to simply perceive these compounds as

nature's way of adding appeal, variety and delight to the (aquatic) environment,

which undoubtedly would have been monotonous, drab and dreary otherwise.

However, as will be shown in the following pages, these compounds serve

several very fundamental and crucial functions within the species they occur in.

For instance, some of them serve as mating signals in certain species; some

provide camouflage and concealment for prey from their predators; some others

potentiate crucial biological processes like photosynthesis whereby complex

biomolecules (carbohydrates) are fabricated from simpler molecules (CO2 and

H2O); while others function as antioxidants; and yet others participate in vision

or protect skin and membranes against the ravaging effects of sunlight and

radiation.

19

Pigments from by-products of seafoodprocessingB. K. Simpson, Department of Food Science and AgriculturalChemistry, Canada

From time immemorial, humans have derived benefit and delight from these

naturally occurring pigments present in aquatic species, and this tendency does

not show any signs of abating any time soon. One is rather inclined to predict

that our reliance on these compounds for good health and well-being will

continue to increase in light of new discoveries about their health and related

benefits, as well as the general preferences by consumers for all things `natural'

versus their synthetic or artificial counterparts.

It is not possible to fully and extensively cover all the different pigments in a

single chapter of this book. Thus, the focus will be placed on carotenoid pig-

ments from crustacean processing discards, due to current interest in these

biological molecules for commercial use in health, food/feed and related appli-

cations, as well as the relative abundance of the source material that currently

represents an environmental pollution problem ± in great need to be put to more

profitable use.

19.2 Pigment types and sources

There are several classes or types of pigments known at the present time. These

include the carotenoids ± responsible for the bright red, orange and yellow

coloration of the flesh and skins of species like salmonids (e.g., salmon and

trout), as well as the exoskeletons of crustacea (e.g., shrimp, lobster, krill,

crayfish and crab); melanins ± the brown to black pigments formed by enzyme

catalyzed oxidation of phenolic compounds that are found in the skins, eyes and

peritoneal lining of certain species; the green chlorophyll pigments that occur in

all photosynthetic organisms to enable these species to function as primary

producers in the food chain. The chlorophylls are invariably found together with

carotenoid pigments including fucoxanthin (the brown and dominant pigment in

brown seaweeds or brown algae) and accessory pigments like phycocyanin and

phycoerythrin. As indicated earlier, this chapter will cover carotenoid pigments

more extensively, while the other pigments will be briefly mentioned to

highlight their significance or potential.

19.3 Carotenoid pigments

The carotenoids are synthesized in plants, bacteria and microalgae (Rodriquez-

Concepcion and Boronat, 2002) from the simple precursor molecules, pyruvate

and acetyl CoA, via a 5-carbon intermediate compound known as isopentenyl

pyrophosphate (C5H9O7P2) or IPP (Fig. 19.1) for short, via the mevalonic acid

(MVA) and the deoxyxylulose (DOXP) pathways (Kasahara et al., 2002). The

IPP formed from pyruvate and acetyl CoA, is converted by a series of enzyme

mediated steps to a 40-carbon polyunsaturated hydrocarbon compound with nine

double bonds known as phytoene (Fig. 19.2). It (phytoene) has a molecular

formula of C40H64, and after it is formed, it undergoes four desaturation or


dehydrogenation reactions catalyzed by the enzyme phytoene desaturase to form

lycopene, C40H56 (Fig. 19.3), a hydrocarbon carotenoid compound with thirteen

double bonds. Lycopene may then undergo isomerization and cyclization by

carotene isomerase and lycopene cyclase, respectively, to form other hydro-

carbon carotenoids like �-carotene, �-carotene and -carotene (Fig. 19.4). Thesecyclic hydrocarbon carotenoids may then be hydroxylated by hydroxylase

enzymes (carotene hydrolases) to form oxygenated carotenoids like crypto-

Fig. 19.1 Isopentenyl pyrophosphate.

Fig. 19.2 Phytoene.

Fig. 19.3 Lycopene.

Pigments from by-products of seafood processing 415

xanthin, lutein and zeaxanthin (Fig. 19.5). Other oxygenated carotenoids such as

astaxanthin and canthaxanthin (Fig. 19.6) may subsequently be formed from the

carotenes or hydroxylated carotenoids by epoxidation and de-epoxidation

reactions. Unlike plants and microorganisms, animals are incapable of de novo

synthesis of these carotenoid compounds. Rather, animals must obtain these

molecules preformed in their diets; and they have the capacity to modify dietary

carotenoids for storage in their tissues for various physiological functions. This

capacity by animals to modify carotenoids from the diets is exemplified by the

conversion of �-carotene into canthaxanthin in species like the Artemia (Tanaka

et al., 1976).

Fig. 19.4 (a) �-carotene, (b) �-carotene and (c) -carotene.


19.3.1 Properties and functions

Carotenoid pigments serve several functions besides imparting beautiful bright

red, orange and yellow colors to these species. For instance, carotenoids have

antioxidant properties by virtue of their highly unsaturated nature, which enable

them to lend themselves to (sacrificial) oxidation instead of other molecules.

Carotenoids like �-carotene, �-carotene, zeaxanthin and �-cryptoxanthin are

cleaved by dioxygenase in the gastrointestinal tract to release, at least a

molecule of vitamin A ± thus these carotenoids are said to have provitamin A

activity. Carotenoids also participate in energy transfer reactions during

photosynthesis and in singlet oxygen quenching. As a result of these properties,

carotenoids play crucial roles in vision and are effective in: curtailing age-

related eye disorders such as cataracts; preventing the oxidation of low density

Fig. 19.5 (a) Cryptoxanthin, (b) lutein and (c) zeaxanthin.


lipoprotein (LDL) that culminates in platelet formation and aggregation leading

to cardiovascular diseases (Hadley et al., 2003); prevention of various cancers

via free radical scavenging; as well as protection of tissues against damage from

exposure to light. Carotenoids also enhance/regulate the immune system in

several ways, including increasing the activities of lymphocytes, as well as

protecting macrophages and the immune system against oxidative damage from

exposure to ultraviolet light and X-rays.

Fucoxanthin, shown in Fig. 19.7, is an oxygenated carotenoid pigment found

in brown algae. It is yellowish-brown in color and has a molecular formula of

C40H60O6. Fucoxanthin is mentioned here because of its demonstrated health

benefits. Various studies have shown it to induce apoptosis and enhance anti-

proliferative effects on certain cancer cells (Kotake-Nara et al., 2005; Hosokawa

et al., 2004). An example of economically important brown algae with high

fucoxanthin content is the kelp that is consumed as food by humans.

Fig. 19.6 (a) Astaxanthin and (b) canthaxanthin.

Fig. 19.7 Fucoxanthin.


19.3.2 Preparation and stabilization

Crustacean processing waste is a rich source of carotenoid pigments. However,

the use of this abundant resource as source material for carotenoid pigments in

aquaculture feeds and similar products is not widespread and also not very

practical because the material deteriorates rapidly in the raw form. Other

disadvantages with direct incorporation of the offal in feeds include bulkiness,

variable pigment levels and high content of calcium and chitin. Various methods

have been devised to either extract the carotenoid pigments from crustacean

waste, or modify the offal into semi-stable or stable forms. These include the

organic solvent extraction process described by Bligh (1978). The organic

solvent extraction process involves soaking the waste in an acetone-petroleum

ether-water mixture (75:15:10, v/v/v) and then filtering out the de-pigmented

residue. The pigment trapped in the acetone-petroleum ether-water mixture is

then transferred to petroleum ether, and then dried with anhydrous sodium

sulphate. Crustacean offal has also been processed into meals and used in feeds

(Ruthledge, 1971), although this approach invariably requires decalcification of

the meals prior to use as feed ingredient.

The acid ensilage method developed in Norway for producing fish protein

hydrolysates from processing discards and underutilized by-catch (Torrissen et

al., 1981) has also been adapted for use with crustacean waste. The acid ensilage

process involves treatment of comminuted heat processed crustacean waste with

formic acid, and the process successfully recovers a product that is high in

carotenoid pigments, low in calcium and chitin, and is also stable at ambient

temperature. However, the drawback with the acid ensilage method is the high

acidity of the product and the need to adjust the pH prior to use (Meyers and

Chen, 1982a). Vegetable (soy) oil has also been used to strip carotenoid

pigments from crustacean offal into the oil. This involves blending the vegetable

oil with comminuted heat-processed crustacean waste, and heating the con-

coction with continuous stirring at a temperature ranging between 40 and 50ëC

in the dark. The process achieves high yields of the pigment that may be

stabilized by the addition of suitable antioxidants such as ethoxyquin, �-toco-pherol and butylated hydroxytoluene (BHT) (Shahidi and Synowiecki, 1991;

Meyers and Chen, 1982b); however, this approach recovers only the free

pigment, and excludes valuable protein nutrients from the offal. Another process

is based on the treatment of the raw offal with proteolytic enzymes and chelating

agents to co-extract the carotenoid pigments with proteins (Cano-Lopez et al.,

1987; Simpson and Haard, 1985); this last approach recovers valuable protein

nutrient with the pigment. The advantages with the latter approach include the

fact that chelating agents used in the process together with the proteins co-

extracted with the pigments help to stabilize the pigments. As well, the

carotenoprotein product is depleted in ash and chitin, and also achieves

substantial reduction in the bulk of the offal to ease storage, transportation and

distribution.


19.3.3 Uses in food and feed products

The global market for carotenoid pigments is estimated at about US$935 million

(Fraser and Bramley, 2004). Carotenoid pigments are used as colorants for food,

drugs and cosmetics, and as animal feed and nutritional supplements. In the food

industry, carotenoid pigments are used to impart color to confectionery, as well

as in bakery and dairy products (Britton, 1996). They are also used as colorant

for butter, egg yolk, salmon and lobster. There is no gainsaying the fact that

aquaculture feeds represent the leading user sector of natural carotenoid

pigments from crustacean waste. This fact may hold true for the foreseeable

future, given the steady increase in salmon and trout farming in the leading

producer countries like Norway and Chile and elsewhere in the world.

Salmonids, crustaceans and other animals all have a demanding requirement

for carotenoid pigments for various functions including provitamin A activity,

antioxidant effects, as hormone precursors, in immune response, and in growth,

maturation and reproduction (Lorenz and Cysewski, 2000). However, because

animals are incapable of de novo synthesis of carotenoid pigments (Fraser and

Bramley, 2004), they must have these compounds in their diets for the various

functions listed above. As mentioned above, microorganisms and plants first

synthesize carotenoid compounds such as lycopene, �-carotene and cantha-

xanthin from pyruvate and acetyl Co A via the isoprenoid pathway. The

carotenoids from these primary sources are subsequently ingested by animals

with their diet and converted enzymatically to other carotenoids like astaxanthin.

In some of these animals, deficiencies in carotenoids lead to undesirable defects

like the blue color syndrome in shrimp. The strategies that have been used to

obviate such defects and/or impart the desirable bright orange or red pig-

mentation in these animals reared in captivity include supplementation of the

diets with synthetic colorants like NatuRoseTM astaxanthin (Lorenz, 1998) and

CarophyllÕ pink, or carotenoid pigments derived from crustacean meals. These

materials have also been incorporated in poultry feed to impart desirable

coloration to egg yolks of various poultry animals.

19.3.4 Comparison with synthetic colorants

Carotenoid pigments are available for commercial use in both `natural' and

synthetic forms. The natural source materials for carotenoid pigments include

krill oil (White et al., 2003), shrimp waste (Saito and Regier, 1971), crayfish oil

extract (Peterson et al., 1966), crab meals and oil extracts (Spinelli et al., 1974),

Spirulina and paprika (Meyers, 1994), the microalga Haematococcus sp.

(Johnson and An, 1991), and the yeast Phaffia rhodozyma (Parajo et al., 1998).

The commercially available synthetic forms include canthaxanthin (�,�-carotene-4,40-dione), astaxanthin (3,30-dihydroxy-�-carotene-4-40-dione) and

astaxanthin dipalmitate (Storebakken et al., 1987). Synthetic astaxanthin and

synthetic canthaxanthin are also known as CarophyllÕ pink and CarophyllÕ red,

respectively. Both have been approved for food use by the US Food and Drug

Administration.


Carotenoids from different sources may occur either in the free or the

esterified form. For example krill oil astaxanthin is found predominantly in the

diester form (Yamaguchi et al., 1983); astaxanthin from Haematococcus sp.

occurs predominantly as monoesters (Harker et al., 1996; Breithaupt, 2004);

while Phaffia rhodozyma astaxanthin occurs in the free non-esterified form

(Johnson and An, 1991; Breithaupt, 2004). The form in which astaxanthin occurs

is believed to influence the uptake of the pigment from the diet and its subsequent

incorporation into the flesh of the animal (White et al., 2003). The view is that the

free and the monoester forms of the pigment are taken up more rapidly and more

extensively than the diester forms, thus lending credence to the notion that

astaxanthin is deposited in the flesh of the animal in the free unesterified form, so

that the esterified forms need first to be hydrolyzed in the guts before they can be

absorbed and assimilated (Choubert and Heinrich, 1993; White et al., 2003). The

differences in the degree of esterification of the carotenoids in feed supplements

from various sources is believed to be one of the main reasons for the observed

differences in the extent of deposition and coloration of the flesh of cultured

salmonids and crustaceans. Other studies have shown that astaxanthin in the free

form is also found to color the flesh of salmonids more extensively than

canthaxanthin (Negre-Sadargues et al., 1993). Furthermore, fish flesh color

achieved with canthaxanthin tends to be more yellowish, while astaxanthin

imparts a more pinkish/orange color (Bjerkeng, 2000).

There have been a number of studies that have compared coloration achieved

by feeding astaxanthin from `natural sources' with their synthetic counterparts.

Examples of those studies include the feeding of rainbow trout with diets

supplemented with CarophyllÕ Pink (unesterified synthetic astaxanthin),

astaxanthin monoesters and astaxanthin diesters extracted from the microalga

H. pluvialis. This study suggested an influence of the degree of astaxanthin

esterification on pigment uptake and deposition in the fish flesh (White et al.,

2003). In another study, rainbow trout (Oncorhynchus mykiss) were fed diets

supplemented with shrimp carotenoproteins or CarophyllÕ Pink and compared

with control fish samples that were maintained on the non-pigmented ration

(Nguyen et al., 2003). The overall mean fish size at the start of the feeding trials

was 166.6 � 31.2 g. The final average mass values for each treatment were: fish

fed carotenoprotein supplemented ration, 375.1 g; fish fed CarophyllÕ Pink

supplemented ration, 390.4 g; and the control fish fed the non-pigmented ration,

391.7 g. The fish fed CarophyllÕ Pink and control diets showed slightly higher

specific growth rate (SGR) than those fed the carotenoprotein diet. The

carotenoprotein fed fish showed a slight aversion to the carotenoprotein diet at

the beginning of the trial, but rapidly overcame this aversion, as evidenced by

the very minor differences in fish size at the end of the trial. The flesh from the

fish samples that were fed carotenoprotein or CarophyllÕ Pink diets had similar

intense orange color compared with the control fish samples. Thus, shrimp

carotenoprotein was as effective in coloring the rainbow trout flesh as the

synthetic product (CarophyllÕ Pink) and did not appear to adversely affect the

overall growth of the animal.


In general, these studies have shown that carotenoid pigments from `natural'

or synthetic origins were both effective in achieving flesh coloration, and there

were no significant differences in the absorption and deposition of the pigments

from these two sources. However, considerably larger quantities of the `natural'

source material were required to achieve the same effect that is achievable with

much smaller amounts of the synthetic pigment, because the pigment levels in

the natural source materials tend to be quite low. Similar observations were

made when the diets used in the studies by Nguyen et al. (2003) were used to

feed brook trout, Salvelinus fontinalis (Finn, 2004). Other limitations with some

of the `natural' source materials (such as crustacean offal/meals, and Phaffia)

include high contents of moisture, ash and/or chitin.

19.3.5 Methods for measuring carotenoid pigments

Carotenoid pigments (as astaxanthin) may be quantitated spectrophotometric-

ally, by measuring the absorbance at 485 nm (Saito and Regier, 1971). Other

methods that have been used to study the molecular properties of carotenoids

include field desorption mass spectrometry (Takaichi et al., 2003), thin layer

chromatography (TLC) separation followed by transmethylation and analysis by

gas chromatography (GC) (Renstrom and Liaaen-Jensen, 1981). High pressure

liquid chromatography (HPLC) has also been used for the separation and

identification of astaxanthin esters and chlorophylls in the Japanese fresh water

algae, Haematococcus lacustris (Yuan et al., 1996). More recently, detection

and measurement of carotenoid pigments was accomplished by the negative ion

liquid chromatography-atmospheric pressure chemical ionization mass

spectrometry (negative ion LC-[APC]-MS) method described by Breithaupt

(2004) for studying astaxanthin esters in shrimp and algae. In practice, the most

commonly used instrumental methods for measuring fish flesh color are the

Hunter L, a, b, and the Commission Internationale de l'Eclairage/International

Commission on Illumination (CIE) L*, a*, b* systems.

19.4 Other pigments

Apart from carotenoids, there are other natural pigments also found in various

species that inhabit the aquatic environment. Examples of these other natural

pigments include the green chlorophyll pigments, the blue and red

chromoproteins (phycocyanin and phycoerythrin) from in the blue-green algae

and cyanobacteria, and melanins. There are also other minor pigments such as

flavins, pterins, quinones and porphyrins.

19.4.1 Chlorophylls

The chlorophylls are the green pigments whose primary function is photo-

synthesis ± whereby they permit the species that harbor these pigments to


synthesize complex biomolecules (carbohydrates) from simpler sources (carbon

dioxide and water). Chlorophylls occur abundantly in green algae, also known as

chlorophytes. They are predominantly freshwater, with only a small percentage

inhabiting the marine environment. Chlorophylls are tetrapyrolle compounds ±

i.e., four pyrolle groups are linked together by a central Mg++ ion to form a

porphyrin ring, which together with phytol (a 20-carbon hydrocarbon chain)

makes the chlorophyll molecule (Fig. 19.8). The chlorophyll pigments are best

known as the basis of all plant life because of its functions as light trapping

pigment and electron donor in photosynthesis. Perhaps, a lesser mentioned

property of chlorophylls is their demonstrated benefits to human health. For

example, as far back as 1936, Patek demonstrated that chlorophyll (as wheat-

grass) rebuilds the bloodstream (Patek, 1936), and studies using various animals

showed that dispensation of chlorophyll restored red blood cell counts to normal

levels within 4 to 5 days, even in extremely anemic animals. Chlorophyll is

nontoxic even in large doses when administered intravenously, intramuscularly,

or orally and as a colon implant to animals and humans without toxic side

effects. It is anti-bacterial and can be used inside and outside the body as a

healer (Bowers, 1947). Furthermore, the high magnesium content in chlorophyll

is thought to enhance fertility by building up the enzymes that modulate the sex

hormones. Chlorophyll is believed to cleanse drug deposits from the body,

neutralize toxins in the body, purify the liver, and alleviate blood sugar problems

(Colio and Babb, 1948). Other uses for chlorophyll include clearing up foul

smelling odors, neutralizing streptococcus infections in the buccal cavity,

promoting wound healing, hastening skin grafting, reducing varicose veins,

healing rectal sores, and reducing typhoid fever (Offenkrantz, 1950).

Excellent sources of chlorophylls (in commercial quantities) include the

green algae chlorella, the blue-green algae spirulina, the string lettuce (Entero-

Fig. 19.8 Chlorophylls.


morpha) and the sea lettuce (Ulva). Spirulina has both chlorophyll (green) and

phycocyanin (blue) pigments in its cellular structure. All four major sources of

chlorophyll listed above are consumed as food by humans for their known health

benefits (Ayehunie et al., 1996). They are used in salads and soups. Also

because of their intense green color, chlorophylls are used in many applications

in coloring soaps, oils, creams and body lotions, oral hygiene products as well as

confectionery.

19.4.2 Phycocyanin and phycoerythrin

The blue pigment, phycocyanin is found in blue-green algae and cyanobacteria.

The red pigment, phycoerythrin is found in the red algae commonly referred to

as the rhodophytes. Phycocyanin and phycoerythrin pigments are conjugated

chromoproteins and are composed of a number of subunits, each having a

protein backbone covalently attached to open chain tetrapyrrole groups. Their

molecular weights range from 44 000 daltons (monomers) to 260 000 daltons

(hexamers) (Boussiba and Richmond, 1979). They function as light-absorbing

substances together with chlorophyll during photosynthesis.

An example of red algae of commercial relevance is the nori or Porphyra. It

is consumed as food and is used as wraps for sushi and in several other Japanese

dishes. The two pigments are produced commercially from the blue-green algae

Spirulina platensis. Spirulina grows well in warmer climates and warm alkaline

waters. There are several Spirulina species, although Spirulina platensis and

Spirulina maxima are the best known. The former is cultivated in California

while S. maxima are cultivated in Mexico. Phycocyanin and related compounds

found in Spirulina are believed to have antiviral and anticancer properties as

well as the ability to stimulate the immune system, and also promote formation

and development of red blood cells, and thereby curtail the incidence of anemia

(Jensen and Ginsberg, 2000; Jensen et al., 2001; Mani et al., 2000; Mathew et

al., 1995; Samuels et al., 2002; Shih et al., 2003). They are also thought to

promote the development of healthy skin. Thus, it is used to treat skin disorders

like eczema and psoriasis. Phycocyanin and phycoerythrin are found together

with carotenoids and chlorophylls in the blue-green and red algae. The red

pigment, phycoerythrin, in particular, is believed to facilitate red seaweed

subsistence at greater depths in the ocean where hydrostatic pressures are high,

unlike the other seaweed species (e.g., brown and green algae) that can thrive

only in shallow waters.

Studies with various experimental animals have demonstrated the potent

antioxidant, free radical scavenging and anti-inflammatory properties of

phycocyanin, as well as antiviral activity of the pigment against herpes simplex

and anti-arthritic effects (Bhat and Madyastha, 2000). Some other studies have

also revealed protective effects by phycocyanin against neuronal damage and

oxidative damage to DNA (Bhat and Madyastha, 2001; PinÄero Estrada et al.,

2001). Phycocyanin is used as a natural coloring agent in several food products

including dairy products (yoghurts, milk shakes and ice creams), alcoholic and


non-alcoholic beverages, desserts, and also in cosmetic products. The

phycoerythrin pigment is one of the brightest dyes and is commonly used as

fluorescent dyes for FACS analysis (Hardy, 1986). Phycocyanin is used in

medicine to facilitate selective destruction of atherosclerotic plaques or cancer

cells by radiation with little or no damage to the surrounding cells or tissue

(Morcos and Henry, 1989).

19.4.3 Melanins

Melanins are partly responsible for the dark brown and black colorations found

in aquatic species. Melanins are formed from the amino acid, tyrosine, and

phenolic compounds by enzymatic oxidation through a series of intermediates

like dihydroxyphenolics and quinones, followed by polymerization to form the

large molecular weight melanins. Melanins occur in the eyes, skins and

peritoneal lining of these species, and they play the role of protecting tissues

against light/UV radiation, among others. In particular, species living in clear,

shallow waters are exposed to the damaging effects of ultraviolet radiation from

the Sun, and the melanins these species are endowed with help to curtail such

damaging UV effects. Damage from UV radiation may come about in various

ways, such as thymine dimerization in DNA, or via the effects of reactive

oxygen species such as singlet oxygen and superoxide radical (Jagger, 1985;

Tyrrell, 1991). The capacity of aquatic species to subsist in these habitats

suggests that they have the mechanism(s) to protect themselves against the

adverse effects of the radiation from the Sun. This protective ability derives

from naturally present melanins or melanin-like substances present in these

species.

Simple life forms such as algae and cyanobacteria make these melanin-type

compounds as part of their normal metabolism, and these compounds sub-

sequently pass along the food chain to provide similar protective effects in

higher forms of aquatic life. The enzymes responsible for the oxidation steps are

the phenolases or phenoloxidases. In crustacean species such as shrimp, prawn,

crab and lobster, the formation of melanins is referred to as melanosis or

`blackspot' formation. Although these `blackspots' formed on the animals are

not toxic, consumers nevertheless find them unappealing. The `blackspot'

phenomenon in crustaceans has been extensively studied by various researchers

with the goal to curtail this undesirable effect of polyphenoloxidases in these

animals (Benjakul et al., 2005; Chen et al., 1993; Ferrer et al., 1989; Ogawa et

al., 1984; Yan et al., 1989). Nevertheless, these same melanins may be

recovered and put to very good use. For instance, in humans melanins protect

against skin damage from UV radiation from sunlight and also minimize glare

within the eyes. This property of melanin has been exploited in making superior

quality sun lenses that better filter colors to reduce their damaging effects, and

thus mitigate the risks of macular degeneration and cataracts. A company in the

US, PhotoProtective Technologies, produces melanins for incorporation in a

myriad of lenses including sunglasses, reading glasses, computer glasses, pilot


glasses and other special purpose glasses (Anon, 2005). Melanin has also been

incorporated in a formula known as Melancor-NH for reducing gray hair in men

and women (www.911healthshop.com/melancornh.html).

19.5 Economic, environmental, and safety considerations

Agricultural harvesting and processing lead to the production of large quantities

of residual materials that are either underutilized or not utilized at all. Invariably

such unutilized material ends up as waste, polluting the environment. For

instance, most commercial shrimp processing operations entail semi-

mechanized peeling operations to remove the heads, viscera, carapace and

legs. These parts account for between 70 and 80% of the whole animal and are

commonly not consumed in certain cultures or communities, and end up as a

major waste disposal problem. Although there are efforts by several companies

to make products like chitin, chitosan, glucosamine, etc., from crustacean waste,

there is still a large bulk of it that is dumped as waste. Because of growing

concerns for environmental health and safety, stringent environment regulations

have been promulgated aimed at curtailing the dumping of such processing

discards back into the ocean or in landfills. So in some countries like Canada,

disposal of agricultural harvesting and processing waste is becoming quite

costly. This high waste disposal cost is making processors more amenable to the

idea of converting processing discards into profitable by-products. Fortunately,

these processing discards have high levels of useful nutrients and other bio-

ingredients that may be recovered by relatively simple procedures to increase

profits.

As alluded to elsewhere in this chapter, animals such as salmonids and

crustaceans have a demanding requirement for carotenoids for several functions,

yet are incapable of making these compounds on their own from scratch. Apart

from the normal metabolic functions of these pigments in the live animal, these

compounds also impart colors to foodstuffs ± for example, the reddish orange

colors associated with salmonids are due to the carotenoid pigments derived

from the diet (Fox, 1957). Consumers have come to associate these animals with

these colors, and in some products (like salmon, trout, crab, lobster and shrimp),

these colors are used (rightly or wrongly) as one of the measures of quality and

acceptability. For instance, the market value of prawn is determined largely on

the visual appeal of its body color (Lorenz, 1998).

For some time now, the wild salmon harvest has been in steady decline. The

annual wild salmon harvest worldwide is estimated at less than a million tonnes,

while the world production of farmed salmon has been increasing for the same

period. The 1999 harvest of farmed salmon was greater than 750 000 metric

tonnes, and this figure is projected to reach about 1.3 million tonnes for 2005

(Lorenz and Cysewski, 2000). For cultured salmonids, carotenoid pigments in

either the `natural' or synthetic forms, are incorporated in the diet to impart the

desired flesh color. According to Lorenz and Cysewski (2000), more than 95%


of the carotenoid pigments used to supplement aquaculture diets is synthetic.

Because of consumer aversion to the use of synthetic products in foodstuffs, fish

farmers desire to use carotenoid pigments from `natural' sources. Furthermore,

the synthetic colorants currently available for use as feed supplements are quite

expensive and constitute from 40 to 60% of the total operating costs in intensive

aquaculture operations (Meyers, 1994). Synthetic astaxanthin, for example,

retails for about US$2,500 per kilogram, and the global market for astaxanthin

was estimated in the year 2000 at US$200 million (Lorenz and Cysewski, 2000).

Thus, there is great interest in crustacean processing discards and

microorganisms (e.g. Phaffia yeast and the Haematococcus microalgae) as

cheaper sources of carotenoid pigments for food, feed and health use; and for

various reasons such as their being perceived as more `natural', putting discards

from the crustacean harvest to profitable use, as well as reducing environmental

pollution.

Some of the factors that augur well for crustacean waste versus micro-

organisms as `natural' source of carotenoid pigments include:

· the pressure on the fishing industry by environmentalists and regulatory

agencies to convert the discards into high value products instead of simply

dumping them into the environment to aggravate the pollution problem

· the added profits that could accrue to the fishing industry from recovering

useful bioingredients from these sources for use in human food,

nutraceuticals and animal feed

· the relatively high production cost in producing the pigments from

microalgae (e.g., Haematococcus pluvialis) and yeast (Phaffia rhodozyma)

that is estimated at about US$5 to US$20 per kg dry weight (Lorenz and

Cysewski, 2000)

· the presence of tough cell walls in microalgae and yeast that could limit the

bioavailability of carotenoid pigments from these sources (Burcyk, 1987).

Crustacean offal has been used in both the fresh and frozen forms in

aquaculture diets (Saito and Regier, 1971). Other forms in which crustacean

offal have been used include its processing into meals to reduce bulkiness prior

to incorporation in aquaculture feeds (Spinelli et al., 1974); differential screen-

ing of the crustacean meal to exclude chitin and calcium before feeding it to

farmed animals (Rutledge, 1971); acid ensilage of the offal (Torrissen et al.,

1981); stripping the carotenoid pigments from the crustacean offal with soy oil

for incorporation in aquaculture rations (Meyers and Chen, 1982a); or by co-

extraction of the carotenoids as pigment-protein (carotenoprotein) complexes

(Cano-Lopez et al., 1987; Simpson and Haard, 1985).

19.6 Future trends

This discussion has mentioned various sources of natural pigments. Carotenoid

pigments from crustacean offal were treated at some length compared with the


other natural pigment sources because of their importance as antioxidants, and

as colorants for food and farmed fish. Various algae such as chlorophyll,

fucoxanthin, phycocyanin and phycoerythrin were mentioned as sources of

pigments. So far, the foremost use of algae or seaweeds is as source material for

(food) hydrocolloids such as agar, alginates and carrageenan for food and

cosmetic uses. The recovery of these ingredients is carried out without much

regard for the pigments present with the result that the bulk of these pigments

are wasted. This neglect is bound to change with the growing awareness of the

health benefits that could be derived from these pigments, as well as their uses to

color food, cosmetic and related products. This is especially so given that

pigments from `natural' sources are gaining in importance over their synthetic

counterparts, as the former are perceived to be relatively non-toxic and non-

carcinogenic.

The literature is also replete with conflicting reports on the effects of degree

of esterification of carotenoid pigments and their uptake by fish flesh. More

studies aimed at clarifying this situation would help to improve feed

supplementation/formulation to assure better bioavailability of the pigments.

Several companies based on bioingredients or by-products from marine algae

are sprouting all over the world and are all touting the spectacular health benefits

of these products. When it comes to foods or ingredients that we ingest through

our mouths into our bodies, most consumers see `natural' products as better and/

or safer. Nevertheless, it is known that excessive intake of iodine from high-

iodine-content macro algae can upset thyroid function (Teas et al., 2004). Some

consumers are wary about heavy metal levels in their foods, and these toxicants

may either be absorbed by (or accumulated in) some of these seaweeds, or co-

extracted with useful bioingredients from the seaweeds (Ethus, 2003). Further-

more, with the continued disposal of waste (including sewage) into oceans and

landfills (that may seep into rivers, streams and oceans), and the incidence of

nuclear accidents, there are justifiable concerns about the possible contami-

nation of these `natural' bioingredients or by-products with environmental

toxicants and hazardous radioactive fallout materials (Barnaby and Boeker,

1999). Thus far, food and health regulatory agencies such as the US Food and

Drug Administration and Health and Welfare Canada do not stringently regulate

seaweeds and similar herbaceous products used as dietary supplements. Thus,

aspects such as levels of incorporation, purity and safety, as well as the

interactive effects of these products with various prescription drugs need to be

verified by more research.


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Part III

Non-food uses of marine by-products

20.1 Introduction

There is a rich history associated with fish by-product utilization both abroad

and in the United States of America. The classic reference in this area is the

1981 book Introduction to Fishery By-products by Windsor and Barlow (1981).

In the USA, research in this area was conducted by the National Marine

Fisheries Service at their Charleston, South Carolina Laboratory and focused on

products from the menhaden fishery until the program area was phased out. The

interesting history of Alaska marine by-products production and utilization from

1882 through 1989 was compiled by Meehan et al. (1990). Currently in Alaska

there are a number of on-shore fish meal plants located close to large fish

processing operations. These plants produce fish meals and oils from by-

products of fish processed for human consumption.

Fish processing by-products refer to tissues that remain after much of the fish

muscle has been removed and include heads, frames, viscera, and skin, among

others. Edible parts such as heads, milt, and stomachs are on occasion collected

and sold and some fish skin is made into gelatin or fish leather. By-products can

be used to make fertilizer and other products; however, most of the fish by-

products produced in large shore-side fish processing operations are used to

make fish meal and fish oil. Primary uses of fish meals and oils are as

aquaculture feed ingredients for fish and shrimp, and as livestock and poultry

feed ingredients.

This chapter deals with making aquaculture and animal feed ingredients

from fish processing by-products and focuses on by-products currently

produced from marine finfish. The first section deals with the rationale for

20

By-products from seafood processing foraquaculture and animal feedsP. J. Bechtel, University of Alaska Fairbanks, USA

making feed ingredients from processing by-products. The second section deals

with different by-product components obtained from the processing of finfish.

The third and fourth sections briefly describe the process used to produce

protein meals, solubles and oils from fish processing by-products and the

production of hydrolysates and silages. This is followed by a discussion of the

nutritional value and issues associated with feed ingredients made from fish

processing by-products.

20.2 Driving forces for utilization of by-products

Ocean fish are used for human consumption as well as the production of feed

ingredients for aquaculture and livestock. The world fish harvest from the

oceans has been stagnant or in decline for a number of years. There is concern

over the availability of high quality fish meal and oil for the world aquaculture

industry. Hardy and Tacon (2002) have stated that the annual fish meal

production has been more or less constant over the past 15 years at approxi-

mately 6.2 million metric tons (MMT). However, the proportion of global fish

meal production utilized in feeds for farmed aquatic species has increased

dramatically, because of the worldwide growth of aquaculture. Aquaculture is

predicted to expand two to three fold over the next decades and create a large

need for aquaculture feed ingredients (Hardy and Tacon, 2002). Most fish meal

and fish oil are produced from the harvest of whole fish with high oil contents

such as anchovy, capelin, and menhaden and less than 10% comes from white

fish offal (Pike and Barlow, 1999). An additional supply of fish meal and oil can

be recovered from seafood processing by-products. New (1996) has estimated

that if seafood processing by-products and by-catch were converted into fish

meal, the supply would be equivalent to a significant portion of the current

global fish meal production.

There can be practical problems in utilizing seafood processing by-products

to make meals and oils and these can include seasonal availability, large

volumes of by-products during a limited number of processing days, and remote

fish processing locations. Also, because seafood by-products differ from whole

fish in composition, due to the removal of much of the muscle flesh, modified

production methods are required to make products that can compete with

traditional fish meal made from whole fish. If aquaculture growth continues and

supplies of fish meal and oil remain constant, fish meal and fish oil in

aquaculture feeds will shift from being primary sources of protein and energy to

being specialty products (Hardy, 2003). This trend is already underway for many

aquaculture species.

Federal and state regulations such as the Magnuson-Stevens Fishery and

Conservation Act of 1996, The American Fisheries Act of 1998, and the permits

required for discharging waste of on-shore processing plants, are encouraging

the utilization of fish processing by-products. Producing fish meal is often the

most economical solution for handling fish wastes produced from on-shore


processors. Without the development of effective means in dealing with fish by-

products, the fish processing industry will face increased costs associated with

running their businesses.

As an example of the potential to utilize fish processing by-products to make

feed ingredients, Crapo and Bechtel (2003) estimated the total amount of Alaska

by-products produced on a dry matter basis in 2000 was 208 599 MMT and that

about 40% of the solids were reported to be recovered as fish meal and fish oil.

On-shore processors recovered over 60% of the solids from their by-products,

while catcher-processors recovered less. Increased utilization of fish wastes

would allow for the production of more aquaculture fish feeds without increas-

ing the harvest from the oceans. Other benefits include a cleaner environment

and greater resource utilization.

20.3 By-product components

Substantial amounts of seafood by-products are available for processing into

feed ingredients and for other products. The largest amount of these by-

products are from marine finfish and include viscera, frames, heads, skin and

fins, among others. In addition, there are by-products available from squid,

shrimp, crab, and other seafood industries that are used to make feed

ingredients that have unique properties. Given the proper economic incentive it

could be desirable to collect selected fish processing by-product components

(e.g., viscera, heads, milt, liver) to make high quality feed ingredients that

would command a premium price.

Obtaining precise numbers for the amount of waste and by-products produced

is difficult due to the proprietary nature and competitive aspects of the industry.

The volume of processing by-products varies significantly by species, type of

processing, and many other variables such as the time of year. Examples of

variation in the percentage of by-product produced are 90% from fish harvested

only for their roe to 30% for fish that are simply eviscerated-headed and frozen

for the markets. As a broad generality the overall amount and quality of

available fish processing by-products is being reduced as improved fish

processing technology is developed, which removes more of the muscle tissue

and leaves an increasing percentage of bone behind (Babbit and Stevens, 1996;

Kelleher and Hultin, 2000, Gildberg et al., 2002). In addition, new technologies

are being developed that increase the utilization of fish such as the Arrowtooth

flounder and other species (Crapo et al., 1999).

A broad guideline for recoveries and yields of common pacific fish and

shellfish has been compiled (Crapo et al., 1993). There are good estimates of the

harvest from different fisheries; however, there is less data on what happens to

the fish wastes after processing. When fish by-products from food processing

lines are made into fish meals and oils, much of the raw material is initially of

the higher quality, e.g. human food grade. Advantages of seafood by-product

derived from processing fish for human consumption include:

By-products from seafood processing for aquaculture and animal feeds 437

1. fresh food grade seafood by-products are available at the time of processing,

because seafood quality must be maintained from the time of harvest

through processing to meet edible food standards

2. separate components can be obtained from the mechanical processing of

these fish (e.g., heads removed first followed by viscera, frames, and skin

during processing of boneless fillets).

Rather than mix by-products together and make a protein meal from all the by-

products, an active research area is the development of specialty feed ingredients

and products from variety parts including skin, trim, frames, heads, viscera,

liver, and milt, among others.

There are chemical and biochemical differences in whole fish composition

between fish species (Stansby, 1976; Krzynowek et al., 1989). This translates

into species differences in fish waste composition and by-products made from

wastes. There has been little evaluation of fish processing by-products such as

heads, frames, viscera and skins beyond proximate analysis. The chemical

composition of parts such as heads, viscera, frames and skin from a number of

species has been determined (Gunasekera et al., 2002; Dong et al., 1993;

Freeman and Hoogland, 1956; Kizevetter, 1971; Krzynowek et al., 1989;

Montero et al., 1991; Nagai and Suzuki, 2000; Stansby, 1976; Olley et al., 1968;

Bechtel, 2003; Bechtel and Johnson, 2004; Oliveira and Bechtel, 2005).

20.4 Overview of different products produced from fishby-products

20.4.1 Fish meal

In the production of fish meal from the by-products of seafood processing, most

large plants employ a wet reduction process (Hardy, 1992; Babbitt et al., 1994).

This process includes cooking, pressing, and drying operations. The first step in

the process of making fish meal involves heating the fish and/or fish waste to 95±

100ëC, which denatures the protein and facilitates the separation of oil and liquid

from the solids. The cooked material is then passed through a press to produce

both a press cake and a liquid fraction. The press cake is dried to less than 10%

water and the resultant fish meal is milled to the desired particle size and usually

stored and transported in bulk containers. The liquid, which contains the aqueous

phase and associated water soluble proteins plus the fish oil, is usually passed

through a centrifuge to separate the fish oil from the aqueous phase (stick water).

Stick water contains substantial amounts of soluble protein, which is

concentrated and added back to the press cake. Proteolytic enzymes are added

during the concentration step to decrease viscosity and improve evaporator

efficiency. Concentrated stick water can be added back to the press cake before

drying or dried separately and sold as fish solubles. Marine fish lipids have a high

degree of unsaturation and in the presence of oxygen will oxidize and reduce the

quality and palatability of fish meal. An antioxidant such as ethoxyquin is added

to many meals at levels of 130±150 ppm in the finished product (Hardy, 1998).


Most fish meal processing technologies have been developed for use with

high-oil whole fish from the large industrial fisheries. Adaptations that have

been devised for making white fish by-product meals include the potential for

eliminating oil recovery (depending on the percentage lipid of the specific fish

by-product) and the removal of bone fragments to decrease the ash content in the

final product. In order to make fish meals with 65% protein and less than 20%

ash from fish processing wastes, it has often been necessary to remove some of

the bone from the dried meal. This is usually accomplished by using a gyrating

sieve separator that removes bone and other fragments in the meal. This method

has been successfully employed in a number of plants in Alaska. Another

method of reducing ash content in the final product is to remove bone from the

raw fish waste using a mechanical bone remover (Rathbone et al., 2001).

Although the general operations for meal manufacture have changed little

over the past 30 years, there have been improvements in the process conditions

and equipment resulting in increased efficiencies and better quality products (see

review by Tarr, 1982). Improved meal quality resulted from replacing direct

drying of fish meal with indirect steam drying. Additional quality improvements

have been made when low temperature (LT) drying had been employed, which

results in products with higher digestibility than steam dried meals. Other

advancements have been made in the development of more efficient evaporators

for concentration of stick water. An alternative to using the press to separate

solid and liquid fractions after the initial cooking step is to substitutes the use of

a decanter type centrifuge for the separation (Windsor and Barlow, 1981).

Over the years, fish meal quality has continued to improve due to a number of

factors including an emphasis on raw material quality, and low-temperature

drying. Recognized premium quality fish meals with high protein and low ash

are now produced that have both manufacturing and product specifications. One

example is the Norwegian reference meal (Norse-LT 94), which is approxi-

mately 74% protein, 11±13% fat, 10% ash and 7% moisture. Products that

increase feed efficiency and have higher protein content generally have a higher

market value.

The importance of raw material freshness and low temperature processing

have been documented in growth trials with fast-growing salmon, halibut, sea

bream and Penaeus shrimp. Freshness in the raw material can be monitored

using total volatile nitrogen, content of the main biogenic amines (histamine,

cadaverine, putrescine and tyramine) and free fatty acid content. Koning (1999)

and Hardy and Dong (1995) provided insight into the measurement of quality in

fish meals and guidelines for the consistent production of high quality feed

ingredients starting from the quality and composition of the raw materials to the

storage properties of the feed ingredient.

20.4.2 Fish oil and bone

From a historical perspective fish oil has been an important commodity with

food, feed, fuel, and industrial applications. Processing of fish oils has been


described by Bimbo (1990) and a host of others. Global fish oil production in

2000 exceeded 1.3 MMT and around half the production was projected for use

in aquaculture diets. The requirements for the long chain omega-3 fatty acids in

marine and freshwater aquaculture diets have been reviewed by Sargent and

Tacon (1999). In spite of the importance of fish oil in aquaculture diets the price

of fish oil remains relatively low. Refined fish oils from a variety of fish species

including salmon are currently available in world markets.

To make many fish meals produced from by-products competitive in the

market place it has been necessary to remove bone in order to reduce the ash

content below 20%. Removing the bone results in a bone by-product that is useful

as a fertilizer soil amendment, and as a supplement in animal feeds; however, fish

bone meal has a relatively low value. The profitability of a marginally-valued

commodity can be a problem when transportation from remote areas is involved.

A potential solution lies in the development of new, high-value uses for bone-

derived products. If organic certification can be obtained there may be additional

markets for the product. The composition of fish bone meal depends on the species

and the process used to clean the bone. However, the ash content of dried fish bone

is high, often in the range of 40 to 55%.

20.4.3 Solubles

The stick water fraction is usually 50±70% of the initial weight of the raw

material and contains approximately 5±10% solids, most of which is protein. A

limited number of studies have focused on concentration of stick water (Gaude,

1994; Valle, 1990). When stick water is concentrated and added to the fish meal

press cake during drying, it can account for 20% or more of the solids in the meal

(Pedersen LD et al., 2003; Soares et al., 1973; Ammu et al., 1986; Zarkadas et

al., 1986). When stick water is concentrated and sold separately it is referred to as

fish solubles. The price of fish solubles has been significantly below that of fish

meal; therefore, it has been economically advantageous to sell the fish solubles as

fish meal. This is often done by concentrating the fish solubles to 30±45% solids

and then adding the concentrated solubles to press cake and drying to the desired

endpoint. For some feed applications it is desirable to have a defined soluble

protein content, which can be achieved with the inclusion of concentrated stick

water in fish meal. A major problem is that the concentration of stick water

traditionally requires a lot of energy-expensive evaporation systems. There are

few studies on the biochemical characterization of stick water, although this

soluble fraction should contain a rich assortment of biomolecules. Bechtel (2005)

has reported that dried stick water contains substantial amounts of

hydroxyproline, an amino acid abundant in connective tissue.

20.4.4 Protein powders

Most protein powders and specialized protein ingredients will be produced for

human use; however, there are some products such as insoluble protein fractions


that can be used as aquaculture, farm animal and pet animal feed ingredients.

Protein powders have been made from fish by-products by Sathivel et al. (2004),

in a process that involves grinding, heating to denature enzymes and release

lipid, sieving to remove bone and large tissue fragments, centrifugation to

remove lipid, and drying of both the soluble (supernatant) and insoluble protein

fractions (pellet). Another method of extracting and concentrating protein from

by-products (e.g., heads, frames, etc.) involves using pH extraction and

isoelectric precipitation (Underland et al., 2002; Kristinsson and Demir, 2003;

Choi and Park, 2002). In this process the ground tissue is mixed with water and

the pH adjusted to an alkaline or acid pH to solubilize the protein. Bone and

other unsolubilized materials are removed by centrifugation and then the pH is

adjusted to 5.5, to precipitate the protein, which is collected by centrifugation.

20.5 Methods of producing hydrolysates and silage

An alternative to making fishmeal from seafood processing wastes is to make

fish hydrolysates (Kristinsson and Rasco, 2000; Hardy, 1992; Raa and Gildberg,

1982; Sathivel et al., 2003; Shahidi, 1994). Fish hydrolysates are made by

proteolytic digestion of the fish wastes. After proteolytic digestion, bones,

undigested solids and often oil are separated from the hydrolysate. The liquid

hydrolysates are usually concentrated and sometimes dried. Hydrolysates can be

acidified to reduce microbial growth and oxidation retarded by adding an

antioxidant. During the concentration process, protein hydrolysates tend to

clump and become viscous. One solution has been to mix hydrolysates with dry

ingredients (often plant materials) and then co-dry the mixture. There are three

basic methods of producing protein hydrolysates from fish wastes: addition of

acids or bases (the product is often referred to as silage), addition of proteolytic

enzymes, and the use of microbial fermentations.

20.5.1 pH

Producing silage (hydrolysate) using acidification can be accomplished by first

decreasing fish waste particle size and then reducing the pH to approximately

3.5 with either organic or mineral acids (Espe et al., 1992; Skrede and Kjos,

1995). The low pH inhibits growth of microorganisms but allows proteolytic

enzymes from the fish wastes to digest the wastes. Often the temperature is

elevated to increase the enzymatic activity. Digestion is stopped by heating the

hydrolysates to denature the proteolytic enzymes. Most microbes will not grow

in the hydrolysate due to the low pH; however, chemical reactions still proceed

and an antioxidant, such as ethoxyquine, reduces oxidation problems. The liquid

material can be stored for later use, concentrated, or mixed with other dry

materials. The advantages of the acidification procedure are low cost, minimal

equipment requirements and low technical manpower requirements. The dis-

advantages are a lack of product uniformity and protein quality, increased levels


of free fatty acids and ammonia in the product, increased length of time required

for the process (days), large holding tank capacity required for multiple day

production, and the handling of acids.

20.5.2 Commercial enzymes

Hydrolysis can proceed faster by adding commercial proteolytic enzymes or

adding by-products with high concentrations of proteolytic enzymes (Rebeca et

al., 1991; Diniz and Martin, 1997; Ferreira and Hultin, 1994; Benjakul and

Morrissey, 1997; Liceaga-Gesualdo and Li-Chan, 1999; Shahidi et al., 1995;

Onodenalore and Shahidi, 1996). In this process the fish waste is ground and

mixed with a defined amount of proteolytic enzyme and the time, temperature

and other enzymatic reaction conditions are controlled. After the desired degree

of hydrolysis has occurred, the reaction is stopped by heating to denature the

proteolytic enzymes and an antioxidant added to reduce lipid oxidation. The

hydrolysate can be acidified and stored, or concentrated immediately. Dis-

advantages include additional cost of the enzymes and the greater process

control required to regulate the enzymatic activity. Advantages are fast pro-

cessing (hours), uniform product, and ability to customize hydroysates for

specific uses. Enzymatic hydrolysis of fish processing by-products offers a

potentially less expensive technological solution to utilizing by-products than

the relatively capital intensive process used to make fish meal. There are a

number of variables that need to be controlled including the species and

composition of fish used, the enzyme system used for the hydrolysis reaction,

determining whether endogenous proteolytic enzymes should be inactivated

prior to addition of the commercial enzymes, and determining the optimal

degree of hydrolysis needed to obtain the desired product (Mackie, 1982;

Rebeca et al., 1991).

20.5.3 Microbial fermentation

The use of microbial fermentations to hydrolyze fish wastes is the third method

used for producing hydrolysates. This method introduces and maintains a

microbial culture to obtain hydrolysis of the ground waste material

(Dapkevicius, 1998; Faid et al., 1997). There are two general methods:

1. use of microorganisms that produce an acid and thus lower the pH and allow

the endogenous proteolytic enzymes to hydrolyze the wastes, and

2. use of microbes that have additional proteolytic enzymatic activity to

enhance by-product hydrolysis.

In either case conditions must be employed that support the microorganism

population (microbial feed source, pH, temperature, etc.). After reaching an

appropriate degree of hydrolysis, a heating step is used to destroy the microbes

and denature the proteolytic enzymes. Hydrolysates can be preserved with the

addition of acid to a pH below 4.5 and the addition of mold inhibitors such as

sodium benzoate or formic acid.


20.6 Nutritional benefits and other properties of fish andanimal feeds made from seafood processing wastes

Soybean meal and corn gluten meal dominate the protein feed ingredient

business and global production of soybean meal exceeds 130 MMT. Animal

proteins derived from the rendering industry and those derived from capture

fishing, e.g. fish meal and solubles, constitute only a small portion of the world's

total protein meal production. Total world production of fish meal and solubles

is approximately 6±7MMT depending on the production from the large

industrial fisheries of Peru and Chile (Barlow, 2003). To put the amount of fish

meal produced from fish processing by-products in perspective, Alaska harvests

over 2MMT of fish for human consumption; however, the fish meal produced in

Alaska accounts for only 1 to 2% of the total world production of fish meal

(Crapo and Bechtel, 2003).

Fish meals are used in many types of aquaculture feeds (Hardy and

Masumoto, 1990; Pike and Hardy, 1997; Li et al., 2004; Hardy et al., 2005). Fish

meal is a good aquaculture feed ingredient because it is a high quality protein,

and compliments most vegetable proteins in feed formulations. In addition, fish

meals usually have a high content of the long chain omega-3 fatty acids and

minerals, and have good palatability characteristics. Fish meal quality has

improved due to a number of factors including an emphasis on raw material

freshness, and low-temperature drying. Babbitt et al. (1994) and Rathbone et al.

(2001) evaluated the nutritional characteristics of meals made from white fish

by-products. The digestibility of meals from fish processing by-products and

whole fish have been reported in numerous studies including trout and salmon

(Sugiura et al., 1998). The compositions of thirteen commercially available fish

meals made from fish processing by-products compared well with other high

quality fish meals (Smiley et al., 2003; Forster et al., 2004). The addition of

stick water to fish meal from white fish processing by-products was evaluated in

shrimp diets by Forster et al. (2003).

Hydrolysates are often of lower nutritional value when used as feed

ingredients than equivalent whole protein fish meal products (Stone and Hardy,

1986, 1989). However, the high level of digestibility of protein hydrolysates can

be of importance in diet formulation for very young animals with immature

digestive systems. Hydrolysates are potentially valuable aquaculture feed ingre-

dients as feed binding agents and for their attractant and palatability properties

(Lieske and Konrad, 1994). In addition, hydrolysates have been reported to

stimulate an immune response in fry (Gildberg and Mikkelsen, 1998). Protein

hydrolysates are commercially produced for use in animal milk replacers, and as

animal feed and pet food ingredients that have unique palatability and functional

properties. Hydrolysates made from seafood by-products are commercially

available.

Fish oils from cold water species are especially rich in long chain omega-3

fatty acids (Gruger et al., 1964) that are required as essential nutrients in marine

and freshwater aquaculture diets (Sargent and Tacon, 1999). There are large


seasonal changes in the lipid content of livers from number of species, and

Aidos et al. (2002) reported large seasonal changes in the amount and

composition of lipid in by-products. Oils extracted from fish by-products are

utilized in aquaculture diets. The addition of a smaller volume of omega-3 rich

fish oils to less costly vegetable oils can lower diet costs while providing other

benefits associated with omega-3 fatty acids. Another strategy used to increase

the omega-3 fatty acid content in aquaculture fish fillets is to feed diets rich in

omega-3 fatty acids during the finishing phase of the production cycle.

Freeman and Hoogland (1956), Olley et al. (1968), Dong et al. (1993) have

reported nutritional values for fish viscera, and Ferreira and Hultin (1994) for

liquefied cod frames. Recently, Gunasekera et al. (2002) reported the nutritional

evaluation of selected by-products from three species of fish including carp

offal, fish frames and trout offal. The chemical and nutritional properties of the

individual pollock, cod, and salmon by-products have been determined (Bechtel,

2003; Bechtel and Johnson 2004; Oliveira and Bechtel, 2005). Small basic

proteins associated with chromatin have been extracted from fish processing by-

products and reported to have anti-viral properties (Pedersen GM et al., 2003,

2004). It has been reported that high dietary nucleotide levels can have a

prophylactic effect on salmonids when challenged by viral disease (Burrells et

al., 2001a,b). Although yeast nucleotides were used, there is potential for using

material derived from fish by-product.

Fish meals are often used in the diets of young pigs, and feed ingredients

developed from hydrolysates have been used in aquaculture and in the diets of

young pigs and calves. Early weaned pigs require special dietary ingredients

until their digestive system is fully developed. It has been suggested that cheaper

specialty fish meals can replace expensive spray-dried animal plasma (Dijk et

al., 2001) that is currently being used as a minor dietary ingredient during the

early weaning period (BergstroÈm et al., 1997). Studies have suggested that

inclusion of marine fish oils in diets for pregnant sows improves fetal survival

rate (Rigau et al., 1995). In addition to uses as feed ingredients for livestock, fish

by-products have been used in pet foods as sources of protein and oil and there is

interest in using products made from fish by-products to enhance the health of

pets.

20.7 Future trends

There are many exciting areas that are being explored in the field of by-product

utilization for making feed ingredients; other uses include the following:

1. Continue to provide ingredients that will reduce the overall aquaculture feed

costs by increasing the use of cheaper plant-derived protein and oil

ingredients. Although fish meal and oil are ideally suited for use as

aquaculture feed ingredients, efficiencies can be gained by using cheaper

sources of protein and oil during part of the production cycle and using fish


meals and oils to retain palatability and attractant properties and improve

the nutritional profile.

2. Develop new products from fish by-products and separated components of

the by-product stream such as separation of high valued viscera components

for human use, higher valued protein and oil aquaculture feed components,

unique nutritional ingredients for different segments of the life cycle for

aquaculture, farm animal and pets, and mineral, protein and oil

supplements.

3. Extract interesting biomolecules and fractions from fish processing by-

product and hydrolysates that have unique applications for animal health

and well-being.

4. Develop economically viable processes and methods for utilizing and

enhancing the value of fish by-products from small volume processors,

seasonal processors, and processors in remote locations that cannot support

traditional by-product processing operations.

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21.1 Introduction

The concept of utilizing bioactive compounds of marine origin in cosmetic,

medical, and pharmaceutical applications is an ancient tradition. Historical

records indicate that marine organisms were once items of commerce for

biomedical uses to cure exotic diseases.1 For some time, bioactive compounds

from the sea were used as drug carriers, demulcents, and nutritional supple-

ments. Advances in marine biotechnology have increased the efficiency and

profitability of the seafood industry. The USDA has estimated that by the year

2025 global aquaculture will provide 50 to 60% of the world food supply.2

Increasing food and human health products will originate from the sea for

several reasons including the outbreak of bovine spongiform encephalopathy

(BSE) also known as mad cow disease and the need for more efficient healthcare

products from the sea.3 The search for novel antibiotics that can overcome the

problem of multi-drug resistant bacterial strains is a well enunciated reason for

investigation of the bioactivity of marine bioactive compounds. Currently,

harvested seafood products generate significant amounts of by-products from

which the seafood industry, scientists, government agencies, conservationists,

consumer advocates and seafood processors would like to obtain high value fine

biochemicals.3±6

The disposal of seafood waste is a problem at every one of the seafood

processing plants around the world. All seafood processors always stress that this

is one of the larger problems they face. For instance, in 2002, disposal rates in the

New Orleans, Louisiana area were $30/ton, and it was not uncommon for a large

21

Using marine by-products inpharmaceutical, medical, and cosmeticproductsJ. Losso, Louisiana State University, USA

plant to spend $2,000 to $3,000 per month in disposal fees.7 Very often, local

landfills refuse seafood as well as fishery wastes because of the nitrogenous runoff

that they can produce, and the fouling of truck scales. As a result, alternative uses

of the by-products that can provide economic benefits to processors and reduce the

waste disposal issue have been investigated. The following paragraphs describe

some of the most studied bioactive compounds from edible marine food products.

21.2 Squalamine

Squalamine (7-24-dihydroxylated-24 cholestane sulfate; Fig. 21.1) is an

aminosterol present in the liver, gallbladder, intestines, testes, and stomach of

dogfish shark (Squalus acanthias).8±11 Squalamine is a cationic water-soluble

steroid and is effective against both gram-negative and gram-positive bacteria,

fungi, angiogenesis, and tumor activities.8,12±15 Squalamine has shown promise

for the treatment of lung and ovarian cancer.8 Phase I clinical trials using

squalamine in patients with a variety of solid malignancies who had failed

conventional therapies showed that squalamine was well tolerated by humans.16

Bayes et al.17 reported that squalamine is still among the bioactive compounds

undergoing clinical trials for various types of cancer. Clinical trials are also

underway for the regression of retinopathy.17 When used as inhibitor of age-

related macular degeneration, squalamine at a concentration as low as 1 ppm

adminsitered once intravenously promoted shrinkage of the choroidal neo-

vascularization lesion associated with macular degeneration and an improvement

of 3 to 8 lines of vision with most patients having stabilization of vision.18±21

Another aminosterol with antimicrobial and anticancer activities similar to

squalamine is MSI-1436 which differs from squalamine by the presence of a

spermine side chain at C-3 on the cholesterol A ring8,14 (Fig. 21.2). Appetite

suppression is another health enhancing property ascribed to squalamine and

MSI-1436.8,22 MSI-1436 decreases body fat content by decreasing mRNA levels

of agouti-related peptide and neuropeptide Y in the hypothalamus of mice;

human trials are awaiting. Squalamine and MSI-1436 selectively inhibit bacteria

with low minimum inhibition concentration of about 5�g/ml.8

Fig. 21.1 Structure of squalamine.

Marine by-products in pharmaceutical, medical, and cosmetic products 451

Squalamine is a very attractive bioactive compound for several reasons. First,

as an inhibitor of cancer progression, the opportunities are enormous. Second, as

an antimicrobial compound of marine origin, it offers a marketing advantage

because there is no current evidence to suggest that viral or sub-viral particles,

that are adapted to cold-blooded (poikilothermic) organisms, can be transmitted

to humans. Third, as an appetite suppressing molecule, the possibilities are

enormous because obesity and diabetes are on the rise worldwide. Obesity can

lead to diabetes and possibly to cancer.23 Whereas squalamine has interesting

biomedical properties, only microgram quantities of the amino steroid can be

isolated from one shark. Tissue concentration of squalamine is very low (liver

and gallbladder: 10±20�g/g; spleen and testes: 2�g/g; stomach: 1�g/g; gill:0.5�g/g; and bowels: 0.02�g/g). Chemical synthesis is being used to produce

analogs of squalamine; however, yields have been very low so far.

21.3 Collagen

Collagen is unique among body proteins because it is the single most important

protein of connective tissues. Collagen molecules are classified into 21

different types and differ in their sequences, molecular weights, structures, and

functions, but they can be broadly subdivided into families. Type I and II

collagens are mostly found in the skins and bones whereas type IV, VI, VIII,

X, and dogfish egg case collagens belong to the network-forming family.

Collagen molecules are covalently cross-linked into fibrils that may swell, but

do not dissolve.

Commercially available collagen has several applications in the cosmetic,

medical, and pharmaceutical industries. Successful medical and pharmaceutical

applications of collagen include the treatment of urinary incontinence and pain

associated with osteoarthritis, scaffolding biomaterials for ligaments replace-

ment, cartilage engineering to repair join cartilage defects, other implants in

humans, and inhibition of angiogenesis as means of preventing cancer meta-

stasis.24±28 A matrix of collagen containing calcium phosphate (CaP-Gelfix(R))

was produced to create a cartilage via tissue engineering.24 Fibrocartilage

Fig. 21.2 Structure of MSI-1436.


formation and bone invasion was observed in 20 weeks. Cells maintained their

phenotype in the matrix; the matrix had a good healing response, was effective

in cartilage regeneration, and showed potential for use to repair defective joint

cartilage. Collagen-based tissue-engineered blood vessels (TEBVs) containing

elastin have been suggested as a better mimic of arterial physiology with im-

proved mechanical properties for use as bypass grafts in in vivo investigations.29

The cosmetic industry uses collagen extensively in personal care products. Anti-

angiogenic activity of type I collagen has been reported.30 Oral administration of

type II collagen to adjuvant arthritis (AA) rats alleviated both distinct articular

and general symptoms of arthritis.26 The authors suggested that the effectiveness

of collagen was associated with downregulation of both IFN- and TNF-�, andsuppression of cell immunity.

Traditionally, collagen has been obtained from the skins of land-based

animals such as bovine and porcine. In light of recent reports on mad cow

disease around the world, the use of collagen and collagen-derived products

from land-based animal skins has been called into question.31,32 As a result,

marine collagen and its by-products are in very high demand. Ogawa et

al.33,34 isolated and purified type I and II collagens from warm water black

drum (Pogonias cromis) and sheepshead (Archosargus probatocephalus)

skins and bones and identified biochemical and thermal properties that were

similar to bovine collagen. In an effort to find additional non-land-based

sources of collagens, cartilages from farm-raised alligators have been used as

sources of collagen (Losso, unpublished). However, the availability of

collagens from the skins of warm water fish will not offset the huge demands

for collagen in biochemical, biomedical, functional food, and pharmaceutical

industries.

Shark skin collagen is one of the most abundant and important sources of

marine collagen because large numbers of sharks are caught as by-product of

tuna fishing. The collagen from shark skin is of type I with similar properties as

land-based animal type I collagen. However, shark skin collagen contains fewer

imino acid residues and has a lower denaturation temperature than collagen

from land vertebrates.35 Shark skin collagen hydrolyzates are sold as cosmetics

and dietary supplements for the treatment of osteoporosis in Japan.35 Shark

cartilage is known for its anti-angiogenic properties. Trade names such as

Neovastat and AE 941 have been used by manufacturers to suggest probably a

similar product obtained from shark cartilage. AE 941 [Arthrovas, Neoretna,

Psovascar] inhibits angiogenesis by at least four molecular mechanisms:

blockade of endothelial cell signaling via inhibition of vascular endothelial

growth factor binding to its receptor vascular endothelial growth factor

(VEGFR); modulation of matrix metalloproteinase MMP-2 and MMP-9

pathways; induction of endothelial cell apoptosis; and stimulation of angiostatin

production.36,37 AEterna, the company which appears to own the exclusivity for

the commercialization of AE 941 is right now focusing on non-small cell lung

cancer as target for AE 941.37


21.4 Elastin

Elastin is a cross-linked protein in the extracellular matrix that provides

elasticity for many tissues. Elastin is a component of fish skin along with

collagen and dermatan sulfate. Elastin fibers consist of an amorphous material as

well as of 10±12 nm microfilaments which provide unique mechanical proper-

ties to those body tissues where they are present: the lung, skin, cartilage and

vessels walls.38 The unique physicochemical property of elastin can be attri-

buted to its characteristic amino acid composition, rich in glycine, proline and

other hydrophobic amino acids. The high hydrophobicity and cross-linked

character confers to the protein a high resistance to protease degradation.39

Elastin-based therapeutic approaches showed that soluble elastin and its

peptide VAPG at 1mg/ml inhibited lung colony formation of human melanoma

HT168-M1 and the whole elastin protein, �-elastin was a strong inhibitor of

murine lung carcinoma.40 Elastin peptides have the unique property of being

thermosensitive at high temperature and become insoluble by coarcervation.39

This unique property of elastin is an attractive new field of clinical research that

is being exploited in cancer treatment to create hyperthermia to improve the

permeability of the tumor cell and enhance the delivery of circulating anti-

cancer agents.41 Commercial applications of elastin include skin care products,

treatment of stretch marks (striae gravidarum) in pregnant women, and protein

hydrolysates added to hair-care products to repair broken hair.

21.5 Proteoglycans

Proteoglycans contain more than 90% polysaccharides with about 5% poly-

peptide bound along the linear carbohydrate chain. Other members of the family

include heparin, heparin sulfate, chondroitin sulfate, keratan sulfate, dermatan

sulfate, and hyaluronan. Proteoglycans are found extracellularly. Proteoglycans

are major molecules involved in angiogenesis. Dermatan sulfate (DS) is one of

the most important antithrombotic agents that has been approved for the

prophylaxis and treatment of deep vein thrombosis (DVT) in patients under-

going elective hip replacement surgery, and for the treatment of disseminated

intravascular coagulation (DIC).42 DS has important anticoagulant and anti-

thrombotic activities.43,44 DS accelerates heparin cofactor II mediated inhibition

of thrombin.45,46 Based on these findings, there are a number of future applica-

tions for DS including tissue transplantations, preparation of medical devices

and artificial tissues.46±48

DS is commonly obtained from porcine skin as well as porcine and bovine

intestinal mucosa. However, the ongoing episodes of BSE in cattle around the

world is forcing researchers, manufacturers, and government officials to find

new and safe sources of DS and other bioactive compounds that were tradi-

tionally obtained from land-based mammals. Because of the phylogeny distance

between fish and mammals, it is so far assumed that bioactive compounds from


marine-based resources may be less prone to viral infections that could be

pathogenic to humans.

Proteoglycans possess a growth factor-dependent activity and in the presence

of growth factors such as fibroblast growth factor -1, they modulate endothelial

cell proliferation and migration. Fucosylated chondroitin sulfate (FucCS), a

glycosaminoglycan obtained from sea cucumber (Ludwigothurea grisea) has the

same structure as mammalian chondroitin sulfate, but some of the glucuronic

acid residues display sulfated fucose branches.49 FucCS inhibits smooth muscle

cell proliferation as heparin and has a potent enhancing effect on endothelial

cell proliferation and migration in the presence of heparin-binding growth

factors. The sulfated fucose branches are the structural motif for the proangio-

genic activity of this chondroitin sulfate. FucCS also prevents venous and

arterial thrombosis, in animal models. FucCS may be a promising glycosamino-

glycan and as a promising molecule with possible beneficial effects in

pathological conditions affecting blood vessels such as the neovascularization

of ischemic areas.

21.6 Protamine

Protamine, a naturally occurring cationic polypeptide of about 30±65 amino acid

residues is found in mammals, fish, birds, and reptiles. Protamines package

DNA of most vertebrates sperm in a highly condensed and genetically inactive

state.50 The protein has a very high content of arginine residues which inhibits

both Gram positive and Gram negative bacteria.51 The protein is also highly

basic protein, lysine deficient, high in cysteine, pI 12±13, and structurally

diverse from species to species. The amino acid sequence from different species

is given in Table 21.1. Human and experimental gliomas spread and grow in

response to both paracrine and autocrine release of endothelial, fibroblast and

platelet growth factors. Protamine dose-dependently reduced tumor volume,

mitotic index, vascular density, and cell viability of highly malignant C6

glioblastoma in Wistar rats at a dose lower than toxic dose of suramin.52 Many

types of carcinoma accumulate large numbers of degranulating mast cells which

will release heparin. Protamine binds to heparin and neutralizes heparin anti-

coagulant effects and may therefore induce selective tumor cell thrombosis.

Intravenously injected protamine induced selective thrombosis in tumors, and

the effect lasted for several hours.53 Antithrombic compounds can also prevent

lipid rich plaque rupture in diabetic and dyslipidaemic patients. Ornithine

decarboxylase (ODC) which is associated with the onset and progression of a

variety of cancers including colon, prostate, and breast was inhibited by

protamine.54 Inhibition of ODC leads to polyamine depletion in cells, a

cytostatic effect on proliferating endothelial cells, and the inhibition of

angiogenesis.55 Nitric oxide (NO) is a pivotal factor for gastric ulcer healing.

Protamine sulfate has been reported to stimulate NO production and potentiate

the effect of heparin.


21.7 Future trends

Searching for bioactive compounds from the sea is an ancient tradition that will

not end anytime soon. Bioactive compounds from marine origin appear to

possess, in some cases, stronger biological activities than their land-based

counterparts. Because of phylogeny differences, disease resistance appears to be

a remote concern. However, supply of these marine bioactive compounds may

become a problem in the future. Genetic engineering is already looking into

designing and biosynthesizing analogs of bioactive compounds in short supply.

Elastin and squalamine are two prototype products for which laboratory

synthesis and biological engineering of analogs are already underway.8,56

Protamine bioactivity is mostly associated with the repeated chains of arginine.

Arginine is a nitric oxide precursor. Biosynthesis of polyarginine molecules that

mimic protamine bioactivity may provide a way to replace bioseparation of the

polypeptide from aquatic sources.

21.8 References

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7. SCHEXNAYDER M. LSU AgCenter. Personal Communication 2002.

Table 21.1 Amino acid sequence of protamine from different species

5 10 15 20 25 30Stallion ARYRC CRSQS QSRCR RRRRR RCRRR RRRSV RQRR--

Bull ARYRC CLTHS GSRCR RRRRR RCRRR RRR- F GRRR

Salmon PRRRR RASRP VRRRR RARRS TAVRR RRRVV RRRR

Perch PRRRR HAARP VRRRR RTRRS SRVHR RRRAV RRRR


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22.1 Introduction

Food processing by-products are largely organic in nature and can be potentially

utilized for production of valuable products. For example, many by-products

produced by the seafood industry may be used for production of food or feed

products and energy ± a very desirable outcome from both environmental and

sustainability points of view.

The seafood industry produces many kinds of by-products. In addition to

liquid effluent, solid materials are also produced, which include crab, prawns,

lobster body parts, damaged shellfish, heads, tails, frames, offal (guts, kidney,

liver, etc.) of fish as well as fish that do not satisfy the quality standards (Mhara,

2005). Lindsay (1975) stated that in the processing of most fish species for food

purposes, the by-products represent 30 to 80% of the raw material. The seafood

by-products typically have high contents of organic matter and salts and are

highly putrifiable. Some of these materials are treated prior to discharge, used

for fish meal production or directly disposed of by landfilling, land application,

or marine dumping. According to Champ et al. (1981), the impacts of ocean

disposal of fish by-product can include:

1. high oxygen demand on receiving water

2. visible surface slicks

3. turbidity plumes

4. organic enrichment and

5. the attraction of undesirable predator species such as sharks.

Regulations in many countries have become more restrictive, with the aim of

preventing or reducing the contamination caused by discharging seafood

22

Bio-diesel and bio-gas production fromseafood processing by-productsR. Zhang and H. M. El-Mashad, University of California Davis,USA

processing by-products into the sea. In 2001, the Northwest regional office of

the United States Environmental Protection Agency issued a new, stricter permit

system for Alaska's seafood processing facilities, which requires that seafood

processing by-products be discharged at least one nautical mile (1852m) from

shore in waters at least 36.6m deep. However, some environmental impact is

still expected from such ocean disposal.

Fish oil is one of the major by-products produced by the seafood industry. It

is generally produced in conjunction with the production of fish meal; it is the

lipid fraction that can be extracted from fish or fish by-products (Aidos, 2002).

Fish oil can be produced both from fish species specifically harvested for this

purpose and from the offal and waste resulting from processing higher value

species such as salmon, halibut and hake (Boyd et al., 2004). For example,

Alaskan seafood processing operations produce approximately 30 million liters

of fish oil annually. Fish oil has many food and non-food uses.

Fish oil and other seafood processing by-products can be used as a source of

renewable energy. The main objective of this chapter is to identify the

theoretical and practical issues concerning utilization of seafood processing by-

products as valuable resources for energy production, and review the conversion

technologies that could be used to convert such materials into bio-fuels in the

forms of bio-diesel and bio-gas. Both bio-diesel and bio-gas are renewable bio-

fuels. Many studies have been carried out to evaluate the potential of using

seafood processing by-products for bio-diesel and bio-gas production. The bio-

diesel can be used as a fuel directly or blended with petroleum diesel and used in

conventional diesel engines with few or no modifications. Bio-gas can be used

as a fuel for internal combustion engines, turbines, or fuel cells for heat and

electrical power generation. In the latter application, part of the heat produced

by the electric generator is recovered and used to heat digesters. Removal of

moisture and other constituents, such as hydrogen sulfide and ammonia, may be

required prior to the end use. Many articles have been published on the

importance and methods of bio-gas purification. The bio-gas can also be purified

to produce methane gas that can be used as a fuel for transportation vehicles.

The detailed descriptions of bio-gas cleaning, purification, and utilization

methods are provided by Ravishanker and Hills (1984) and Jensen and Jensen

(2000).

22.2 Quantity and quality of various seafood processingby-products

The amount and characteristics of effluent streams produced at seafood

processing plants largely depend on the type of animals being processed, the

parts of animals that are left over, and amount of water added into the by-

products as a result of animal handling and plant facility wash-down. In addition

to the quantity, the following characteristics of by-products are important for

initial evaluation of their suitability as substrates for production of bio-diesel and

Bio-diesel and bio-gas production from seafood processing by-products 461

bio-gas: moisture content, oil content, volatile solids or ash content, and protein

content. Due to the large variability among different seafood by-products, it is

recommended that laboratory or pilot trials be performed to determine the bio-

diesel or bio-gas yields from actual materials to obtain accurate information for

performing the energy production calculations and designing the conversion

processes.

22.2.1 Characteristics of fish oil

Many factors affect the characteristics of fish oil, including species of origin, the

season and the unit operations used by the processors. According to Hardy and

Masumoto (1990), fish oils do not have significant differences among species in

caloric content but they do differ in the content of essential fatty acids. Steigers

(2003) cited the characteristics of fish oil, produced from many species, such as

pollock, produced in Alaska: color is amber to light orange; density is 923 kg/

m3; sulfur content is 0.004±0.0084% by weight and the gross heat of combustion

is 36.4±36.8MJ/l. Garcia-Sanda et al. (2003) found that the oil separated from

the waste streams of the seafood canning industries had very good charac-

teristics as a fuel. The characteristics of fish oil and fuel-oil No. 1 in Spain, as an

example, are compared in Table 22.1. The seafood oil has lower contents of

sulfur and ash. It also has a relatively high caloric value, though lower than the

value of fuel-oil No. 1. It should be mentioned that while the gross caloric value

represents the total heat available in combustion of material, net caloric value

represents the energy value after subtracting the energy required for water

evaporation in the reaction from the total caloric value.

22.2.2 Characteristics of fish wastes

The average composition of various parts of fish carcasses for Pacific cod,

pollock and salmon, as reviewed by Babbitt (1990), is shown in Table 22.2. As

can be seen, all materials have relatively high moisture contents (>70%) and

very low ash contents (<4.1%), indicating that these materials could be highly

Table 22.1 Comparison of oil separated from seafood processing by-products and fuel-oil No. 1 in Spain (Garcia-Sanda et al., 2003)

Parameter Seafood oil No. 1 fuel oil

Gross caloric value, kJ/g 38.82 42.29Net caloric value, kJ/g 36.49 40.19Sulfur, % 0.09 2.7Carbon and hydrogen, % 89 ±Ash, % 0.28 10Freezing point, ëC 6.7 ±Density at 15ëC, kg/m3 957 ±Viscosity at 23ëC, N.sec/m2 0.08 ±


desirable for use as substrates for biological conversion processes. Salmon heads

have the highest oil content among the three species examined. There is no

significant composition difference between hand and machine filleting.

22.2.3 Characteristics of wastewater from seafood processing plants

Carawan (1991) mentioned that the major types of by-products found in seafood

processing effluent are blood, offal products, viscera, fins, fish heads, shells,

skins and meat `fines'. These constituents contribute significantly to the sus-

pended solids concentration of the effluent stream. The characteristics of liquid

effluents (wastewater) from several seafood products are shown in Table 22.3,

indicating high organic and oil contents in most streams. The high-strength

wastewater shows high turbidity, strong greenish yellow color and strong odors.

Moreover, seafood processing wastewater may contain high concentrations of

organic nitrogen (up to 0.300 g/L), sulfate (0.6±2.7 g/L), chlorides (8±19 g/L)

and brine solution from processing water (Carawan et al., 1979; Omil et al.,

1995). Fortunately, these effluents, unlike many industrial effluents, normally do

not contain toxic or carcinogenic substances (Afonso and BoÂrquez, 2002).

22.3 Theories and technologies for production of bio-dieseland bio-gas fuels

22.3.1 Bio-diesel production

Bio-diesel (fatty acid methyl esters) is attractive because it is made from renewable

resources, giving it many environmental and sustainability benefits. Chemically,

bio-diesel is made up of alkyl esters of long-chain fatty acids (Meher et al., 2004).

The required properties for bio-diesel are specified in the ASTM D6751-03a

Standard (ASTM, 2003). According to this standard, some of these properties are

required to obtain the best engine performance (e.g., flash point, viscosity, cetane

number, carbon residue and free glycerin). Others are needed to assure

environmentally friendly fuel usage (e.g., phosphorus content). According to the

ASTM D6751-03a standard, a maximum value of 10 ppm is not problematic.

Table 22.2 Average composition (% wet base) of some fish by-products (averages fromdata reviewed by Babbitt, 1990)

Fish species Protein Oil Ash Moisture

Pacific cod Hand filleting 15.0 4.1 4.1 79.2Machine filleting 14.1 3.8 3.8 79.4

Pollock Hand filleting 11.3 3.6 3.6 81.3Machine filleting 12.5 3.7 3.7 82.0

Salmon Head 14.2 3.9 3.9 71.4Viscera 17.1 1.8 1.8 78.3


Many types of oils and fats have been used for bio-diesel production,

including vegetable oils, food grade cooking oils, off-quality and rancid

vegetable oils, used cooking oil and animal fats such as lard, tallow, chicken fat

and fish oils (Coltrain, 2001). Basically, there are at least three methods for

converting oils and fats into bio-diesel, namely, microemulsions, pyrolysis (i.e.,

thermal cracking) and, the most common, transesterification (Ma and Hanna,

1999; Ghadge and Raheman, 2005). The main purpose of these methods is to

lower the viscosity of oils and fats so that they can be combusted in engines

without the problems that have been experienced with direct combustion of

vegetable oils and animal fat, such as choking of fuel injectors and build-up of

carbon deposit in the combustion chambers of engines.

Microemulsions and pyrolysis processes

Microemulsions made with solvents such as methanol, ethanol and ionic or non-

ionic amphiphiles have been studied as methods to overcome the high viscosity

of vegetable oils (DemirbasÎ, 2003). Amphiphiles are defined as molecules that

have an affinity for both aqueous and non-aqueous media.

Pyrolysis is the thermal degradation, at high temperature, of vegetable oils by

heat in the absence of oxygen, which results in the production of alkenes,

Table 22.3 Characteristics of wastewater from fish processing plants (Carawan et al.,1979)

Subcategory BOD5 COD TSS Oil and grease(mg/L) (mg/L) (mg/L) (mg/L)

Farm-raised catfish 340 700 400 200Conventional blue crab 4400 6300 620 220Mechanized blue crab 600 1000 330 150Alaskan crab meat 270 430 170 22Alaskan whole crab andcrab section 330 710 210 30

Dungeness and Tanner crab 280±1200 550±2000 60±130 28±600Alaskan shrimp 1000±2000 2000±3700 1300±3000 100±270West coast shrimp 2000 3300 900 700Southern non-breaded shrimp 1000 2300 800 250Breaded shrimp 720 1200 800 ±Tuna processing 700 1600 500 250Fish meal 100±24000 150±42000 70±20000 20±5000All salmon 253±2600 300±5500 120±1400 20±550Bottom and fin fish 200±1000 400±2000 100±800 40±300All sardines 1300 2500 921 250All herring 1200±6000 3000±10000 600±5000 600±800Hand shucked clam 800±2500 1000±4000 600±6000 16±50Mechanized clam 500±1200 700±1500 200±400 20±25All oysters 250±800 500±2000 200±2000 10±30All scallops 200±10000 300±11000 27±4000 15±25Abalone 430±580 800±1000 200±300 22±30


alkadienes, carboxylic acids, aromatics and small amounts of gaseous products.

Depending on the operating conditions, the pyrolysis process can be divided into

three subclasses: conventional pyrolysis, fast pyrolysis and flash pyrolysis

(DemirbasÎ, 2003). Ma and Hanna (1999) have mentioned that the equipment for

pyrolysis can be costly. In addition, pyrolysis has produced more bio-gasoline

than diesel fuel. Thus the product should be examined for its suitability as a fuel

for either diesel or gasoline engines.

Transesterification

As stated above, transesterification is the common method for bio-diesel

production; it is also called alcoholysis, and when methanol is used it is called

methanolysis (Meher et al., 2004). Although blending of oils with other

solvents, and microemulsions lower the viscosity, engine performance problems

(e.g., carbon deposit and lubricating oil contamination) still occur (Ma and

Hanna, 1999).

In transesterification, bio-diesel is produced by chemically reacting vegetable

oil or animal fat with an alcohol in the presence of a catalyst (Gerpen, 2005).

Several parameters influence the reaction. Among them are the mole ratio of oil

to alcohol, reaction temperature, type and concentration of catalyst, mixing

intensity, reaction time and free fatty acid and moisture content of the reactants

(Madras et al., 2004; Meher et al., 2004).

The stoichiometry of the reaction requires 3mol of methanol and 1mol of

triglyceride to give 3mol of fatty acid methyl ester and 1mol of glycerol

(Vicente et al., 2004). In practice, the amount of methanol needs to be higher in

order to drive the equilibrium to a maximum ester yield (Ma and Hanna, 1999).

The general equation of transesterification can be formulated as follows:

RCOOR1 + R2OH ÿÿ!CatalystRCOOR2 + R1OH

Ester Alcohol Ester Alcohol

A transesterification process for biodiesel production using methanol as the

reacting alcohol is shown in Fig. 22.1 (Gerpen, 2005). Typically for oils having

low levels (less than 1%) of free fatty acids, the one-step esterification reaction

is used, in which alcohol, oil and catalyst are combined in a reactor and agitated

for some time (e.g., 0.5±1 hour) at an elevated temperature (e.g., 50±60ëC).

While for oils having high concentrations of free fatty acids, a two- or three-step

process is used. In the two-step process, oil and alcohol react first in the presence

of acid catalyst, and then more alcohol is added and allowed to react in the

presence of a caustic catalyst. During the three-step process, oil reacts with

alcohol first, and then an acid catalyst is added to allow for more reaction.

Afterwards, more alcohol is added to react with the product of the first two steps

in the presence of a caustic catalyst (Canakci and Van Gerpen, 2001).

Following the esterification reaction, glycerol is removed from the alkyl

esters using either a settling tank or a centrifuge. After separation, alkyl esters

pass through a methanol stripper (either vacuum flash process or falling film


evaporator) for alcohol removal. Then alkyl esters enter a neutralization step,

where acid is added to the bio-diesel to neutralize any residual catalyst and to

split any soap that may have formed during the reaction. The salts are removed

during the water washing step and the free fatty acids stay in the bio-diesel. The

water washing step is intended to remove any remaining catalyst, soap, salts,

methanol, or free glycerol from the bio-diesel. Following the wash process, any

remaining water is removed from the bio-diesel using a vacuum flash process. In

some systems the bio-diesel is distilled in an additional step to produce a

colorless bio-diesel.

Methanol and ethanol are commonly used alcohols, with methanol being the

most often used because of its low cost, and its physical and chemical advan-

tages. The reaction can be catalyzed by an alkali (e.g., sodium methoxide,

sodium ethoxide, sodium propoxide and sodium butoxide), acids (sulfuric acid,

sulfonic acids and hydrochloric acid), or enzymes such as lipases (Ma and

Hanna, 1999). The mechanisms of the different catalyzed transesterification

processes are described by Meher et al. (2004). Alkali-catalyzed transesterifica-

tion is much faster than acid-catalyzed transesterification and is most often used

commercially.

One limitation to the alkali-catalyzed process is its sensitivity to the purity of

reactants. It is very sensitive to both water and free fatty acids (Zhang et al.,

2003), and has some other drawbacks, including the difficulty of recycling

glycerol, as it has some impurities, and the need for removal of the catalyst and

treatment of wastewater produced during the production and washing of bio-

diesel. In particular, several steps such as the evaporation of methanol, removal

Fig. 22.1 Typical procedure for bio-diesel production (adapted from Gerpen, 2005).


of saponified products, neutralization, and concentration are needed to recover

glycerol as a by-product.

22.3.2 Bio-gas production

Bio-gas is produced via anaerobic digestion processes and mainly consists of

methane and carbon dioxide. Depending on the chemical composition of sub-

strate and the operating conditions of the digestion process, methane content of

bio-gas can range from 50 to 80%. The anaerobic digestion process involves

transformation of organic material by a mixed culture of bacteria in the absence

of oxygen. It consists of a number of sub-processes that occur in series and

parallel manner (Pavlostathis and Giraldo-Gomez, 1991; Batstone et al., 2002) as

shown in Fig. 22.2. The particulate organic matter is hydrolyzed by extracellular

enzymes of microorganisms to soluble compounds such as amino acids, sugars

and long-chain fatty acids. Then the products of the hydrolysis step are fermented

into short-chain volatile fatty acids (VFAs), alcohols, ammonia and hydrogen

sulfide. The VFAs (other than acetate) and alcohols are further converted by

acetogenesis bacteria to acetic acid, hydrogen and carbon dioxide, which are then

converted by methanogenic bacteria to methane and carbon dioxide.

The anaerobic digestion process is normally carried out in an anaerobic

digester (bioreactor) that is a closed vessel, which is gas-tight, thermally

insulated in most cases, and equipped with a heating and a mixing device

(Demuynck et al., 1984). The digesters are often cylindrical for achieving good

mixing and some of them use cone-shaped bottoms to facilitate sludge removal

(Metcalf and Eddy, 2003). The digester top can be fixed or floating. A floating

Fig. 22.2 Main sub-processes involved in anaerobic digestion process (adapted fromPavlostathis and Giraldo-Gomez, 1991; Elmitwalli, 2000; Batstone et al., 2002).


top provides expandable gas storage with pressure control but is more expensive

and difficult to manage (Erickson et al., 2004).

The major factors that need to be considered when designing and operating

anaerobic digesters include temperature, pH, organic loading rate, biodegrad-

ability and nutrient availability of substrate, and retention time. Anaerobic

digestion occurs in a wide range of temperatures. It can occur at a temperature as

low as 4ëC, but the digestion rate increases with the increase of temperature.

Three temperature ranges have been explored for anaerobic digestion, including

the psychrophilic range (10±20ëC), the mesophilic range (20±45ëC, typically

35ëC), and thermophilic range (45±60ëC, typically 55ëC). Psychrophilic

digestion has been primarily associated with covered lagoon digesters operating

at ambient temperature. Anaerobic digesters, however, are commonly designed

to operate in either mesophilic or thermophilic range. Digestion at these higher

temperatures handles higher organic loading rates and requires shorter solids

retention time and is therefore more space-efficient. Also, a higher temperature

increases the destruction of pathogens that may be present in wastewater. To

reduce the heat loss and increase net energy production, digesters should be

insulated. Many types of insulation materials, including both natural and

synthetic materials, are used (Demuynck et al., 1984).

The schematics of some commercially available anaerobic digesters used for

wastewater treatment are shown in Fig. 22.3. Conventional anaerobic digesters

used for wastewater treatment include batch or fed-batch, completely mixed, and

plug-flow digesters. These digesters are suitable for treating concentrated waste.

To improve the economics of treating dilute wastewater, a number of new biomass-

retaining digesters, often called high-rate digesters, are being developed. These

biomass-retaining digesters are designed to provide special mechanisms to keep

bacteria and solids in the digesters longer than the treated liquid fraction. The

biomass-retaining mechanisms include internal solids settling, external solids

separation and recycling, and biomass immobilization with a fixed or suspended

medium. Major types of biomass-retaining digesters include anaerobic contact

reactor (Defour et al., 1994; MasseÂ and Masse, 2000), anaerobic sequencing batch

reactor (ASBR) (Zhang et al., 1997, 2001; Dugba et al., 1999), upflow sludge

blanket reactor (UASB) (Lettinga et al., 1980; Boardman et al., 1995; PunÄal and

Lema, 1999; Field, 2004), anaerobic filter (MasseÂ and Masse, 2000; Frankin,

2001), fluidized bed reactor, and anaerobic mixed biofilm reactor (Romano and

Zhang, 2005). For space limited areas, these digesters are more suitable for treating

dilute wastewater, for example, such as the wastewater from fish canning plants

(Palenzuela-RolloÂn, 1999). According to the survey conducted by Demuynck et al.

(1984) in Europe, 41% of the wastewater is treated using UASBs and 19% of

industrial bio-gas plants use ACRs. Anaerobic biofilter and completely mixed

digesters represent 15 and 14%, respectively. These percentages are changing, as

the application of different digestion technologies evolves.

Compared to anaerobic digesters that are designed for wastewater treatment,

there are fewer digester designs available for treatment of solid wastes, which

typically have less than 90% moisture. Different digester designs for treating


solid wastes are shown in Fig. 22.4. The simplest design is the batch digester with

leachate recirculation. In such a system, the feedstock is loaded with a certain

amount of inoculum at the beginning of the digestion time. Then the digester is

closed and maintained at the desired temperature. During the digestion course,

leachate is recalculated to provide the mixing. At the end of the digestion period,

the digester is emptied, keeping a certain amount of the digestate as inoculum for

the next batch. An improvement of batch digester is the sequential batch

anaerobic composting (SEBAC) (Chynoweth et al., 1991) which consists of three

batch digesters working in sequential order. The new batch digester is inoculated

with the liquid from the old batch digester. Lissens et al. (2001) described the

Fig. 22.3 Schematics of different designs of anaerobic digesters for wastewatertreatment.


designs of Kompogas, Dranco, and Valorga digesters. In the Kompogas process,

the wastes move via horizontal plug-flow in cylindrical digesters. The flow is

aided by slowly rotating impellers. In the Dranco process, the plug-flow occurred

vertically. Mixing is provided by recirculation of digesting waste between the

reactor bottom and top. The flow of digesting waste in the Valorga process is

circular plug-flow in cylindrical reactors. The mixing in this system is provided

by bio-gas injection from the reactor bottom.

The anaerobic phased solids digester (APS-Digester) as shown in Fig. 22.5 is a

new digester design which combines the features of batch and continuous digesters

(Zhang and Zhang, 1999; Hartman, 2004). The system typically consists of five

reactors, including four hydrolysis reactors and one bio-gasification reactor.

Feedstock is loaded into each of the hydrolysis reactors, acted on by extra-cellular

Fig. 22.4 Schematics of different designs of anaerobic digesters for solid wastetreatment.


enzymes and acidogenic bacteria, thereby liquefied and converted to simple

organic acids. These acids are collected and transferred to the bio-gasification

reactor, where they are reduced further into bio-gas by methanogenic bacteria.

Multiple hydrolysis reactors allow for a time separation between the beginnings of

different batch hydrolysis reactions. This time separation contributes to a relatively

constant level bio-gas production rate despite the batch loading and operational

schedule. After digestion is complete for each batch feedstock, the digested solids

and liquid are removed from the respective hydrolysis reactor. The APS-Digester

has been tested in the laboratory with a variety of organic solid wastes and is now

being scaled up for commercial applications.

22.4 Potential yields and quality of bio-diesel and bio-gasfuels

22.4.1 Yields, and chemical and physical properties of bio-diesel

The literature on production of bio-diesel from fish oil is scarce. Recently

research was conducted at the University of California, Davis, to produce bio-

Fig. 22.5 Schematic of anaerobic phased solids digester system.


diesel from the oil separated from salmon hydrolysate. The bio-diesel yields

from the two- and three-step procedures were compared as shown in Table 22.4.

In the two-step procedure, sulfuric acid was added with methanol in the first

step. Then potassium hydroxide was added later. In the three-step procedure, no

acid was added during the first step because the pH of the salmon hydrolysate

was about 3.7 due to formic acid addition during its production. In the second

step, sulfuric acid was added to reduce the level of free fatty acids, via

converting the free fatty acids to esters, to a level at which soap is not formed

during the alkaline treatment (Canakci and Van Gerpen, 2001). In the third step,

potassium hydroxide was added as a caustic catalyst. All experiments were

performed at a temperature of 52±55ëC with a mixing intensity of 600 rpm. The

oil and chemical reagents used, and the bio-diesel and glycerol yields are shown

in Table 22.4. There was no significant difference in the bio-diesel yield

between the two procedures. The bio-diesel produced was orange-red in color. It

should be mentioned that the yield presented here is the yield without washing

of the bio-diesel; a slightly lower yield is expected after a washing step, as some

of the soap and other impurities will be removed. A more detailed study is under

way to optimize the yield using the minimum amount of catalysts and to

determine the characteristics of bio-diesel produced from salmon oil.

Meher et al. (2004) showed the general characteristics of bio-diesel in

different countries. Some selected properties of No. 2 diesel and biodiesel are

compared in Table 22.5. Tyson (2004) stated that the biodiesel characteristics

(Table 22.5) are not based on the specific raw materials or the manufacturing

process used to produce the bio-diesel. However, the ASTM D6751 standard is

based on the physical and chemical properties needed for safe and satisfactory

Table 22.4 Bio-diesel yield from salmon oil separated from fish hydrolysate

Two steps Three steps

First stepOil, (g) 30.1 30.1Methanol, g (% w of oil) 9.58 (31.8) 6.25 (20.8)H2SO4, g (% w of oil) 0.27 (0.9)

Second stepPretreated oil, g 31.5 32.8Methanol, g (% w of oil) 5.25 (16.7) 3.3 (10.1)KOH, g (% w of oil) 0.24 (0.7) ±H2SO4, g (% w of oil) 0.27 (0.8)

Third stepPretreated oil, (g) ± 31.0Methanol, g (% w of oil) ± 5.32 (17.2)

KOH, g (% w of oil) ± 0.24 (0.8)Biodiesel yield, g (%) 29.4 (97.5) 29.1 (96.6)Glycerol yield, g (%) 5.33 (17.7) 4.85 (16.1)


diesel engine operation. No data are available in the literature about the

characteristics of bio-diesel produced from fish oil.

22.4.2 Yields, and chemical and physical properties of bio-gas

Many studies have been made of methane production from fish by-products.

Folkecenter (2005) identified some values for the bio-gas yield from fish oil and

different fish by-products (Table 22.6).

Some of the published data on bio-gas production from anaerobic digestion

and codigestion of fish by-products under different experimental conditions are

given in Table 22.7. The bio-gas yield and composition varied with the actual

material digested. Lanari and Franci (1998) studied the anaerobic digestion of

solid materials removed from fish-farm effluents, under psychrophilic

conditions (24±25ëC). Their results showed that methane yield ranged from

0.4 to 0.46 L/gVS.

Table 22.5 Selected properties of typical No. 2 diesel and bio-diesel fuels (Tyson,2004)

Fuel property Diesel Bio-diesel

Fuel standard ASTM D975 ASTM D6751Net heating value, MJ/L ~36.0 ~33.0Kinematic viscosity, mm2/sec at 40ëC 1.3±4.1 4.0±6.0Specific gravity, at 15ëC 0.85 0.88Density, kg/m3 at 15ëC 848 878Water and sediment, vol % 0.05 max 0.05 maxCarbon, wt % 87 77Hydrogen, wt % 13 12Boiling point, ëC 180±340 315±350Flash point, ëC 60±80 100±170Cloud point, ëC ÿ15 to 5 ÿ3 to 12Pour point, ëC ÿ35 to ÿ15 ÿ15 to 10Cetane number 40±55 48±65

Table 22.6 Some values of bio-gas and methane yields from fish oil and fish processingby-products (Folkecenter, 2005)

By-product type Volatile solids content Methane yield Bio-gas production% L/gVS m3/ton

(wet substrate)

Fish oil/meal industry 8±24 0.36 43±136Fish filleting industry 7±20 0.45 47±135Herring cannery 8±11 0.55 66±91Mackerel cannery 17±23 0.55 140±190Shellfish industry 20±26 0.75 225±281Smoke fish industry 8±44 0.59 71±389


Table 22.7 Bio-gas production from fish by-products under different experimental conditions

Feedstock Reactor type Tempera- HRT, SRT, OLR, VS Bio-gas Bio-gas Methane Referenceture, ëC day day g[VS]/L/day destruc- production yield content,

tion, % rate (L/g[VS %(L/L. day) added]

Fish by-product ASBR 35 12 68 0.7 75.9 1.1 1.57 78.6 Hartman et al. (2001)Fish by-product ASBR 35 5 70 1.6 87.9 2.3 1.44 76.5 Hartman et al. (2001)Fish by-product ASBR 35 7 66 0.83 89.2 1.0 1.21 81.3 Hartman et al. (2001)Fish by-product + wood ASBR 35 7 52 1.5 89.7 1.1 0.73 79.3 Hartman et al. (2001)Saline fish by-products Semi-continuous 35 24 2.2 48.2 0.81 0.39 50.9 Gebauer (2004)Saline fish by-products Semi-continuous 35 27.5 1.8 48.2 0.79 0.43 51.7 Gebauer (2004)Saline fish by-products Semi-continuous 35 27.9 1.8 47.4 0.58 0.33 48.9 Gebauer (2004)Saline fish by-products Semi-continuous 35 41.2 1.1 59.0 0.51 0.43 50.0 Gebauer (2004)Saline fish by-products Semi-continuous 35 60.0 0.9 61.9 0.42 0.45 54.1 Gebauer (2004)Sisal pulp (SP) and fish Batch 27 24 0.31 61 Mdhandete et al.by-products (FW) (2004)Sisal pulp (SP) and fish Batch 27 24 0.62 64 Mdhandete et al.by-products (FW) (2004)Sisal pulp (SP) and fish Batch 27 24 0.48 65 Mdhandete et al.by-products (FW) (2004)Sisal pulp (SP) and fish Batch 27 24 0.44 58 Mdhandete et al.by-products (FW) (2004)Solid by-product removed Upflow 24±25 38 0.227 about 98 0.13 0.46 80 Lanari and Francifrom fish farm anaerobic reactor (1998)Solid by-product removed Upflow 24±25 31 0.345 about 96 0.21 0.45 80 Lanari and Francifrom fish farm anaerobic reactor (1998)Solid by-product removed Upflow 24±25 22 0.751 about 93 0.38 0.4 80 Lanari and Francifrom fish farm anaerobic reactor (1998)

During anaerobic digestion of blue crab cooking wastewater in laboratory-

scale, upflow anaerobic reactors, Rodenhizer and Boardman (1999) found that

bio-gas production ranged from 6.6 L [gas]/L [feed] to 10.0 L [gas]/L [feed]. In

the bio-gas, methane, carbon dioxide and hydrogen sulfide comprised 68, 28 and

1.5% of the gas, respectively. O'Keefe et al. (1996) studied the feasibility of

using a hybrid sludge-bed filter (HSBF) reactor for anaerobic composting of

crab residuals at 35ëC. It should be mentioned that the HSBF is a tank containing

granular sludge in the bottom and biofilters are installed on the top. Thus it has

the advantages that come with both the UASB and anaerobic filter. The reactor

system consisted of a leach bed reactor and an HSBF reactor. A leachate volume

of about 4 liters was maintained in the HSBF reactor. Two volumes (4 L and

10.5 L) of leachate were maintained in the leach bed reactor. With the smaller

leachate volume, the operation was characterized as a percolating operation,

whereas with the larger leachate volume, the operation was characterized as a

flooded operation. During the percolating operation, the methane yield of

0.25 L/gVS and VS destruction of 78% were obtained after 47 days. During the

flooded operation, an average methane yield of 0.29 L/gVS and a VS destruction

of 50% were obtained after 28 days. The reactors were stable, producing a bio-

gas with high methane content (>70%).

Hartman et al. (2001) studied the codigestion of fish (salmon) and wood

wastes in an anaerobic sequencing batch reactor (ASBR) at 35ëC. They found

that stable bio-gas production could be obtained at loading rates of less than 2 g

VS/L/day. At loading rates of 2 g VS/L/day, reactor failure occurred. The

ammonia concentration was 700mg/L. The authors attributed this failure to the

high level of long-chain fatty acids in the fish oils. Adding the steam exploded

wood waste resulted in a 10% increase in bio-gas production from 1.0 to 1.1 L/L/

day (Table 22.7). The methane content of the biogas was 81.3% for fish waste,

and averaged 78.5% for the fish and wood waste. The addition of wood waste

may contribute to reactor stability without necessitating an increase in reactor

volume, which could allow a higher loading rate, thereby decreasing the reactor

volume required. During batch codigestion at 35C, Callaghan et al. (1999) found

that codigestion of fish offal with cattle slurry resulted in an increase in the

methane yield compared with the digestion of cattle slurry alone. VS reductions

of 47.3 and 31.1% were achieved, respectively.

Palenzuela-Rollon et al. (2002) treated a synthetic wastewater process,

simulating that from canning sardines and tuna, using an upflow anaerobic

sludge blanket (UASB) reactor operated at 30ëC. Total ammonia concentration

ranged from 243±299mg/L. A methane yield of 0.23 L/g was calculated. Achour

et al. (2000) studied the anaerobic digestion of tuna processing liquid effluent in

an upflow anaerobic cylindrical fixed bed reactor operated at 30ëC. The results

showed that a methane yield of 0.25m3/kg COD degraded (0.18m3 CH4/kg VS

degraded) was achieved.

Fish-oil by-products were used as an additional feedstock during the

anaerobic codigestion. According to Francese et al. (2000), fish oil by-product is

a residue of the manufacturing of fish oil having 32.8% TS and 91.2% VS.


During the anaerobic codigestion of pig manure (97% v/v), fish oil by-product

(2% v/v) and bentonite-bound oil (1%v/v), a bio-gas yield of 0.184 L/g VS with

65% CH4 in the bio-gas was obtained at 30ëC temperature and a 15-day

hydraulic retention time (HRT).The HRT is defined as the average time that a

liquid substrate is retained in the reactor and calculated by dividing the reactor

volume by daily feeding rate. The codigestion of fish oil by-product and

bentonite-bound oil with pig manure increased the net daily bio-gas production

four-fold compared to digestion of pig manure alone.

22.5 Problems encountered and possible approaches forovercoming them

22.5.1 Problems concerning bio-diesel production

Generally, free fatty acids in the oil are known to react with the alkaline catalyst

and form saponified products during transesterification reactions. They also lead

to longer production processes and an increase in the production cost (Meher et

al., 2004). Therefore, purification of the bio-diesel produced is essential.

To overcome the drawbacks of using alkali-catalysis, enzymatic processes

using both extracellular and intracellular lipases have recently been developed

(Fukuda et al., 2001). A comparison between the alkali-catalysis and lipase-

catalysis methods for bio-diesel fuel production is shown in Table 22.8. A

lipase-catalysis process seems to be superior in terms of the quality of the final

product and recovery of glycerol. However, the application of enzymatic

catalysis on an industrial scale may not be feasible because of the high cost

associated with enzymes (Fukuda et al., 2001; Jaeger and Eggert, 2002).

Use of the supercritical methanol method, without any catalyst, is proposed

as a way to solve the problems encountered in alkali-catalysis method for bio-

diesel production. The study performed by Meher et al. (2004) on the trans-

esterification of rapeseed oil showed that the supercritical methanol method had

a higher bio-diesel yield than conventional methods, due to the higher

conversion (95%) of free fatty acids to methyl esters. The saturated fatty acids

Table 22.8 A comparison between alkali-catalysis and lipase-catalysis transesterification(Fukuda et al., 2001)

Alkali-catalysis process Lipase-catalysisprocess

Reaction temperature 60±70ëC 30±40ëCFree fatty acids in raw materials saponified products methyl estersWater in raw materials interference with the reaction no influenceYield of methyl esters normal higherRecovery of glycerol difficult easyPurification of methyl esters repeated washing noneProduction cost of catalyst cheap relatively expensive


were completely converted to methyl esters at temperatures above 400ëC, while

the unsaturated fatty acids required a lower temperature of 350ëC. Thus it is

expected that use of the supercritical methanol method at a temperature below

350ëC will be suitable for bio-diesel production from fish oil due to the

relatively high content of unsaturated fatty acids in fish oil. According to Aidos

(2002), the saturated fraction of fish oil ranged from 22.5 to 35.8%. The amount

and variety of fatty acids present in fish oil depend on fish species and the

biological stage, as well as fish diet, fishing location, ocean temperature, and

nutritional and spawning state.

Cao et al. (2005) studied the production of bio-diesel via non-catalytic

transesterification using supercritical methanol with propane as a co-solvent. The

reaction could be completed in a very short time. They found that at 280ëC more

than 98% of triglyceride was converted to methyl esters after 10 minutes, while at

300ëC, the total conversion was achieved within 5 minutes. Compared with the

catalytic processes, purification of products is much simpler and more

environmentally friendly. However, the process requires high temperatures and

pressures, which means higher production costs and energy consumption.

Research is still being pursued by various researchers to determine the most

effective method for producing a high quality and low cost bio-diesel from fish oil.

22.5.2 Problems concerning biogas production

Anaerobic digestion has been proven to be an excellent method for treating high-

strength wastewater from the economic and sustainability points of view

(Lettinga, 2001). However, anaerobic treatment of marine wastewater can be

inhibited by high concentrations of sodium and sulfide (Soto et al., 1991; Vidal

et al., 1997). Feijoo et al. (1995) mentioned that seafood-processing wastewater

contains high concentrations of different ions, mainly Na+, Clÿ and SO42ÿ. The

sodium concentration can be as high as 12 g Na+/L in seawater. Thus careful

operation of the digester and selection of the appropriate anaerobic inoculum are

required (AspeÂ et al., 1997).

Boardman et al. (1995) studied the effect of different sodium concentrations

on the specific methanogenic activity (SMA) during digestion of clam-

processing wastewater at 32ëC. Their data showed that methanogenesis was

inhibited by NaCl added to the wastewater. A ten-fold decrease in methanogenic

activity was observed for a three-fold increase in Na+ concentration: the SMAs

were 0.4, 0.24, 0.18, 0.06 and 0.04 g CH4 COD/g VSS/day at salt concentrations

of 4.2, 5.3, 6.3, 8.4 and 12.6 g Na+/L, respectively. Also, total cumulative

methane production was 78, 55, 47, 10 and 5% of the theoretically possible

production at the various Na+ concentrations above. Sodium levels at about

5250mg/L significantly impacted the performance of a UASB reactor operated

at 32ëC. Boardman et al. (1995) also found that the methane production

efficiency of the UASB reactor was somewhat better at an organic loading rate

(OLR) of 13.8 g COD/L/day than at an OLR of 16.3 g COD/L/day. The methane

content in biogas ranged between 70 and 80%.


Gebauer (2004) studied the mesophilic (35ëC) anaerobic treatment of sludge

from saline fish farms under different HRTs (24, 27.5, 27.9, 41.2 and 60 days) in

a semi-continuous digester. Using undiluted influent having a salinity of 35%

and Na concentration of 10.2 g/L, an average methane content of 51.1% could be

achieved. The process was stable with a VFA concentration of 4.2±7.2 g/L as

acetic acid, indicating that the digestion was inhibited. Methane yield ranged

from 0.16 to 0.24 L/gVS. While using water-diluted substrate that had 17.5%

salinity and 5.3 g [Na]/L, a stable operation was obtained at 30-day HRT with

total the VFA concentration being 0.6 g/L as acetic acid and a methane yield of

0.22 L/gVS. The methane content of the biogas was 57.6%. With this diluted

substrate the hydrogen sulfide content of the biogas was almost half (1±1.6%) of

that obtained with the undiluted substrate, which is an advantage for the final use

of the bio-gas.

Vidal et al. (1997) studied the anaerobic digestion of wastewater from a fish

meal processing factory. An anaerobic filter was used, which was started with

marine sediments as an inoculum. The reactor was operated at 37ëC under

OLRs. Concentrations of 30 g [NaCl]/L in this wastewater had no toxic effect as

indicated by the methane production. The authors attributed this either to the use

of marine sediment as the inoculum or to the adaptation of bacteria to

concentrations over 10 g Na+/L. Methane production yield expressed as L/g

COD are calculated from the data of Vidal et al. (1997) at different OLRs (Fig.

22.6). The maximum methane yield was obtained at an OLR of 5.7±7.1 g/L/day.

Sulfate abatement also increased with greater OLRs. A reduction of methane

Fig. 22.6 Methane yield from anaerobic digestion of fish meal wastewater in ananaerobic filter (calculated from Vidal et al., 1997).


yield per unit of converted COD was observed due to the effect of sulfate on

methanogenesis. AspeÂ et al. (1997) mentioned that marine sediment adapted

better and faster to the saline substrate at 37ëC. They observed a 50% inhibition

of methanogenic activity at 0.22 g [H2S]/L, 53 g [Na+]/L and 10 g [SO4ÿ2]/L.

According to Ward and Slater (2002), high levels of ammonia are released

during anaerobic digestion of fish by-products, which then inhibits the digestion

process. High ammonia concentrations coincide with high pH values and cause

inhibition or total cessation of anaerobic digestion, especially at high operating

temperatures (Van Velsen, 1981). This is due to the fact that at high digestion

temperatures and pH values, the free ammonia concentration increases. McCarty

(1964) reported that free ammonia concentrations exceeding 150mg/L were

toxic to methanogenesis. However, the adaptation of the inoculum and many

other factors could affect the response of bacteria to ammonia.

It is important to have adapted inoculum, which can handle high concen-

trations of ammonia, sulfate and salts, for starting an anaerobic digester to treat

seafood processing by-products that normally have high concentrations of these

constituents. According to Omil et al. (1995), the adaptation to high salinity,

with the antagonistic effects on sodium caused by the presence of other ions,

makes it possible to operate reactors at high sodium concentrations (5±12 g/L).

Moreover, marine sediment inoculum appears to be a good option as well. There

are other techniques to overcome the problems of inhibition in anaerobic

digesters. One of them is dilution of the feedstock with fresh water. However,

this option needs to be carefully assessed, because increased wastewater volume

may require greater reactor volume to treat, which in turn increases the total cost

of the reactor and perhaps affects the total economics of the digester systems.

Besides the aforementioned challenges, there could be other constraints on

producing bio-gas and bio-diesel from seafood by-products. In fact, these

constraints apply to other biomass technologies as well. Among them are the

high capital cost of the processing plant and lack of financial incentives.

22.6 Future research needs

More research is needed in the future to study and optimize various parameters

involved in the bio-diesel production from fish oil and develop cost-effective

technologies. Optimum quantities of alcohol and the best particular catalyst for

producing bio-diesel from fish oil via transesterification still need to be

determined. A continuous production process needs to be developed. Yields and

quality of bio-diesel produced using different catalysts should be evaluated. An

economic analysis of production in both batch and continuous reactors is

required.

Compared to fish oil, other seafood processing by-products are much more

variable in their moisture content and chemical composition. Application of

anaerobic digestion technologies is often site-specific. More research is needed

to demonstrate the application of various anaerobic digesters for treatment of


both liquid and solid streams. Process parameters, such as organic loading rate

(OLR), and hydraulic and solid retention times, should be investigated for

specific materials. Scientific documentation and report of by-product charac-

teristics and the performance of anaerobic digesters in terms of bio-gas and

methane yields, total solid and volatile solid destruction, and stability of the

process, will benefit the further development of science and engineering

involved in anaerobic digestion technologies.

22.7 Summary

Utilization of various seafood processing by-products as valuable resources

instead of throwing them away as wastes is important from the standpoint of both

sustainability, and environmental and public health protection. Many seafood by-

products have a high organic content and are biological degradable, and

therefore, are desirable substrates for the production of bio-fuels. Fish oil can be

converted into bio-diesel and many liquid or solid materials can be converted into

bio-gas, though some unique characteristics of such materials, such as high salt,

sulfur and nitrogen contents, need to be considered. In the decision-making

process for assessing the costs and benefits of bio-diesel or bio-gas production,

performing energy and mass balance calculations is often necessary.

As pointed out in this chapter, many factors influence the characteristics of

fish oil and other by-products and should be considered during the selection of

an existing conversion technology or development of new technologies. As the

seafood industry puts more emphasis on the collection and utilization of their

by-products, the demand for efficient and cost effective conversion technologies

will grow. There are only limited research data available in the literature on bio-

diesel production from fish oil. Most of the past research on biogas production

has been concentrated on the treatment of wastewater generated at seafood

processing plants. Based on the energy density of the materials, the by-product

steams that have lower moisture content are preferred substrates for bio-gas

production. More research is needed to investigate the conversion of these

materials for energy production. More scientific publications that document the

characteristics of specific seafood by-products and performance of conversion

technologies are needed to increase the knowledge and data bases, which will

aid in the development and application of conversion technologies for bio-diesel

or bio-gas production.


Among the useful books on the basics of anaerobic digestion and solving

problems in the operation of bio-gas plants are Demuynck et al. (1984) and

Metcalf and Eddy (2003). More information about anaerobic digestion of

industrial wastewater using UASB reactors can be found on the website


developed by Field (2004). Advice on implementing sustainable and robust

environmental protection technologies (e.g., anaerobic reactors) can be obtained

from the Lettinga Associates Foundation for Environmental Protection &

Resource Conservation, at Wageningen University, The Netherlands. Guidance

and recommendations on the application of anaerobic reactors, especially the

anaerobic phased solids digester system, can be obtained from the research

group of Prof. Ruihong Zhang, Department of Biological and Agricultural

Engineering, University of California, Davis.

More information about the benefits of bio-diesel, methods for bio-diesel

production, making bio-diesel at a lab-scale, applications of bio-diesel,

environmental impacts of bio-diesel applications, and suppliers in different

countries can also be found on: http://journeytoforever.org/biodiesel_meth.html.

http://journeytoforever.org/biodiesel_link.html

Many articles have been published in journals and magazines such as

Bioresource Technology, Transactions of the ASAE, and Biocycle. Among them,

two excellent review articles (Ma and Hanna, 1999; Meher et al., 2004) which

contain much valuable theoretical and practical information about biodiesel

production. More information about bio-diesel production and purification can

be found in Biodiesel Industry Directory (2005), on the website of http://

bdid.texterity.com/bdid/2005/. Many useful links to this site can be found

regarding many aspects of bio-diesel production, consulting and application.

22.9 List of abbreviations

ACR anaerobic contact reactor

AMBR anaerobic mixed biofilm reactor

APS-Digester anaerobic phased solids digester

ASBR anaerobic sequencing batch reactor

BOD biological oxygen demand

COD chemical oxygen demand

CSTR constantly stirred tank reactor

HRT hydraulic retention time

HSBF hybrid sludge-bed filter

LCFA long chain fatty acids

OLR organic loading rate

SEBAC sequential batch anaerobic composting

SMA specific methanogenic activity

TS total solids

TSS total suspended solids

UASB upflow anaerobic sludge blanket reactor

VFA volatile fatty acids

VS volatile solids


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23.1 Introduction

In many areas around the world where commercial fish processing is conducted,

large amounts of seafood solid wastes are created. Some reports indicate that as

much as 60% of the finfish catch is not used, and a potentially higher amount of

waste can be found in shellfish processing (Green and Mattick, 1979). Although

a fraction of seafood waste and by-products is recovered and processed to make

products such as fishmeal, this is not always possible, owing to economic and

geographical considerations. Because of the biological characteristics of the

waste, it rapidly decomposes; therefore, much of this waste is disposed of by

returning it to the sea which, in addition to serving as a nutrient for marine life,

in some circumstances results in potential pollution problems.

With its high nitrogen composition, seafood waste has the potential to be used

as a plant fertilizer. However, its sensory characteristics, i.e., odoriferous

nitrogen compounds work against this use, and this type of application is only

done on a small scale. Some processing of solid seafood waste, either by adding

acids or lactic acid bacteria, results in the production of liquefied fish silage,

which can be used as either feed or fertilizer. Although fish fertilizer products

have been developed and are commercially available, the economics of their

production and marketing do not meet the needs of all seafood processors.

Moreover, because many fisheries activities are located in remote areas, on

many occasions there are few alternatives to dumping the wastes at sea or to

disposing of them in landfill sites. However, environmental concerns and

increasing regulations regarding the disposal of organic wastes have resulted in

the need to find better alternatives to these practices, based on ecologically

responsible methods.

23

Composting of seafood wastesA. M. Martin, Memorial University of Newfoundland, Canada

This chapter will present and discuss the composting of seafood wastes,

including vermicomposting operations, as a potential economic and environ-

mentally-friendly way of solving the problem created by the accumulation of

fisheries biomass from offal, by-catch, and from undesirable by-products of

further processing. The characteristics and potential uses of the compost

produced will also be presented.

23.1.1 General characteristics of seafood wastes

Seafood processing wastes can account for a large proportion of the seafood

biomass processed. In some cases, well-ground and dispersed waste added to

some environments might be beneficial by enhancing the nutrient content of the

waters. However, as indicated above, the traditional way of disposing of this

waste in the sea, harbours, landfills or rivers is becoming unsustainable because

of their biological characteristics, which results in a high organic content at the

disposal sites. Subsequently, high values of biochemical oxygen demand (BOD)

and chemical oxygen demand (COD) at those sites will result from the dumping

of non-treated solid seafood wastes. Treatment of these wastes could be

considered a more acceptable option from an environmental point of view, if a

simple and inexpensive treatment is employed (Veiga et al., 1994).

The fisheries industry has been recognized as a potential large source of

waste materials (Martin, 1998). Fish frames, guts, heads and fins are commonly

not used, and generally constitute the largest quantity of seafood wastes. In the

filleting industry, the frame, which is the portion left after the fillets have been

removed, could amount to over half of the initial biomass. Martin and Patel

(1991) reported that in 1988, in the Canadian Province of Newfoundland alone,

from approximately 351 000 tonnes of landed ground fish, approximately

140 000 tonnes of fish frames and 32 000 tonnes of fish guts became wastes. In

general, large amounts of seafood wastes can be found in any fishing area

around the world. However, given the nutrient value of fisheries by-products and

their potential use in feed formulations or compost (Shahidi, 1992), there are

opportunities for finding uses for those wastes.

23.2 Biodegradation of seafood wastes by composting

23.2.1 Composting

Among the many definitions of composting available in the literature, Mathur

(1991) stated: `Composting is the biological conversion of waste materials,

under controlled conditions, into a hygienic, humus rich, relatively biostable

product that conditions soils and nourishes plants'. Also, in general terms,

composting has been defined as a `low cost, environmentally beneficial and

potentially profitable use of fisheries products' (Anonymous, 1992). Several

comprehensive works about the different aspects of composting operations,

including those for seafood wastes, have been published (Minnich and Hunt,

1979; Mathur 1991, 1998; Miller 1992; Gershuny and Martin, 1992), including

Composting of seafood wastes 487

practical handbooks such as that published by Kreith (1994). Table 23.1 presents

a synthesis of the main characteristics of the composting process, raw materials

and final product.

Owing to their nature as a high-concentration animal-protein product, seafood

wastes have specific characteristics that could affect the success of a composting

operation. Among these, the presence of products from fish protein decomposition,

such as ammonia, have important potential effects on the biodegradation reactions.

A typical composting process is the microbial degradation of biological

organic material under aerobic conditions. Thermophilic microorganisms will do

much of the biodegradation. Heat will be generated during the process, which

should end when the materials added to the composting process have been

transformed into a useful product. It is expected that this product can be used as

a soil conditioner, given its physicochemical characteristics such as porosity and

water retention, as a soil enhancer by adding organic matter to the soil, or as a

fertilizer if it contributes substantial nutrients to the soil (generally, by

supplementing the compost with other nutrient sources). Also, it has been

pointed out that the final product from a composting process should be free of

viable plant seeds and human, animal, and plant pathogens to avoid lessening its

beneficial impact when applied to land (Haug, 1993).

In addition to the benefits from avoiding pollution caused by the alternative

disposal of waste materials, the production of compost from seafood wastes

could be of interest to regions with both fisheries industries and agriculture

activities. This is specifically true, for example, in areas with limited amounts of

soil, or with poor soil characteristics, which could benefit from the application of

products such as compost.

23.2.2 Other alternatives for the use of seafood wastes

A number of studies have been published regarding alternatives for seafood

waste and by-product utilization, with an emphasis on solid wastes, such as

Table 23.1 Main characteristics of the composting process, raw materials and finalproduct

Process description Raw material features Impact on final compost

Carbon dioxide production Appropriate carbon to Able to enhance soilnitrogen ratio conditions

Heat generation Appropriate moisture content Appropriate nutrientcontent for plants

Microbial degradation Appropriate pH Free of pathogens

Mineralization Organic substrate Mature and stable organicmatter

Oxidative reactions Solid state

Thermophilic reactions Wastes and/or by-products


those by Green and Mattick (1979), Martin (1994), and Shahidi (1995). Martin

(1998) presented the options available for the bioconversion of seafood wastes

to industrial products. The following paragraphs will briefly introduce processes

that result in the degradation (by chemical, physical and biological methods) of

seafood waste biomass, as some of them are treated extensively elsewhere in this

book.

Discarding wastes on land

In many areas of the world, seafood wastes have been used directly, without

further processing, to add nutrients to cultivated land. For example, in the island

of Newfoundland, Canada, the direct use on gardens and fields of capelin

(Mallotus villosus), harvested close to shore during their yearly migration to

spawn, has been a practice for many years. This kind of spreading of undigested

biological material rich in protein and lipid components has, as expected, many

drawbacks, including negative odour effects produced by their sensory charac-

teristics. Also, much of the nutrient value of the fish waste is lost using this

method. Some components of the waste fish biomass, such as viscera and even

muscles, are rich in proteolytic and other hydrolytic enzymes, some of which are

psychrophilic (Martin and Patel, 1991). As a result, even under low-temperature

conditions, enzymatic activity in the waste fish biomass will rapidly result in the

release of volatile ammonia from protein-rich wastes, and high ammonia con-

centration could overcome the capacity of microorganisms present to produce

nitrates. Moreover, calcium from the fish bones will result in an alkaline

medium, facilitating the loss of ammonia gas created (Hayes et al., 1993).

Nevertheless, this alkaline characteristic could be welcome in acidic soils.

Landfilling

Landfilling implies the deposition of waste on specially prepared sites where the

wastes are accumulated on top of previous loads, sometimes with the addition of

layers of soil between loads. Therefore, landfilling means burying the seafood

wastes. Under these conditions, anaerobic degradation will prevail, with sub-

sequent production of bad-smelling gases rich in reduced nitrogen and sulphur

compounds. Hayes et al. (1993) reported the generation of compounds with very

descriptive names such as `cadaverine' (also, `putrescine'), and the well known

`rotten egg gas', hydrogen sulphide. Although well-designed landfill operations

can minimize this negative effect, this operation interferes with the recovery of

valuable organic materials. Many of these materials could be transformed by

aerobic composting into a valuable product, rich in humic compounds.

Production of fishmeal

Using seafood wastes as a raw material for the production of fishmeal is a

common practice in many countries. Fishmeal production involves heat treat-

ment of the fish biomass, among other process operations. Although fishmeal is

more valuable than compost in terms of nutrient content, its production could be

limited by economic factors such as energy costs and, in some cases,


geographical ones, which limit its potential as a solution to the disposal of

seafood waste biomass.

Other technologies

Martin (1998) refers to the considerable work that has been conducted on the

production of fish protein concentrates, fish protein hydrolysates, and fish silage,

from seafood wastes, by-products, and species that are caught but have no

market. Also, significant work has been done with minced fish (Regenstein,

1986). These products, some of them based on the liquefaction of seafood

wastes by chemical or biological hydrolytic processes, have been developed

with the aim of recovering valuable protein present in seafood biomass. Their

intended use has been as ingredients in food or feeds, with the only probable

exception being that of liquid fertilizer products. Given these objectives, it is

obvious that the materials involved in the formulation of food products for

humans and feed products such as pet food or feed for aquaculture operations

should be of acceptable quality and sanitary standards (in the case of fishmeal,

although it is mostly marketed as a feed component, the heat treatment involved

in its production allows, in some cases, for the use of materials of lesser quality).

Therefore, the previously mentioned technologies generally exclude the use of

rotten, semi-decomposed, smelly fish offal and the like, as raw materials. These

are the kind of materials more properly destined to composting, spreading on

land, and landfilling operations.

An overview of the main alternative uses of seafood wastes is presented in

Table 23.2.

23.3 Composting operational parameters

A composting operation can be interpreted as the combination of a series of

physical, chemical and biological reactions occurring in the materials being

composted. These reactions depend on a number of variables or parameters, the

most important of which will be presented in the following subsections. In

addition to these parameters, others such as the particle size of the materials

being composted, are important if a comprehensive optimization of the process

is required (Mathur, 1998). It is important to point out that this section refers to

composting due to the action of microorganisms, which can be defined as

`microbial composting.' The characteristics of what is known as `vermi-

composting' will be presented in another section of this chapter.

23.3.1 Aeration

Composting is an aerobic process (although an ànaerobic compost' process

could be considered as equivalent to what happens in operations such as

landfilling, some of them are more complex than that and are inoculated with

specific microbial cultures to optimize the operation; many aerobic composting


systems probably have a fair bit of anaerobic activity, also). Consequently, the

presence of air is required as a provider of oxygen, which is needed for the

growth, and other metabolic reactions of the aerobic microorganisms responsible

for the biodegradation of the waste material being composted. An insufficient

Table 23.2 Alternative uses of seafood wastes

Technology Advantages Disadvantages References

Discarding ofwastes on land

Simplicity Foul odours fromrotten wastes

Hayes et al.(1993)

(spreading) Ammonia loss to air

Invasion of pests suchas rodents

Habitat for insects

Landfilling orburial

Simplicity Fetid odours fromanaerobicdecomposition

Production offishmeal

Product can becommercialized

Requires relativelyhigh capitalinvestment

Martin and Patel(1991)

Not economical forsmall seafoodoperators

Production of fishproteinconcentrates andhydrolysates

Potential for highquality product

Cost, requires precisecontrol of the process

Martin (1998)

Production of fishsilage

Low capitalinvestment

Needs to be driedunless used nearproduction site

Martin (1996)

Can be used as feedor fertilizer

Composting Simplicity Potential loss of muchof the nitrogen

Mathur (1991,1998), Martin

Generally, a lowcapital investment

Lower value thanalternatives

(1998)

Product· Adds nutrients tothe soil

· Enhances soilproperties

· Can becommercialized

· With hygieniccharacteristics

Requires carefulmanagement ofproduction


supply of air will result in the composting mixture adopting some of the

characteristics of an anaerobic degradation process, including the release of

malodorous gases. Aeration contributes to maintaining an adequate moisture

level (by water being evaporated into the air inside the mixture), to water vapour

at equilibrium conditions. Excessive aeration could dry out the composting

mixture. Aeration also acts as a temperature control mechanism, by dissipating

and transporting the heat generated in the degradation reactions (excessive

aeration can also result in temperatures too low for the composting process).

23.3.2 Carbon to nitrogen ratio

The carbon to nitrogen (C/N) ratio is an important parameter, which will relate

the composting reactions to the relative concentrations of essential chemical

constituents required for the growth and metabolic reactions of the microbial

population. Compounds such as carbohydrates, in addition to being sources of

carbon for the microbial biomass, will generate energy required for the

microbial metabolic activity. Nitrogen is an essential component of proteins and

amino acids required for the growth of the microbial biomass. Generally, it is

recommended that, to maintain an active microbial population in a composting

operation, the available carbon to nitrogen ratio should be kept at appropriate

levels. Lower ratios will result in losses of nitrogenous compounds, while higher

ratios will retard the composting reactions (Inbar et al., 1991).

23.3.3 Level of moisture

Water is essential to the viability of microbial populations and is a medium for

the biodegradative reactions in the compost mixture. While low moisture levels

will affect the speed of decomposition in the compost, flooding of a compost site

will interfere with the gas exchange required in aerobic processes. For example, if

the air-filled empty or free spaces inside the compost mixture are filled with

water, the amount of oxygen available to be transferred to the compost biological

phase could be affected. Hobson and Wheatley (1993) indicated a range of 40±

60% moisture content as the appropriate one for composting operations.

23.3.4 Temperature

Temperature is a significant parameter due to its role in the regulation of the

composting reactions. These reactions are multiple, some occurring in parallel

and others in a sequential way. Therefore, the range of adequate temperatures for

the composting process is wide. Some biodegradation can occur at low ambient

temperatures, such as 20ëC, however, most of the composting reactions will be

conducted at higher temperatures, including thermophilic conditions of as high as

65ëC. Temperature has an important effect in controlling the kind of microbial

population present and the reaction rates of the degradation taking place during

the composting process (Nakasaki et al., 1985a; Golueke and Diaz, 1996).


23.3.5 pH levels

Biodegradation in composting depends on microbial activity, and similar to the

effect of temperature, pH contributes to regulating microbial reactions.

Microbial resistance to acidic and alkaline conditions is a function of the type

of microbial population present, with individual species having optimum values

of pH for growth and for their metabolic reactions. In general, the optimum pH

for many microbial species, including bacteria and fungi, are, approximately, in

the range of pH values from 5 to 8.

Extremes values of pH affect the composting process. However, the organic

nature of the wastes composted, specifically if they contain protein compounds

(such as is the case of seafood wastes), will provide buffering qualities to the

composting mixture, i.e. the materials themselves will establish the pH. The

release of carbon dioxide and ammonia during the degradation of the wastes will

impart, respectively, acidic and alkaline characteristics, which tend to neutralize

the pH value of the mixture without the need of external adjustment (Haug,

1993). Table 23.3 presents a summary of the most important operational

parameters for composting.

Table 23.3 Composting operational parameters

Parameter Effect on process References

Aeration Provide oxygen to aerobic microorganisms Lau et al. (1992),Haug (1993)Control water content

Control temperature by removing heat

Carbon content Source of carbon and energy formicroorganisms

Haug (1993)

Nitrogen content Necessary for microbial biomass growth Decker (2000)

C/N ratio Active microbial aerobic metabolism aidedby an appropriate ratio

Inbar et al. (1991),Haug (1993)

Higher ratio slows process

Too low ratio leads to loss of nitrogen

Moisture content Necessary to microbial activity Haug (1993),Hobson andWheatley (1993)

If too high, can block airspace, reducingoxygen transfer

Oxygen Required by aerobic microorganisms Haug (1993)

Prevent anaerobic conditions

PH pH control not required due to compostbuffer properties

Haug (1993)

Temperature Influences type of microbial populationand biodegradation rates

Golueke and Diaz(1996), Stentiford(1996)Sanitizes compost


23.4 Characteristics of the composting of seafood wastes

Fisheries waste biomass contains valuable macronutrients including protein and

elements such as phosphorus, among others. The degradation of materials rich

in protein results in the production of gases rich in ammonia and other

compounds, some of them producing putrid smells. Moreover, the release of

ammonia and other nitrogen-containing gases will make the process pointless

from the point of view of nutrient recovery, as the main element contributed by

the protein, nitrogen, will be lost in the gas. Another characteristic of protein-

rich composting mixtures is that, owing to its chemical composition, it tends to

have a low C/N ratio. To avoid these problems, another main ingredient is

required for successful composting of seafood wastes: what is known as a

`bulking agent'. Bulking agents have a major role in composting operations, as

their presence will facilitate the aeration of the mixture, avoid the release of

offensive gases, and contribute the required carbon for an adequate carbon to

nitrogen ratio (Frederick et al., 1989; Imbeah, 1998). Examples of suggested

bulking agents that could accomplish these objectives are forestry and wood

processing wastes (such as size-reduced or shredded brush, bark, wood chips,

sawdust), agricultural wastes, and peat. Some materials, if used as bulking

agents, will fulfil only some of the above-mentioned criteria for a good bulking

agent. For example, some non-biodegradable (non-digestive) material can be

used to facilitate the aeration of the mixture, although it will not contribute

nutrients to it.

The release of ammonia from protein compounds in seafood wastes and of

calcium from fish bones, tends to shift the pH of the mixture towards the

alkaline range. Therefore, it would be convenient if the bulking agent employed,

in addition to increasing the carbon to nitrogen ratio of the mixture, could

contribute to decreasing its pH by having acidic characteristics. Another

requisite for the bulk agent is to be able to absorb water, as seafood wastes

generally tend to have high moisture contents (Mathur et al., 1986).

Therefore, the requirements for an effective bulking agent are complex, and

not all potentially available materials will be successful in this role. For

example, Hayes et al. (1993) reported that, in many cases, the addition to

seafood waste composts of weak acidic materials might fail to avoid the loss of

much of the ammonia. The authors presented, as examples of materials with low

buffering capacity, river mud, citrus wastes, and banana wastes. A study of

several bulking agents for the composting of fish offal was conducted by Liao et

al. (1995), who reported that peat moss, compared to sawdust and wood

shavings, gave the best results from the point of view of nitrogen conservation.

23.4.1 Other substrates of marine origin for composting operations

In this chapter, the term seafood wastes includes wastes from the processing of

shellfish, as the main features for the composting of finfish and shellfish wastes

are the same (Mathur et al., 1986), the only exception being the presence of

chitin in the latter. The processing of shellfish could result in chitin-rich wastes,


which could be composted (Kuo, 1995). Slow chitin degradation rates would

delay the compost process; thus consideration could be given to the separation of

the shells (for which perhaps other uses can be found) from the wastes, before

composting.

In addition to seafood wastes, marine algae biomass can be added to

composting operations to enhance the nutrient value of the final product. Some

seaweed species could contribute elements such as calcium, copper, iodine,

magnesium, phosphorus and potassium (Gershuny and Martin, 1992).

23.5 Technological aspects

23.5.1 Limitations of traditional methods for the composting of seafood

wastes

Through the years, many composting systems have been developed, from the

most unsophisticated ones (such as those in household backyards), to recent

systems that incorporate control equipment and closed environments. In general,

most of the composting technologies have been based on the treatment of

organic materials of plant origin, and in many cases (mostly at the household

composting level) it has been advised not to include wastes of animal origin in

the materials to be composted. The rationale for this is related to the require-

ments for aeration of the composting mixture. Traditional composting operations

are aerated by what can be called àctive aeration' methods, i.e. by periodically

turning of the compost or by forcing air through the mixture. This will result in

increased discharge of gases from the compost, contributing to the loss of

ammonia and if improperly managed, as indicated above, the release of smelly

compounds in the air. Therefore, not only will valuable nitrogen be lost from the

compost, but also the environment surrounding the compost operation will be

affected.

Many of the problems arising when new composting technologies are

introduced are caused by ineffective operational designs. To overcome some of

these problems, quality control methods need to be applied (SereÂs-Aspax and

AlcanÄiz-Baldellou, 1985). Moreover, because of its biological characteristics, on

many occasions it is difficult to predict the behaviour of a composting operation,

as it is dependent on numerous variables, many of them not easy to control. For

example, combinations of organic wastes with sufficient nitrogen content, and

with similar conditions of bulk density, carbon and water content, could result in

different rates of composting (Mote and Griffis, 1980). Weather conditions are

also an important factor for outdoor composting operations.

23.5.2 Passive aeration composting

A compost process can be conducted in enclosed conditions (in barrels, wood

enclosures, or the like), or on the soil in piles (heaps or mounds). In the latter

type, in many cases the system employed is identified as a `windrow' (Kuhlman,


1990). A windrow system, on occasions, could benefit by an appropriate

containment of the system, or enveloping. Because the aim of the passively

aerated system is to increase the retention of the gases produced inside the

composting mixture, it is important to find a good cover for the windrow.

However, by interfering with the mass and heat transfer mechanisms between

the composting material and the environment, covering could result in

overheating of the compost pile (it could limit the flow of fresh air to the

inside of the pile, and of hot gases, produced in the compost operation, to the

outside). The use of covering materials will also add costs to the operation. A

solution to these limitations could be found if the same bulk agent employed in

the composting process is also used to envelope the composting mixture, by

placing it as the most exterior layer of the composting pile. Another possibility is

the use of finished compost as the cover. When the bulk agent or finished

compost are used with this aim, they and the microbial population attached to

them act as a biofilter (Martin, 1991) for the gases produced in the composting

operation.

Generally, the aeration of the composting pile will be achieved by the

penetration of air inside it. To facilitate this process, the bulk material should be

of loose characteristics, to allow for the formation of void spaces where the air

can penetrate the pile. After the decomposing reactions become established in

the composting mixture, it will increase its temperature due to the metabolic

energy liberated by the biophase. If an appropriate flow of air is present, the

temperature rise will be highest roughly in the centre of the pile. This is because

the dissipation of heat from the innermost sections will be more difficult than the

dissipation of heat from the more external layers of the compost mixture.

Therefore, the flow of air, initially mostly caused by gas diffusion, will also be

aided by convective currents, as the hot gases from inside the pile will tend to

move up to colder layers of the pile, being replaced by cooler air from the

external layers of the pile. At this stage, particularly in larger piles, the flow of

air could become the limiting factor in the supply of oxygen to the

biodegradation process, which will result in the development of anaerobic

reactions. Because, in the case of animal wastes, including seafood wastes, no

turning of the pile in aid of the aeration is advised to avoid the release of odorous

gases, new solutions need to be found. One solution, as indicated above, is the

use of cover material with biofilter properties. Another possibility is to enhance

the flow of air by mechanical aeration devices, which will result in forced-

convection conditions. However, this method implies additional capital expendi-

ture (fans, air compressors, pumps, or combinations of them), and operating

expenses (energy). Economic considerations in a composting operation need to

consider the expenses involved in setting up the pile and the cost of maintaining

the operation for a given period of time. Therefore, the latter solution could be

unaffordable. To avoid this problem, the fundamentals of passive aeration

composting have been applied to the design of what is known as Passively

Aerated Windrow System (PAWS), as reported by Mathur (1991, 1998).


23.5.3 Passively aerated windrow system (PAWS)

The PAWS composting system, as its name indicates, avoids any mechanical

action to promote aeration, such as turning, or pumping or blowing air. Instead, the

design of the PAWS incorporates elements that will allow the flow of air inside the

composting pile without forced convention. Therefore, to convey air deep inside

the composting process, pipes with their ends opening outside the pile are placed at

the base. These ends should allow the air to enter the pile, and may be covered with

a mesh or lattice screen to avoid the intrusion of pests to the pile. In addition, the

pipes should have perforations directed towards the top of the pile, through which

the air will diffuse to the composting mixture. To avoid letting liquefied fish waste

go through these pipes, they should be inserted in a section of the pile covered with

the bulking agent. Another possibility is to build the compost pile on top of a

perforated plate or base, which will allow the introduction of air to the system. As

indicated above, the aeration will be further aided by convective currents created

by the differences in temperature between the core of the composting mixture and

the exterior layers, and finally, the surroundings. These added features

characterizing the PAWS should result in avoiding two of the main problems in

composting: overheating, and the development of anaerobic zones.

It has been reported that the PAWS has been successfully applied for the

composting of a number of farming and industrial wastes, including those from

the pulp and paper industry, and from seafood processing wastes (Mathur,

1991). As well as the material to be composted, the selection of the bulking

agent is of paramount importance for the success of the PAWS. The bulking

agent, in addition to its operational role in enveloping the composting pile, can

be used to increase the carbon to nitrogen ratio of the blend (although not all the

carbon in the bulking agent will be available for the composting reactions, so the

full carbon content should not be counted when calculating the ratio). In Table

23.4, some potential bulking agents, and their characteristics, are presented.

Hayes et al. (1993) pointed out the effect of materials such as peat, mature

compost, and by-products and wastes from the forestry and wood industry when

used as PAWS envelope or covering. The authors claim that these materials are

hygienic and free of seeds and pathogens, act as walls for the pile, and function

as a biofilter by retaining vapours liberated from the composting reactions.

Being closer to the outside temperature, the envelope can sustain mesophilic

microbial populations, including those involved in the transformation of

ammonia to nitrates. The proportional amount of bulk material required for a

composting operation needs to be calculated by means of materials balances,

based on its moisture content and the carbon to nitrogen ratio required.

Application of PAWS to the composting of seafood wastes

A composting process has been developed in which seafood wastes are mixed

with peat, which acts as a bulking agent. It has been reported that the compost

produced this way is of good quality and has appropriate concentrations of

nutrients. Moreover, no foul odours were produced in the composting operation

(Mathur et al., 1986). As already mentioned, one of the advantages of this


process is the retention of part of the nitrogen present in the seafood biomass.

This technology is based on some specific properties of the bulking agent

incorporated into the degradation process. When peat is used, its fibres will

interfere, due to sorption reactions, with the loss of the ammonia produced in the

composting reactions. It is possible that both adsorption and absorption

mechanisms take part in this process, resulting in the low pH peat fibres being

able to interact with the alkaline ammonia gas.

It appears that peat is, among the potential bulk materials to be employed in

composting, probably the one which possesses the best characteristics for an

effective operation of PAWS, and specifically for the composting of seafood

wastes. It has been reported that both horticultural sphagnum peat, and light

brown peat that is not used for fuel or horticulture (and therefore, should be less

expensive), do meet the requirements for an appropriate bulk material (Mathur

et al., 1988). The same authors indicated that peat has a higher buffering action

and higher nitrogen concentration than wood waste products, and that peat moss

is a product traditionally employed to ameliorate soil conditions. The use of peat

is fitting in places where it is available (as a local resource or as a commercial

product), such as in some areas of Canada; however, it should be mentioned that

other materials (mentioned elsewhere in this chapter) could be used successfully

as bulking agents.

Use of peat as a bulking agent

Several studies have been conducted on the potential of peat to act as a filter

material by adsorbing organic and inorganic compounds (Mueller, 1972;

Table 23.4 Overview of some potential degrading bulking agents to be used in thecomposting of seafood wastes

Bulking agent Characteristics References

Peat Several types can be employed Dayegamiye and

Established market as a soil conditioner Isfan (1991),

Not available in all areasMathur (1991),

Does not significantly degrade duringcomposting

Martin et al. (1993a),Liao et al. (1995)

Low fertilizer value

Generally needs to be purchased

Wastes and by-products from the

Less acidity and buffering capacity thanpeat

Janssen (1984),Frederick et al.

forestry and woodindustry (sawdust,shavings, tree bark)

Higher bulk density than peat

Low fertilizer value (lower nitrogencontent than peat)

Lignins contribute to humus formation

Tannins, abundant in tree barks, couldslow biodegradation

(1989), Dayegamiyeand Isfan (1991),Mathur (1991),Martin et al. (1993a),Hayes et al. (1993),Liao et al. (1995)


Viraraghavan and Ayyaswami, 1987). A comprehensive review on the role of

peat in waste biodegradation was presented by Martin (1991), who reported that

peat is characterized by being acidic, and by possessing high adsorptive and

absorptive capabilities.

Basically, the solid phase of peat is composed of organic material, with some

ash content. When compared with standard soils, peat ash contents are low. The

chemical composition of peat is the result of the decomposition of organic

materials in the absence of oxygen, which results in a material with a particular

chemical profile, characterized by the presence of humic materials and

bitumens. The degree of decomposition of the peat is defined by its degree of

humification, and this factor is important in the evaluation of the type, quality

and specific applications of the peat.

The determination of the degree of humification of a peat sample is generally

conducted following the von Post system of classification, consisting of ten

levels, from low decomposed, low humification, H-1, to high decomposition and

humification, H-10. Each level represents approximately 10% decomposition.

The extent of decomposition is found by squeezing recently harvested peat and

analysing the water squeezed and the compressed peat (Anonymous, 2005).

Equally, the concentration of ash in peat varies with the degree of humification

(Fuchsman, 1980). As expected, the most decomposed peat is that found at the

lower layers in a peat bog. A peat good for horticultural applications (H-1 to H-

2) has limited humification, with a pale brown colour. Lower layers of peat will

contain darker deposits, and the deepest levels will correspond to values of H-5

and higher, a peat appropriate for fuel applications. The type of peat known as

sphagnum will be found in the higher layers. The characteristics of peat are

related to the biosystem from which it is derived. In general, peat can be

considered a renewable resource, as it accumulates if there is adequate plant

biomass and growing conditions present on site as well as the availability of

water (Fuchsman, 1980).

For a composting process using the technology of PAWS, there is flexibility

in the use of peat. Although horticultural-type peat is generally preferred, peat

from lower layers could be employed in the base of the pile and for enveloping.

However, the peat fibres should have appropriate particle sizes and moisture

contents to avoid clumping, and to facilitate the gas exchange in the pile.

Raw peat is low in nutrients, and its main established commercial use is as a

soil conditioner, which includes its valued capacity for the retention of moisture

in the soil. However, the composting process with seafood wastes should

increase its nutrient content, namely its nitrogen concentration. Mathur et al.

(1986) indicated that the moist, acidic peat fibres adsorb the ammonia from

protein degradation as ammonium ion. Therefore, although not necessarily

possessing the chemical composition of a standard commercial fertilizer

product, the resulting compost will be a nutrient source for plants, which

require nitrogen in nitrate or ammonium forms. In general, composting with peat

is a good technology for nitrogen conservation (Liao et al., 1995). Table 23.5

presents information on the chemical composition of some seafood wastes


composts prepared with peat, and with a combination of peat and sawdust, as

bulking agents.

Phenolic compounds in lignocellulosic materials could act as inhibitors of

biological reactions. Although there are phenolic compounds in peat, Mathur

(1991) indicated that calcium and proteins could deactivate them. Seafood

wastes, in addition to their high protein content, also contain calcium com-

pounds from the fish bones. Martin and Patel (1991) reported that the final

product obtained from peat composted with seafood fisheries could potentially

be a good commercial soil product due to its physical, nutritional, and functional

characteristics. A comparative study on the use of sphagnum peat and sawdust in

the composting of fisheries and other food processing wastes was presented by

Martin et al. (1993a).

Environmental impact

The composting process is a natural way of disposing of solid organic waste and

of producing materials rich in humus with soil-amelioration properties and thus

is an environmentally-friendly operation. As indicated above, potential problems

related to the production of odorous gases, common in ill-designed composting

operations, can be avoided by correctly applying the PAW technology.

Frederick et al. (1989) indicated that a suitable composting pile design with

appropriate bulking agent would result in an efficient, odour-free biodegradation

process.

Another potential problem, the production of leachates, can be dealt with by

building a good base to the composting pile (as indicated elsewhere in this

chapter), by installing impermeable films at the base, or by adding an

appropriate leachate recovery system connected to, for example, an oxidation

lagoon.

However, besides the environmental advantages brought by composting and

related biodegradation operations, consideration should be given to the environ-

Table 23.5 Chemical composition of seafood waste composts with peat and with acombination of peat and sawdust as bulk materials

Components Concentration (% dry weight)a

Bulking agent

Peatb Peat and sawdustc

Ash 19.5 � 2.8 23.9 � 3.7Carbohydratesd 63.9 62.3Lipids 0.9 � 0.0 3.2 � 0.0Nitrogen 2.5 � 0.1 1.7 � 0.0

a Mean values of three determinations � standard deviations.b From Martin and Chintalapati (1989)c Equal proportions of peat and sawdust, from Martin et al. (1993a)d Calculated by difference, assuming a protein content of 6.25 � % nitrogen


mental impact associated with the use of bulking agents, specifically, what kind

of material is to be used, and its source. The quantities of bulking agent required

by a sizeable composting operation have both economic and environmental

consequences, which could limit the viability of the operation. The ideal bulking

agent will be one which is also an industrial waste or by-product available in the

proximity of the composting operation. In this category, the best candidates are,

possibly, lignocellulosic materials including straw and other agriculture

residues, as well as forestry waste organic materials such as sawdust, bark,

and wood chips.

Peat, which possesses very good characteristics as a bulk material for the

composting of seafood wastes, is a material not present in all environments.

However, it is generally abundant in some northern regions, close to locations

with important fish harvesting and processing operations (Canada, northern

regions of Europe, among others). These regions are, generally, characterized by

a restricted amount of cultivable land and a subsequent shortage of agricultural

and in many cases, forestry waste material, factors that hamper the availability

of other bulking agents. Although, as indicated above, peat can be considered as

a renewable material due to its natural regeneration, there have been concerns

about how bogs and wetlands, where peat is deposited, can be affected by its

extraction. However, Hayes et al. (1993) report that peatland ecosystems where

peat has been extracted can be restored, with the new wetland presenting better

ecological characteristics than virgin peatlands due to a diminished production

of methane. The same authors point out the benefits of peat in the restoration of

organic matter in soils, benefits that will add value to a peat-containing compost

product. As an answer to the above-mentioned concerns related to peat

extraction, materials with peat-like characteristics can be used as alternatives.

For example, Briddlestone and Gray (1991) suggested the use of a highly

stabilized product, produced by aerobic processing of organic wastes, as a

substitute for peat.

23.6 Biological aspects

Composting is a very complex biological process, difficult to control even when

the chemical composition of the mixture, including the carbon to nitrogen ratio,

are the result of careful calculations to determine, from the amounts of nutrients

present, how much of them are available (i.e., how much can be metabolized

during the composting process), and the process conditions are monitored. The

biological phase (consisting of multiple biological reactions) of the operation is

ultimately the active agent determining the outcome of the whole operation.

23.6.1 The role of microorganisms in composting

Composting is a biodegradation process, its active element being the microbial

population that does the digestion of the organic materials present, which


generally are composed of wastes of biological origin. A secondary active

element is the presence of hydrolytic enzymes in the wastes, such as those in

plant material and animal tissues. In seafood wastes, this element is of

particular importance, due to the presence of enzymes in fish viscera, which

accounts for a good fraction of the fish-offal mass. Biological degradation is

also contributed by other non-microbial entities such as insects and worms. As

already indicated, composting by worms (vermicomposting) is discussed

elsewhere in this chapter.

Enzymatic contributions from the decaying waste biomass are only relevant

at the beginning of the composting operation, as enzymes will be inhibited and

destroyed as a result of the biological degradation process. Similar fates await

protozoa and insects, mostly due to the heat liberated by bacterial and fungal

degradation, which will be, by far, the main contributor to the composting

process (because these latter organisms can withstand higher temperatures).

Given the appropriate operating parameters, some kinds of microorganisms

(such as bacteria and fungi) have the ability to adapt to the environmental

conditions of the process, and to interact among them in symbiotic and other

types of relationships, which will result in an efficient and complete degradation

or the waste biomass.

Microorganisms, being such a relevant factor in the composting process,

their role should be understood from a basic science point of view (Miller,

1992). However, in terms of practical applications, the use of microorganisms

in composting is studied from what can be called a macro or overall approach

(Davis et al., 1992a,b). Composting, as happens in other biological waste

treatment operations such as sewage treatment, anaerobic digestion, and the

like, is done by a large number of species, working in mixed culture conditions,

and in parallel and consecutive reactions. Therefore, the experimental

methodology employed implies working with mixed populations of micro-

organisms, most of them unknown at a given time, as they develop naturally in

the open environment of the composting operation. This does not exclude the

possibility of seeding with some microbial inoculums specially formulated for

the compost operation. Some authors indicate that inoculation or seeding has

been effective, however some negative results have been also reported

(Nakasaki et al., 1985b). These authors indicate that, in their research, no

clear difference in the overall rate of composting or in the quality of the

compost resulted by seeding.

If it is decided to accelerate the composting reaction by introducing micro-

organisms specialized in the degradation of a given waste material at the

beginning of the process, often the easiest way to accomplish this is to add some

amount of not totally matured (or uncured) compost from another composting

operation.

In vermicomposting, which will be discussed later in this chapter, the actions

of both worms and microorganims are complementary. Bacteria, fungi and

protozoa populate the digestive system of earthworms, and they contribute to

their diet.


23.6.2 Biodegradation of bulk materials

The use of lignocellulosic wastes or by-products as bulking agent will in general

prolong the complete decomposition of all of the materials in the composting

blend, as these bulking agents will degrade more slowly than other biological

wastes, such as those of animal origin. Also, phenolic compounds present in

lignocellulosic materials could inhibit some of the microorganisms present in the

compost. However, Mathur (1998) pointed out that the mixing of this kind of

materials with ammonia-generating wastes would result in the neutralization and

auto-oxidation of the phenolics, due to the ammonia released.

In the case of peat, several studies have been conducted on the use of some

peat hydrolysation products, such as peat extracts, in fermentation processes

(Manu-Tawiah and Martin, 1987; Martin and Chintalapati, 1989; Martin and

Bemister, 1994, Martin et al., 1993b; VaÂzquez and Martin, 1998, among others).

These studies could be a base for future work in understanding the potential

microbial degradation of peat in a composting operation.

23.7 Vermicomposting

Vermicomposting is the use of earthworms in the degradation and stabilization

of organic wastes. This use is based on the properties of some earthworm species

to quickly ingest organic wastes and break them into small particles. In general,

the same principles involved with traditional composting apply to vermi-

composting, although there are some specific characteristics that need to be

taken into consideration due to the presence of worms in the composting pile. A

historical overview of vermicomposting has been presented by Edwards (1995).

Vermicomposting happens in combination with the traditional composting

process based on the degradation of wastes by microorganisms; both processes

complement each other. The microbial population pre-digest the organic

materials upon which the worms feed, and the worms enhance the aerobic

conditions in the composting medium by tunnelling through it. Waste materials

at various degrees of decomposition from microbial action are ingested by the

worms, metabolized and expelled at a higher degree of degradation in the form

of particles identified as castings (Loehr et al., 1985). The presence of degrading

microorganisms in the earthworm digestive system should also be taken into

consideration (Satchell, 1983). Albanell et al. (1988) studied the chemical

changes happening during some vermicomposting processes, and Edwards and

Fletcher (1988) reported on the relationships between earthworms and

microorganisms in this process.

The parallel actions of microbial degradation and worm digestion act in a

mutually beneficial way. However, in spite of the potential advantages of this

symbiotic relationship, the presence of two mechanisms for biodegradation in a

close environment adds complexity to the overall design of the composting

process. For example, the optimum parameters for one mechanism may not

correspond to the optimum for the other, and the metabolites produced by one


type of degradation could be inhibitory to the other. Loehr et al. (1985)

discussed the factors affecting the vermicomposting operation, such as the

worms' requirement for aeration and the need to prevent toxic conditions.

The role of biological mechanisms in the degradation of the wastes in the

compost is also enhanced by the worm digestive enzymes that are present in the

worm excretions or casts (Edwards et al., 1995). Those enzymes, many of them

hydrolytic, continue to act after being expelled from the worm's body (Edwards

and Lofty, 1977).

Many earthworms are found in soils, and indeed some may be present at

times in any compost operation. In general, they thrive and reproduce abund-

antly in the presence of high concentrations of organic matter. An assessment of

earthworms and their relationships with soils was presented by Lee (1985).

Among the earthworm species suitable for vermicomposting, Eisenia fetida, also

known as the tiger or brandling worm (Edwards, 1995) has been found to

possess good attributes such as adaptation to a range of temperatures and water

content in the composting medium. Also, it becomes predominant in mixed

populations, and its metabolic rates are high (Edwards, 1988). Other important

species include Lumbricus rubellus (the red worm), Eudrilus eugeniae (the

African night crawler), Perionyx excavatus (Edwards, 1995), and Eisenia andrei

(Frederickson et al., 1997), among others.

The positive effect of earthworms on soil characteristics has been presented

in several studies (Lee, 1985; Blair et al., 1995; Edwards et al., 1995; Syers and

Springett, 1984; Tomlin et al., 1995). Similar effects also occur with the

compost produced with the aid of earthworms, resulting in the potential for

worms to produce compost with better growth improvement characteristics for

plants (Harris et al., 1991).

The main characteristics of a vermicomposting process and its final product

are presented in Table 23.6.

23.7.1 Vermicomposting process parameters

Aeration

Worms require air in the same way as the aerobic microoganisms involved in

composting do, therefore any improvement in the aerobic conditions of the

composting medium will be beneficial for both composting mechanisms, i.e.

microbial composting and vermicomposting. Indeed, the presence of worms

improves the aeration of the composting operation. Through their digestive action,

worms transform the waste material not only chemically but physically as well,

resulting in reduced sized porous particles with good water-holding capacity. The

presence of these kinds of smaller excreted particles, together with the tunnelling

and turning produced by the earthworms' presence and movement, could eliminate

the need for aeration technologies, either based on forced or passive principles.

This is valid unless the temperature increases to levels that could negatively affect

the worm population. In this case, it would be necessary to introduce aeration

mechanisms to help with the dissipation of the heat generated in the operation.


Chemical composition of the composting medium

Because of the high water content in the worm biomass, the water content

required by a vermicomposting operation is somewhat higher than in traditional

compost, in the range of approximately 70 to 90% moisture content. Dehydra-

tion of earthworms is a possibility if high temperatures and drying of the

medium happens during the composting operation. Also, earthworms exhibit

lesser ability than microorganisms to tolerate and adapt to adverse chemicals in

the medium (Edwards, 1995). In particular, earthworms are sensitive to the

Table 23.6 Main characteristics of the vermicomposting process

Factors Features Benefits Drawbacks References

Aeration Increased due to Enhanced waste Edwards (1995)worm action degradation

pH Affected by Moderate soil Blair et al.secretions from acidity (1995), Mishraworms'calciferous and Tiwariglands (1993)

Temperature Should be kept Pathogens Edwards (1995)below 35ëC are not

killed

Digestion of Increased due to Enhanced waste Neuhauser et al.organic worm action degradation (1988)materials

Casts excreted Supply hydrolytic Enzymatic action Loehr et al.by worms enzymes contributes to (1985), Edwards

waste degradation and Fletcher(1988), Mishraand Tiwari(1993), Edwardet al. (1995)

Final product Finely divided, Mishra andhumus material Tiwari (1993),

Tan (1996)

Improved soil Edwards andstructure, porosity Lofty (1977),

Lee (1985)

High water holding Edwards andcapacity Lofty (1977)

Increased soil Blair et al.respiration (1995)

Nutrients converted Syers andinto forms more Springettavailable to plants (1984), Berry

(1994), Edwards(1995)


presence of ammonia (Edwards, 1998). These characteristics could limit the

amount of organic matter that can be processed by vermicomposting, and could

restrict the use of this method.

Edwards and Bohlen (1995) reported on the relationships between earth-

worms and soil nutrients. Indeed, the spectrum of organic materials ingested by

earthworms is quite comprehensive, including decomposing animal and plant

residues. This makes vermicomposting a suitable step to be added to and

integrated with a process for the degradation of wastes of animal origin.

In addition to organic solid waste, earthworms have been used in the

stabilization of sewage sludge (Neuhauser et al., 1988)

pH

The pH of the composting medium controls the kind of earthworm species

inhabiting a particular environment, and the size of its population. It has been

reported that the optimum pH for E. fetida lies at a pH around neutrality

(Edwards, 1988). Moreover, the same author indicated that this earthworm tends

to migrate to acidic environments of pH 5.0. This could validate the use of some

acidic bulking agents such as peat in composting operations.

Temperature

An important difference between a traditional composting process and

vermicomposting is the range of temperatures at which the reactions take

place. While the former reaches temperatures in the thermophilic range, due to

the heat released by the microbial degradation reactions, the latter needs to adapt

to the viable temperatures for earthworms. It is reported that the maximum

temperature endured by earthworms is 35ëC, while their activity is optimum in

the range of 15 to 25ëC (Edwards, 1995).

The lower temperatures required for vermicomposting compared with those

for thermophilic microbial degradation results in the need to have some phasing

of the process, allowing an initial microbial composting or precomposting phase

without worms, where high temperatures will be able to degrade certain

thermosensitive compounds and inactivate pathogens (to simplify the operation,

this phase is not generally found in home vermicomposting). Afterwards, the

vermicomposting phase should be monitored to avoid increases of temperature

above those tolerated by the earthworms, resulting from high microbial activity.

The temperature can be kept at appropriate levels by ensuring good aeration

conditions to dissipate the heat generated by the microbial activity.

23.7.2 Vermicomposting of seafood wastes

Few studies have been conducted on the application of vermicomposting in the

treatment of solid wastes from the seafood industry. Decker et al. (2000)

pointed out the difficulties in using earthworms to decompose protein matter in

animal biomass wastes because of the potential toxicity to the worms of the

high concentration of ammonia liberated from protein degradation. A solution


to this problem, as indicated above, is to run a precomposting step until the

concentration of ammonia decreases to acceptable levels for the worms.

However, in both the precomposting and vermicomposting stages the nitrogen

to carbon ratio needs to be adjusted by the addition of materials high in carbon

to compensate for the high nitrogen concentration in the seafood waste

materials. This can be accomplished by the addition of, for example,

lignocellulosic by-products or wastes, or peat as bulk material. Mathur et al.

(1986) reported on the favourable characteristics of peat for this kind of

operation, such as high air to water ratio in freshly harvested peat, broad carbon

to nitrogen ratio, and suitable water, heat and odorous-gas retention properties

of peat. Decker (2000) cited these same properties as convenient in the

vermicomposting of seafood wastes. The main objectives of the research

reported by Decker (2000) can be summarized as:

(a) determination of the appropriate amount of peat to be added to a given

amount of fish offal for a vermicomposting operation,

(b) study of chemical changes in the vermicomposting medium, and

(c) determination of maximum concentration of ammonia tolerated by E. fetida

without compromising the survival of the earthworm.

Decker et al. (2000) reported that vermicomposting was an effective method

for stabilizing fresh fish offal, when composted with peat. The authors found

that the maximum amount of fish offal that allowed a 100% survival of Eisenia

fetida amounted to a 13% (dry weight) of the composting mixture, and that the

level of ammonium should be kept no higher than 1.0 ppm.

23.8 Quality considerations

The success of a composting operation can be considered and analyzed from

various points of view: economic, environmental, sociological, and

technological. The main factors in these analyses are:

(a) the elimination of waste materials and pollutant products, and

(b) the creation of a valuable product, the compost.

The first factor is dealt with in a straightforward way when the potentially

pollutant materials are incorporated into the composting operation, and it is

biodegraded as a result of the composting reactions. The second factor depends

on the possibility of marketing and using the compost produced, and is linked to

its quality.

The definition of the quality of a compost product is complex. Not only is the

quality of the compost a function of a number of biological, chemical and

physical factors (Table 23.7), but it will be relative to the raw material used in

the composting and the use intended for the final product. A classification of the

various grades of compost in relation to their intended use has been presented by

van der Werf (2004).


Some important compost characteristics, such as the stabilization of the

biodegradation activities and its degree of humification or maturity, will act as a

common denominator in defining its marketability (Golueke, 1977; Iglesias-

Jimenez and Perez-Garcia, 1989, 1992). These characteristics will be reached

during the last phase of the composting operation, known as curing or aging

(Jenkins, 1999). During curing, the compost should become mature. A mature

compost is the one that is ready to be applied to the soil. The final product

should not develop microbial activity after being added to the soil (which could

subtract nutrients such as nitrogen and oxygen, making them unavailable for

plant growth), should be free of toxic compounds, and be free of ammonia,

which should have been converted to nitrates (Haug, 1993). Additional

considerations for compost quality are the absence of pathogenic organisms

and weed seeds (Hayes et al., 1993). From the point of view of its use, if a

compost is not completely mature, it should at least be stable enough to ensure

that no nitrogen subtraction from the soil will result from its microbial activity.

Standard analytical methods can be applied to evaluate many quality

parameters of the compost. Vinceslas-Akpa and Loquet (1997) reported on the

application of chemical and spectroscopic analyses to find the chemical

composition of the composition mixtures, with the objective of determining the

transformation of organic matter in composting and vermicomposting processes.

Regarding toxicity, Mathur et al. (1986) suggested seed germination tests, which

can be particularly important in the determination of the presence of aliphatic

acids and phenolics (Devleeschauwer et al., 1981; Mathur 1991), which can

inhibit plant growth. To confirm the absence of plant inhibitors, seeds are soaked

with a water extract of the compost and their germination compared with seeds

soaked in pure water; the level of germination should be the same in both groups

if no inhibitors are present in the compost (Mathur et al., 1986). Also, Mathur

and Johnson (1987) reported on the use of tissue-culture tests for detecting

toxins in composts of peat, fisheries wastes and seaweeds.

The level of success of a composting operation can also be measured by the

degree to which the nutrient content of the degraded wastes has been preserved

Table 23.7 Main parameters determining quality in a compost product

Biological Chemical Physical

Absence of pathogens C/N ratio Effect on soil structureand weed seeds

Biological activity Gas generation (odour) Particle size

Maturity Macroelements content Presence of foreign objects

Stability Microelements content Water retention

Nutrient value

pH

Water content


in the final product. However, unless the compost is supplemented with nutrients

before being marketed, the nitrogen, phosphorus and potassium composition (N-

P-K, expressed in %), which is used to characterize fertilizer products, is

generally low. Nevertheless, probably the main value of compost application to

soils is reflected in the term `soil conditioner', which takes into consideration the

extent to which compost products enhance the physical structure of the soil and

characteristics such as water retention and slow release of nutrients (Gershuny

and Martin, 1992).

Reports about seafood waste compost indicate that the product is of high

quality with an earthy odour and good concentrations of organic and inorganic

nutrients (Mathur et al., 1986). As indicated before, shellfish wastes have also

been composted, and Hountin et al. (1995) reported the use of compost prepared

with shrimp wastes and peat on the growth of barley (Hordeum vulgare L.).

Decker (2000) reported that vermicomposting increased the stabilization of

organic matter when compared to experiments without earthworms.

23.9 Future trends

At present, in many areas of the world the use of appropriate technologies for

seafood wastes treatment or recycling is lacking. However, an increasing

awareness of the value of fisheries biomass and a willingness to take measures to

reduce pollution of marine environments should encourage the recovery of

seafood wastes.

Physical and chemical processes for the recovery of seafood wastes and the

production of acceptable products from them have been employed with limited

success. Therefore, it is generally accepted that bioconversion operations should

be applied to the processing of seafood wastes.

For small-scale seafood processing plants, the best option for using wastes is

the application of simple technologies resulting in products to be used as soil

conditioner, fertilizer or feeds. In this context, the biodegradation of seafood

wastes by composting has the advantages of being a low-cost operation. Andree

(1992) discussed practical approaches for the development of composting

applications to fisheries by-products.

As presented in this chapter, appropriate technologies have been designed

based on the study of the composting of seafood wastes with suitable bulk

materials, and on vermicomposting, which have resulted in effective methods

for stabilizing fresh fish offal (Martin and Decker, 2000). Therefore, it is

expected that in the future biodegradation processes, such as the production of

fish silage (Martin, 1996) and seafood waste composting, will be applied more

broadly, particularly in small-scale seafood producing plants, and most probably

in the developing countries.



Composting is a technology that is used across the world. Also, in recent years

interest has increased in many national and international institutions for

environmentally friendly ways to deal with wastes. Therefore, it is possible to

find in many countries both private and governmental agencies dealing with

composting, and many sources of technical and scientific information and

advice are available.

In this section, some sources will be mentioned; however, they do not

represent all the potential sources available. Further information about them can

be obtained by contacting them directly.

Composting Council of Canada

16 Northumberland Street

Toronto, ON M6H 1P7, Canada

Phones: (416) 535-0240; 1-877-571-GROW (4769)

Fax: (416) 536-9892


Web page: http://wwwcompost.org

The US Composting Council

4250 Veterans Memorial Highway, Suite 275

Holbrook, NY 11741, USA

Phone: 631-737-4931

Fax: 631-737-4939

Web page: http://www.compostingcouncil.org/index.cfm

European Compost Network Ecn/Orbit E.V.

Postbox 22 29

D-99403 Weimar, Germany

Phone: +49 (0) 25 22-96 03 41

Fax: +49 (0) 25 22-96 03 43


Web page: http://www.compostnetwork.info

The Composting Association, UK

Avon House

Tithe Barn Road, Wellingborough,

Northamptonshire, NN8 1DH UK

Phone: +44 (0) 870160 3270

Fax: +44 (0) 870160 3280


Web page: http://www.compost.org.uk

Also, institutions dealing with solid wastes will have interest in the development

of composting. One example is the:


Solid Waste Association of North America (SWANA)

1100 Wayne Ave, Suite 700

Silver Spring, MD 20910, USA

Postal Address:

P.O. Box 7219

Silver Spring, MD 20907-7219, USA

Phone: 1-800-GO-SWANA (467-9262)

Fax: (301) 589-7068


Web page: http://swana.org

In the specific area of composting of seafood wastes, The National Sea Grant

College Program of the United States, which includes a number of universities

conducting research and other programmes on the use and conservation of

aquatic resources, is a good source of expertise.

National Sea Grant Office, NOAA/Sea Grant, R/SG

1315 East-West Highway, SSMC-3, Eleventh Floor

Silver Spring, MD 20910, USA

Phone: 301-713-2431

Fax: 301-713-0799

Web page: http://www.nsgo.seagrant.org

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ACE activity inhibition 240±1acetic acid 161±2, 332±3acetolysis 351±2acetyl CoA 414acetylation 347±8deacetylation 345±7degree of 346±7

acid-aided method 75±84, 152±61,199±200, 205±6, 215±16

acid ensilage method 419acid hydrolysis 120, 148±9, 441±2preparation of chitin and chitosan 345,

350±3acid method for collagen 129±31, 283±4,

332±3acid method for gelatin 285±7, 298,

332±3acid-soluble collagen (ASC) 129±31, 132,

282, 283±4acidic processes for protein recovery

161±2see also acid-aided method

acidification 56, 57acidity adjustment 362±3acidolysis 114actin 70degradation and temperature 213

actinidin 121activated charcoal 299active aeration 495aeration 490±2, 493

active 495passive 495±6PAWS 497±501vermicomposting 504, 505

aesthetic deterioration 96affinity chromatography 384agriculture 364alanine 324collagen and gelatin 288, 289, 290,

291, 292, 293, 294Alaska 4, 435, 461Alcalase 123, 124, 125±7, 232alcoholysis (transesterification) 114,

465±7aldehydes 31algaeantioxidants from marine algae

398±403biomass added to composting

operations 495pigments from 424±5, 427, 428

algal oil 268±9, 270alginate 86±7alkali-aided method 75±84, 152±61,

199±200, 205±6, 215±16alkali-catalysed transesterification 466±7,

476alkaline hydrolysis 120, 149alkaline pretreatment 282±3alkaline process for gelatin 285, 286,

287, 298

Index

alkaline processes for protein recovery162

see also alkali-aided methodalkylation 348±9�-carotene 415, 416�-linolenic acid (ALA) 23, 259�-tocopherol 29±30, 32±3amino acids 12antioxidant properties 404collagen and gelatincomposition 288, 289, 290, 291,292, 293, 294

nutritional properties 292±3recommended pattern for childrenand infants and 292, 295

content of fish mince/surimi 216±17essential 80±2hydrophilic 311±12, 320, 321, 323hydrophobic 319, 320, 321, 323seafood flavouramino acid compensation ofhydrolysate 311±12

clam 318lobster 309±10, 311±12, 319±20red hake 320

taste active free amino acids 318ammonia 479vermicomposting and 505±7

ammonium sulphate 382±3amphiphiles 464anaerobic contact reactor (ACR) 468, 469anaerobic digestion 467±71, 479±80problems of bio-gas production 477±9

anaerobic filter 468, 469, 478±9anaerobic phased solids digester

(APS-Digester) 470±1anaerobic sequencing batch reactor 468,

474, 475anchovy 260angiogenesis inhibition 453animal feeds 66, 239±40, 435±49antifungal activity 359±60, 362antimicrobial activity 358±60antioxidants 11, 32, 34, 35, 250, 397±412chitosan 363, 406±7FPH 237±9from marine algae 398±403from marine animals and their

by-products 404±7from other marine sources 407±8

appetite suppression 451, 452aquaculture xxi, 17, 34±5, 61, 88, 145,

171feeds 17, 435±49

FPH 239±40, 443global production 66, 67, 374, 450

aqueous extraction 305arachidonic acid 23argentine 178arginine 456ash 462±3aspartic proteinases 118astaxanthin 134antioxidant 400, 408pigment 416, 418, 420, 421, 422, 427

ATPase activity 161autolysisand FPH 14±15, 118, 120, 231spoilage 48±9, 53, 56see also fish sauce; fish silage

avrainvilleol 402

Baader flesh-bone separator 94±5, 98backbones 4, 5, 6, 7, 8removal prior to flesh-bone separation

99bacteria 408see also microorganisms

baffle flow reactor 469bandages 355batch digester with leachate recirculation

469, 470batch reactor 469, 474beef plasma protein (BPP) 82belly flap/trimmings 186belt and drum flesh-bone separators 93,

94±5, 98, 201�-carotene 415, 416�-N-acetylhexosaminidase 379binary ice (slurry ice) 177bioactive compounds 450±9collagen 452±3elastin 454, 456FPH 128±9future trends 456protamine 455±6protein powder as bioactive ingredient

250proteoglycans 454±5squalamine 451±2, 456see also health; medical applications;

pharmaceutical applicationsbiodegradation of wastes 487±90, 491see also composting

bio-diesel 460±85future research needs 479±80problems concerning production of

476±7

Index 517

quality and quantity of seafoodprocessing by-products 461±3

theories and technologies for producing463±7

yields and properties 471±3bio-gas 460±85future research needs 479±80problems concerning production of

477±9quality and quantity of seafood

processing by-products 461±3theories and technologies for producing

467±71yields and properties 473±6

biological oxygen demand (BOD) 464biological quality of compost 507±8biological separation techniques 92biological value (BV) 69biomass-retaining digesters 468biotechnology 387±8bitterness 236, 308, 315black caviar 182black scabbard fish 174±5`blackspot' formation 425bleaching 269±71blood pressure 240±1blood thinning 240bloom strength 289±90blubber oils 265, 267±8, 271±2blue-green algae 424body weight control 355bone meal 440bones 108, 109, 187±8backbones see backbonescalcium from 330±6

biochemical properties 330±1calcium and phosphorus composition330±1

calcium solubilisation using fishbone peptide 333±5

degradation of bone 331±3in vivo availability of solublecalcium complex 335±6

utilisation of calcium and organiccompound 331±5

enzymatic extraction of collagen129±31, 132

flesh-bone separators 92, 93±5, 98, 99,198, 201, 207

products and feeds 439botargo 183±4bovine spongiform encephalopathy (BSE)

280, 450, 453boxes/boxing, fish 176

branched (cyclic) fatty acids 23bromelain 121bromophenols 401, 402bulking agents 494, 503PAWS 497, 498±501vermicomposting 507

butylated hydroxyanisole (BHA) 34, 250,397

butylated hydroxytoluene (BHT) 34, 250,397

by-catch 107, 144±5, 171±9, 189±90discards, by-products and 171±3using 174±9

By-Catch Bank 173by-products 304±5by-catch, discards and 171±3components and manufacture of feeds

437±8composition of fish by-products 3, 4,

462±3and their applications xxi±xxii

definition 108estimate of available quantities 108±9,

437driving forces for utilisation of 436±7general scheme for utilisation of

by-products 180quantity and quality 461±3and their possible use 109see also waste

calcium xxii, xxiii±xxiv, 188, 328±39biochemical properties of fish bone

330±1in vivo availability of soluble calcium

complex from fish bone 335±6industrial uses 328recommended intake 328±9sources in foods 329utilisation of fish bone calcium and

organic compound 331±5calcium-fortified foods 329, 335±6calcium phosphate 329cancer 451, 454canthaxanthin 416, 418, 420, 421capelinoil 261, 262protein hydrolysates 404±5roe 184

capsules 297capture fisheries, global xxi, 3, 26, 47,

66, 67, 171carbon dioxide 467see also bio-gas

518 Index

carbon to nitrogen ratio 492, 493cardiovascular disease 272Carophyll Pink 420, 421Carophyll Red 420carotenoids xxii, xxiii, 399±400, 414±22,

426±8comparison with synthetic colorants

420±2measurement of colour 422preparation and stabilisation 419properties and functions 417±18uses in food and feed products 420

carotenoproteins xxiii, 133±4carpcultured 5, 6, 7, 13osteocalcin 330, 334±5wild 5, 6, 7, 13

cartilage 131±3, 453cartilaginous fish 292casein phosphopeptides (CPP) 333±6Casson equation 252catalase 101catching methods 61catfish oil 263, 264cattle 293caviar 181±2production using marine enzymes 386see also roe

cellulose 340, 341, 342cellulose acetate 382centrifugation 76±8, 155±6, 299decanter centrifuges 84±5, 203, 206

`champagne' method 177characterisation 16±17cheeks 92±3using 179±81

chemical hydrolysis 119, 120, 148±9see also acid hydrolysis

chemical oxygen demand (COD) 464chemical preservation 53, 55±6chemical processing methods 92, 144±67

chemical hydrolysis 119, 120, 148±9see also acid hydrolysis

fish protein concentrate 146±8fish protein isolates 75±84, 152±62,

163, 199±200, 205±6, 215±16future trends 162±3surimi processing see surimi

processingchemical quality of compost 507±8chilling 52±5, 176±7chitin xxii, xxiii±xxiv, 133±4, 340±73applications of chitin and its oligomers

340±1, 344, 353±64

agricultural 344, 363food 344, 357±63industrial 344, 364medical 344, 354±7

and composting 494±5enzymatic hydrolysis and preparation

of chitin and its oligomers 350±2N-alkylation for improved solubility

348±9production 345±6safety and regulatory status 364±5structure and properties 341±5trapping-retention of heavy metals

349±50chitinase 379chitinolytic enzymes 379chitosan xxii, xxiii±xxiv, 133±4, 340±73antioxidant 363, 406±7applications of chitosan and its

oligomers 340±1, 344, 353±64agricultural 344, 363food 344, 357±63industrial 344, 364medical 344, 354±7

coatings see coatingscomb-shaped 348deacetylation and molecular weight

and its activity 346±7depolymerisation and N-acetylation

347±8enzymatic hydrolysis and preparation

of chitosan and its oligomers352±3

production 345±6safety and regulatory status 364±5structure and properties 341±5trapping-retention of heavy metals

349±50chitosan-alginate treatment 86±7chitosan-oxychitin 357chlorella 423±4chlorophylls 399, 414, 422±4cholesterol 156, 241chondroitin sulphate (CS) 131±3chondroitins xxivchromatin 444chromatography 384chymotrypsin 14, 387citric acid 332±3clam flavour production 316±19clarification agents 295, 297Clostridium botulinum 222coastal fleet 47, 49±50see also on-board handling

Index 519

coatingschitosan edible coatings 361, 406±7chitosan-oxychitin for prosthetic

articles 357cod 5, 6, 7, 12±13, 290, 295composition of by-products 463enzymes from 377, 384, 386oil from 260, 264

coffee 362±3cold-adapted enzymes 375cold-water fish 281, 288, 290collagen 8±9, 188, 279±303, 330enzymatic extraction of collagen and

collagen-derived products 129±31,132

food applications 295gelatinisation into fish glue 15key drivers of marine collagen 279±81manufacture 281±5

stabilising 287medical and pharmaceutical

applications 452±3properties 288±95

chemical 288nutritional 292±5physical 288±91

quality improvement 298±9sources of marine collagen 281

collagenolytic enzymes 377±8colorants see pigmentscolourfish mince 97, 98

masking colour 103whitening methods 99±103

frame mince 209measurement of 422protein isolates and surimi 157±8

comb-shaped chitosan derivatives 348composting 486±515biodegradation of seafood wastes

487±8biological aspects 501±3characteristics of composting of

seafood wastes 494±5future trends 509limitations of traditional methods 495operational parameters 490±3quality considerations 507±9technological aspects 495±501vermicomposting 502, 503±7

computer vision equipment 52, 175±6concentrationmarine enzymes 381±3and seafood flavour 306

quality 322±4connective tissue (stroma proteins) 70consistency index 252consumers 196safety concerns and collagen and

gelatin 280contamination 428continuously stirred tank reactor 469cooking juice 305, 321cook loss 234±6coral 407Corolase 232coronary heart disease 24cosmetic products 450±9covering compost 496crab flavour 321crab sticks, flakes and chunks 149±50,

220±2crustaceans see shellfishcryoprotectants 33, 97cryoprotective properties of FPH 236surimi 150, 151±2, 199, 218

cryptoxanthin 415±16, 417CSW (chilled sea water) 54, 55, 176±7culture media 242curcudiol 408curcuphenol 408cut-offs (trimmings) 4, 5, 6, 7, 8, 186cuttlefish 294cymopol 402cysteine 71cysteine proteinases 118cystoketal 402Cystoseira 398, 399, 401, 402

dairy products 296, 329dark muscle 70, 73, 211±12surimi from 152

deacetylation 345±7decanter centrifuges 84±5, 203, 206degree of acetylation (DA) 346±7degree of hydrolysis (DH) 127, 232±3,

321±2antioxidative properties of FPH 238

degumming 269dehydration see dryingdemersal species 98demineralisation 283denaturationenzymes in enzymatic hydrolysis 233proteins 161protein powders 254±5

denaturation temperature 131, 132deodorising 271

520 Index

depolymerisation 347±8dermatan sulphate (DS) 454deskinning 386deteriorationlipids 30±2see also oxidation of lipids

marine biomass 47±9, 53dewatering 151, 199diesel 472, 473see also bio-diesel

diet (of fish)and fatty acid composition 28±30modification and fish quality 32±3see also feeds

Digestase 386digestive system 2403,4-dimethoxyphenol 408dimethylsulphoniopropionate (DMSP)

4031,1-diphenyl-2-picrylhydrazyl (DPPH)

408discards 171±3see also by±catch; underutilised species

disulphide bond 71docosahexaenoic acid (DHA) 22, 23, 24,

25, 110, 259±61, 272docosapentaenoic acid (DPA) 24, 259±61,

271±2dogfish 292Dranco digester 470dried fish maws 185±6dried heads 58drying 53, 55drying methods and seafood flavour

quality 322±4freeze-drying 322±4, 383gelatin 287low temperature (LT) drying 439

DSC thermograms 254±5

earthworms see vermicompostingeconomic factors 17collagen and gelatin 280±1pigments 426±7

eicosanoids 23eicosapentaenoic acid (EPA) 22, 23, 24,

25, 110, 259±61, 271, 272Eisenia fetida 504elastin 454, 456emulsification properties 237emulsions containing protein powders

251±4flow properties analysis 251±2viscoelastic properties 253±4

endogenous enzymes see autolysisendopeptidase activity 231±2endoproteases 118, 231±2, 306±8environmentdisposal of waste at sea 174impact of PAWS composting 500±1pigments and 426±7, 428

enzymatic hydrolysis 10±11chitin and chitosan 345, 350±3for FPH 118±29, 135, 230±3, 442lipids 31, 111±18, 135seafood flavour manufacture 306±8cooking juice vs hydrolysate in

lobster and crab flavourproduction 321

hydrolysis conditions 321±2, 323seafood flavours from processing

by-products 308±19source of enzymes 307±8source of raw material 308

enzymatic methods 107±43bio-diesel production 476±7by-products extracted by 108±9collagen manufacturing 129±31, 132,

282, 285extraction of antioxidants 403future trends 135±6shellfish industry by-products 133±4skin, bones, fin, scales, cartilage 129±33TGase modification and gelatin 298±9traceability of by-products 134±5see also enzymatic hydrolysis

enzymesand composting 502enzymatic activities in protein-rich

by-products 13±16enzymatic spoilage 48±9, 53, 56marine enzymes see marine enzymes

Escherichia coli (E. coli) 359essential amino acids 80±2esterification 111, 113±18ethanol 146, 147, 148ethylenediaminetetraacetic acid (EDTA)

283European Union (EU) regulations 174exopeptidases 118, 231±2, 306±8

fatty acid methyl esters see bio-dieselfatty acids 22±4, 49composition in selected marine

organisms 260, 261factors affecting fatty acid composition

of fish and by-products 28±30in fish by-products 26±7

Index 521

in fish muscle 25±6marine oils 266

algal oils 269, 270marine mammal oils 266±8

monounsaturated 23, 266, 271omega-3 xxii, xxiii, 22, 23, 75,

258±61, 444omega-6 23, 258±9PUFAs see polyunsaturated fatty acidsunsaturated 23±4, 403see also fish oils; lipids; marine oils

feeds 17, 388, 435±49benefits of feeds made from seafood

processing wastes 443±4by-product components 437±8driving forces for utilisation of

by-products 436±7feed products from fish by-products

438±41FPH 239±40future trends 444±5methods of producing hydrosylates and

silage 441±2pigments in 420, 426±7

fermentation 305±6production of hydrolysates 442using marine enzymes 385

fertilisers 66, 486FPH 241±2spreading waste 489, 491

fillet 108, 109fillet blocks 96filleting waste 91±8by-products from 92, 145recovery of flesh from 92±8

filtration 299membrane filtration 206, 381±2microfiltration 207, 208, 209±10

finfish 304, 308fins 129±33, 188fish bone peptides (FBP)calcium solubilisation using 333±5in vivo effects on calcium

bioavailability 335±6fish frame protein see frame mincefish mince 8, 58, 92, 95±104, 150,

196±228advantages and disadvantages 198by-products 204±5functional properties 210±16future trends 103±4machinery for preparation 200±4manufacturing 197±8nutritional characteristics 216±17

problems of 97±8protein recovery methods 98, 205±7quality and improvement 99±103storage stability 217±19comparison with surimi 218±19factors affecting 217±18mince from surimi by-products 219

use in surimi 210utilisation 219±23

fish oil by-products 475±6fish oils 26, 33, 57, 58, 73±5, 461,

462±3characteristics 462enzymatic extraction methods 109±18,

135feeds 435, 436±7, 439±40, 443±4health effects 24±5, 109±11from isoelectric precipitation and

solubilisation 76±8, 80, 155±6manufacturing process 269±71from processing by-products 261±5see also lipids; marine oils

fish protein concentrates (FPC) 11,146±8, 490, 491

fish protein hydrolysates (FPH) xxii±xxiii,10±11, 146, 229±48, 250, 256,490, 491

antioxidants 404±6feeds 239±40, 443FPC as substrate for making 148future trends 242±3methods of production 441±2autolysis 14±15, 118, 120, 231enzymatic hydrolysis 118±29, 135,230±3, 442

on-board processing 56±8physiological role in humans and

animals 239±41properties 128±9, 234biological activities 128±9functional properties 128

role in food systems 234±9role as growth media for

microorganisms 242role in plant growth and propagation

241±2using marine enzymes 385±6

fish protein isolates 152±62, 163other processes using low or high pH

161±2pH-shift processing 75±84, 152±61,

199±200, 205±6, 215±16fish sauce 10, 58, 118, 188±9, 231, 305,

385

522 Index

fish silage 9±10, 56, 57, 118, 441±2, 486,490, 491

fish solubles 438, 440Fishbase 59fishing regions 28±30fishmeal 58, 91, 338, 489±90, 491and feeds 435, 436±7, 438±9, 443, 444

flavour, seafood see seafood flavourflavour enhancers 324Flavourzyme 122, 232, 307±8, 314seafood flavourclam flavour production 317±19hydrolysis conditions 321±2, 323lobster flavour extracts 309±10, 311

flesh-bone separators 92, 93±5, 98, 198,201, 207

removal of backbone prior toseparation 99

flesh recovery 91±106from demersal species 98from filleting waste 92±8future trends 103±4quality and improvement of fish mince

99±103flocculants 84, 85, 86flow behaviour index 252flow properties analysis 251±2fluidised-bed reactor 468, 469fluorohydrolysis 352Food and Environment Protection Act

1985 (UK) 174food preservation methods see

preservation methodsfood supplements 250chitosan 355±6collagen and gelatin 293±5FPH 241, 243

food systems, role of FPH in 234±9fractionation 383±4frame mince 187, 206±7characteristics 209utilisation 210

frames 4, 5, 6, 7, 8, 487fish flavour from white fish frames

313±15using 187±8

frankfurter analogue 223freeze-dryingflavour products 322±4marine enzymes 383

freezing/freeze-storage 13, 33, 53, 178frozen mince blocks 96stability of fish mince/surimi 217±19surimi 203±4

freshness 212±13freshwater fish 23±4fruit, preservation of 362fruit juices 362fucolls 400±1fucosylated chondroitin sulphate (FucCS)

455fucoxanthin 414, 418fuel oil 462see also bio-diesel

fuhalols 400±1functional foods 249, 256functional propertiesalkali-aided and acid-aided processes

158±61enzymatically hydrolysed proteins 128fish mince/surimi 210±16protein powders 249±57protein-rich by-products 11±16

fungal protease 122

Gadidae species 5, 6, 7gadiform speciesby-products generated during filleting

50, 51utilisation of by-products from 59±61

-carotene 415, 416gear selectivity 175, 189gel formationfish mince/surimi 211gelatin 288±91marine enzymes 386±7pH-shift processes 158±61

gel strength 289±90gelatin 8±9, 15, 129, 188, 279±303, 332±3

food applications 295±7key drivers of marine gelatin 279±81manufacture 281±2, 285±7stabilising 287

non-food applications 297properties 288±95chemical 288nutritional 292±5physical 288±91

quality improvement 298±9sources of marine gelatin 281

gelatin desserts 295±6genetic engineering 456genetic identification method 134±5, 136global catch xxi, 3, 26, 47, 66, 67, 171global fisheries production 66±8, 171,

172, 374glucosamine xxivglutamic acid 318±19, 324

Index 523

glycine 319, 324collagen and gelatin 288, 289, 290,

291, 292, 293, 294grading 175±6Grateloupia filicina 398greater sand eel 178gummy-type confectionery products

296±7gutting 16, 50±1

habitat 11±16haddock 5, 6, 7haem proteins 157, 211±12haemoglobin 239hagfish 292hake 290red hake flavour 313±15, 320

halal food certification 280harp seal 293header/gutter machine 200heads 4, 6, 7, 109dried heads 58fatty acids 27using 179±81

healthFPH and 240±1, 250lipids and 24±5, 109±11pigments and 417±18, 423, 424proteins and 250PUFAs and 24±5, 271±2

health effects and PUFA sources109±11

see also medical applicationsheat treatment 53, 55heavy metals 428chitin, chitosan and

heavy-metal complexes 343trapping-retention 349±50water purification 364

heparin 354, 455herringoil 263, 265roe 184

high-pressure treatment 99Hizikia fusiformis 398homogenisation 76hot-water fish 281, 288, 291hurdle technology 55±6hybrid sludge-bed filter (HSBF) reactor

475hydraulic retention time (HRT) 475hydrocarbons 266hydrochloric acid 283hydrocolloids 97

hydrogen bonds 69, 70±1hydrogen peroxide 99±101, 102hydrolysates see fish protein hydrolysates

(FPH); protein hydrolysateshydrolysation plant 58±9hydrolysischemical 119, 120, 148±9see also acid hydrolysis

degree of 127, 232±3, 238, 321±2enzymatic see enzymatic hydrolysis

hydrolysis conditions 321±2, 323hydrophilic amino acids 311±12, 320,

321, 323hydrophilic colloids 101hydrophobic amino acids 319, 320, 321,

323hydroxyapatite (HA) crystals 3307-hydroxycymopol 402hydroxyproline 288, 289, 290, 291, 292,

293, 294hypercholesterolemic activity 356hyperoxidase (catalase) 101

ice/icing 176, 177ice-water fish 281, 288, 289Iceland 4, 174By-Catch Bank 173Individual Quota System 173

imaging technology 52, 175±6immune system 240impurity removal 299individual variations 11±16indoles 403, 408industrial wastewater treatment 364inoculation (seeding) 502inoculum 477±9interesterification 114interfacial/surface properties 236±7invertebratesantioxidants from 407±8collagen and gelatin from 281, 288,

294iodine 428ion exchange chromatography 299, 383±4ionic strength (IS) 70isoelectric point 72, 73isoelectric precipitation and solubilisation

75±84, 152±61equipment considerations 82±4

isopentenyl pyrophosphate (IPP) 414, 415isopropanol 146, 147

jawless fish 292jellyfish 294

524 Index

kamaboko 220kazunoko 184kazunoko kombu 184kelp 184ketones 314-ketozeaxanthin 408Kjeldahl method 127Kojizyme 122Kompogas digester 470kosher food certification 280krill 67, 68, 260

processing 65proteases 377, 388proteins recovered from 80±2

land, discarding of wastes on 489, 491landfilling 489, 491leachates 500lenses 425±6ling 5, 6, 7linolenic acid 23, 24lipases 48±9, 378±9lipase-catalysed hydrolysis and

esterification 111, 113±18lipase-catalysed transesterification 476

lipids xxii, xxiii, 4, 22±46, 211composition of fish wastes 462, 463,

464deterioration 30±2enzymatic extraction 31, 109±18, 135concentration of n±3 PUFAs 111lipase-catalysed hydrolysis and

esterification 111, 113±18protease-catalysed hydrolysis111±13, 115

factors affecting fatty acid compositionof fish and by-products 28±30

fatty acids found in fish by-products26±7

fatty acids found in fish muscle 25±6future trends 34±5health benefits 24±5, 109±11, 271±2implications for fish fat by-product

valorisation 32±4mincing and degradation of 95±6, 97oxidation see oxidation of lipidsproperties of lipids in seafood 73±5recovery in acid- and alkali-aided

processes 76±8, 80, 155±6see also fish oils; marine oils;

polyunsaturated fatty acids(PUFAs)

lipolysis 31, 48±9liquefaction 306

liver oils 186±7, 266livers 27, 186±7livestock feeds 66, 239±40, 435±49lobster flavour extracts 309±13cooking juice vs hydrolysate 321flavour-imparting compounds and

chemistry 319±20loss modulus 253±4low temperature (LT) drying 439lumpfish 290caviar 183

lungfish 291lutein 416, 417lycopene 415lyophilisation (freeze±drying) 322±4, 383lysine 82

maatjes 385mackerel 291crude proteinases from mackerel

intestine 122macular degeneration 451mammals, marine see marine mammalsmarine-derived tocopherol (MDT) 29±30marine enzymes 374±96chitinolytic 379collagenolytic 378future trends 388±9lipases 378±9polyphenoloxidases 380±1producing from seafood processing

by-products 381±4concentration 381±3extraction/solubilisation 381fractionation/purification 383±4

proteolytic xxiii, 11, 14, 376±7transglutaminases 379±80, 386±7utilisation of 375, 384±8

marine mammalscollagen and gelatin 281, 288, 293oils from 265±8health effects 271±2

marine oils 258±78algal oil 268±9, 270fish oils see fish oilshealth effects 24±5, 109±11, 271±2manufacturing process 269±71marine mammal oils 265±8, 271±2from processing by-products 261±5see also fish oils; lipids;

polyunsaturated fatty acids(PUFAs)

marshmallows 296±7masking colour 101±3

Index 525

maws 185±6MaxFish 60meatpreservation and chitosan 361tenderisation using marine enzymes

387mechanical aeration devices 496mechanical separation see flesh-bone

separatorsmechanised fish filleting 65medical applicationsbioactive compounds 450±9chitin and chitosan 344, 354±7FPH 240±1marine enzymes 387, 388see also health

Mediterranean caviar 183±4megrim 291Melancor-NH 426melanins 414, 425±6melting point 290membrane disruption 381membrane filtration 206, 382menhaden oil 260, 261, 262menopause 335mentaiko 183mercaptan derivatives 349±50mesophilic temperature range 468metallocollagenases 377metalloproteinases 118methane 467problems of bio-gas production 477±9see also bio-gas

methanol 465±6supercritical methanol method 476±7

microemulsions 464±5microfiltration 207, 208, 209±10microorganismsalgal oil 268±9, 270antimicrobial activity of chitin and

chitosan 358±60antioxidants from marine bacteria 408chitosan and preservation of food

360±2FPH as growth medium 242growth/activity and deterioration of

seafood 48, 53, 56microbial fermentation for hydrolysates

442pigments from algae 424±5, 427, 428role in composting 501±2

milk 329milt 4, 6, 7, 9, 185minced fish see fish mince

minerals xxii, xxiii±xxiv, 214content of fish mince/surimi 216

minke whale 293modified atmosphere packaging 33molecular weight 347monosodium glutamate (MSG) 324monounsaturated fatty acids (MUFAs)

23marine mammal oils 266, 271

MSI-1436 451±2muscle 69±70, 73fatty acids in 25±6, 27isoelectric precipitation and

solubilisation of muscle proteins75±84

myofibrillar proteins 70, 211solubility 78, 79

myogen-aggregation phenomenon 205myosin 70, 207, 208degradation and washing cycles 213

N-acetylation 347±8N-alkylation 348±9National Environmental Law Center

(NELC) (US) 86natural savoury flavours 324neurological system 240neutralisation 269Neutrase 124, 126Newlase A 122, 126nitric oxide 455nitrogen 492, 493nitrogen, phosphorus and potassium

(NPK) composition 508±9Norway 3±4regulation of disposal of waste at sea

174Norway pout 178Norwegian reference meal 439nucleotides 318±19, 444nutrients, soil 364, 506nutritionfeeds made from seafood processing

wastes 443±4properties of collagen and gelatin

292±5properties of fish mince/surimi 216±17

o-phtaldehyde (OPA) method 127obesity 451ocean disposal 173±4, 460±1, 486, 487ocean trawlers 47, 50±2see also on-board handling

octopus roe 185

526 Index

odour problems 489, 491, 496, 500off-flavour 363oleaginous microorganisms 268±9, 270oleic acid 23oligomers, chitin and chitosan 340±1applications 344, 353±64preparation 350±3

omega-3 fatty acids xxii, xxiii, 22, 23,75, 258±61, 444

see also marine oilsomega-6 fatty acids 23, 258±9Òmega Bread' 25on-board handling 16, 32, 47±64, 173±4,

189±90conservation and stabilisation 52±6deterioration of marine biomass 47±9future trends 61processing 53, 56±9and sorting 16, 49±52utilisation of by-products from

gadiform species 59±61waste from 173±4

optimisation of extraction process 298Oregon seafood plant 86organic solvent extraction process 419ornithine decarboxylase (ODC) 455osmometry 127osteocalcin 330, 334±5osteoporosis 328, 335±6ovariectomised rats 335±6overfishing 144oxidation of lipids 30±2, 35, 95±6, 97,

115±18, 156±7, 397antioxidants see antioxidantsdeterioration of marine biomass 49, 53,

56implications for by-product valorisation

32±4oyster 260

packaging 33±4papain 121, 125particle settling velocity 83±4particle size 84, 85, 86passive aeration composting 495±6passively aerated windrow system

(PAWS) 497±501application to composting of seafood

wastes 497±8environmental impact 500±1peat as a bulking agent 497, 498±500,

501pastilles 297peat 497, 498±500, 501, 503, 507

pelagic species 144, 145Penzim 387, 389pepsin soluble collagen (PSC) 129±31,

132, 282, 285pepsins 121, 376, 386peptides 48, 251fish bone peptides 333±6peptide size and characterisation of

hydrolysates 127±8pHchemical processing methods for

protein recovery 161±2see also pH-shift processing

and composting 493vermicomposting 505, 506

and protein gel texture 79±80solubility of fish mince 200wash water and surimi/fish mince

properties 214pH-shift processing 75±84, 152±61,

199±200, 205±6, 215±16pH-stat technique 127pharmaceutical applicationsbioactive compounds 450±9gelatin 297see also medical applications

phenolic compounds 400±1, 402, 500phlorethols 400±1phlorotannins 400±1phosphate 218phospholipases 378phospholipids 31, 73±5, 156phosphorus 508±9in fish bones 330±1

photography 297PhotoProtective Technologies 425±6phycocyanin 424±5phycoerythrin 424±5physical quality of compost 507±8phytoene 414±15pigments 413±32carotenoids 414±22chlorophylls 414, 422±4economic, environmental and safety

considerations 426±7future trends 427±8melanins 425±6phycocyanin and phycoerythrin 424±5types and sources 414

pigs 444gelatin from skin of 293, 295

plantsantioxidants from 397, 398growth and propagation 241±2

Index 527

pollack 290, 405±6, 463polyphenoloxidases 380±1polyphenols 400polyphosphates 101polysaccharides 403polyunsaturated fatty acids (PUFAs)

23±5, 26, 29, 31concentration of n±3 PUFA 111health benefits 24±5, 109±11, 271±2marine oils 258±78

algal oil 268±9fish oils 261±5marine mammal oils 266±7

see also fish oils; lipids; marine oilspotassium 508±9potatoes 362power law 252prawns 319precipitation 78enzyme concentration by 382±3isoelectric precipitation and

solubilisation 75±84, 152±61preservation methods 16chitin, chitosan and 360±2on-board 52±6, 176±8

pretreatments, in collagen manufacture282±3

processing 66±7chemical processing methods see

chemical processing methodson-board 53, 56±9physical methods and flesh recovery

92±8recovery of by-products from seafood

processing streams 65±90isoelectric precipitation andsolubilisation 75±84, 152±61

protein recovery from surimiprocessing water 84±7, 145±6,149±52, 154±5, 205±6, 209±10

profitability analyses 60proline 324collagen and gelatin 288, 289, 290,

291, 292, 293, 294propanal 406, 407propyl gallate (PG) 34, 397Protamex 124, 307protamine 11medical and pharmaceutical

applications 455±6proteases 107, 118, 332enzymatic hydrolysis using added

proteases 119±27marine enzymes xxiii, 11, 14, 376±7

protease-catalysed hydrolysis 111±13,115

protein hydrolysatesantioxidants 404±6fish protein hydrolysates (FPH) see

fish protein hydrolysatesprotein powders 249±57, 440±1as bioactive ingredients 250emulsions containing 251±5flow properties analysis 251±2viscoelastic properties 253±4

functional properties 250±1future trends 256thermal properties 254±5

protein±protein interactions 71±3proteins xxii±xxiii, 3±21, 69±73, 211

biological activities of enzymaticallyhydrolysed proteins 128±9

characteristics of by-products fromsurimi wash water 207±8

chemical processing methods 144±67content in by-product fractions 5±8enzymatic extraction methods 118±29,

135, 230±3, 442autolysis vs enzymatic hydrolysis118

enzymatic hydrolysis with addedenzymes 119±27

quantification of proteolysis extent127±8

extraction of carotenoid pigments using419

fish frame protein 206±7fish protein concentrates (FPC) 11,

146±8, 490, 491fish protein hydrolysates (FPH) see

fish protein hydrolysatesfish protein isolates see fish protein

isolatesin fish wastes 462±3functional properties of enzymatically

hydrolysed proteins 128isoelectric precipitation and

solubilisation 75±84, 152±61mincing and 95, 97protein-rich by-products 3±21future trends 17implications for by-products

valorisation 16±17overview 4±11physical and chemical properties

11±16recovery from surimi processing water

84±7, 205±6

528 Index

water-insoluble proteins 209±10Protemax 232proteoglycans 454±5proteolysis 48, 160

marine proteolytic enzymes xxiii, 11,14, 376±7

proteolytic enzyme inhibitors 152quantification of proteolysis extent

127±8psychrophilic temperature range 468puffer fish 291purification 383±4pyrolysis 464±5pyruvate 414

qualityof compost products 507±9drying methods and quality of seafood

flavour 322±4fish mince and 99±103improvement for collagen and gelatin

298±9

rancidity 30±4, 35, 363red algae 424red caviar 182red fish 175red hake, flavour from 313±15, 320refiner 202refrigeration technology 52±5see also chilling; freezing/freeze

storageregulationsregulatory status of chitin and chitosan

364±5waste disposal 174, 426, 436±7, 460±1

response surface regression (RSREG)126

ribonucleotides 324rigor mortis 213roe 4, 6, 7, 8, 9, 12production using marine enzymes 386using 181±5

rosemary 34rotary evaporated flavour products 322±4rotary screen dehydrator 202rotary screen-treated protein 207, 208

roundnose grenadier 178Rozym 386RSW (refrigerated sea water) 54, 55,

176±7

safetychitin and chitosan 364±5

consumers' concerns and collagen andgelatin 280

pigments 426±7, 428saithe 5, 6, 7salmon 5, 6, 7, 290, 426composition of by-products 463enzyme-catalysed process for lipid

extraction 112±13oil 260, 262±3, 264roe 182±3salmon caviar 182

salt soluble collagen 282sand eel 178sarcoplasmic proteins 70and gel formation 160±1solubility 78, 79

sardine 260Sargassum horneri 399Sargassum micracanthum 398, 399saturated fatty acids 23sausage casings 295scales 129±33Schiff base formation 349screw cylinder flesh-bone separators 93screw press 202±3scytonemin 408sea clam processing by-product 316±19sea cucumber 294roe 185

sea lettuce 424sea urchin 294

roe 184±5seafood flavour 304±27aqueous extraction 305enzymatic hydrolysis 306±8enzyme-assisted seafood flavours from

processing by-products 308±19clam flavour 316±19flavour from white fish frames

313±15lobster flavour 309±13

fermentation 305±6flavour-imparting compounds and

chemistry 319±24concentration, drying methods and

flavour quality 322±4cooking juice vs hydrolysate 321formulation of seafood flavour

extract 324hydrolysis conditions 321±2, 323lobster 319±20red hake 320species-specific compounds 324

future trends 325

Index 529

seafood nuggets 222seafood patty 222seafood sausage 223seal 293blubber oils 265, 267±8, 271±2

seasonal variationand fatty acid composition 28, 30functional properties of fish mince/

surimi 212properties of protein-rich by-products

11±16seed germination tests 508seeding (inoculation) 502selective gears 175, 189semi-continuous digesters 474, 478sensory properties, and seafood flavourclam 317±18drying method and 322±4lobster 311±12, 313red hake 314±15

separation 306sequential batch anaerobic composting

(SEBAC) digester 468serine 324serine collagenases 377serine proteinases 118sesquiterpenequinones 408sexual maturity 212shark 292cartilage 131±3, 453liver oil 261±2, 263skin collagen 453

shellfish 185, 304composting wastes from 494±5enzymatic methods for by-products

processing 133±4preparation of chitin and chitosan from

by-products 345±6seafood flavour 308wastes from processing 108, 109

and pigments 426±7shrimp alkaline phosphatase (SAP) 387,

388shrimp fishery 65by-catch 144±5, 172, 174

side chains 70±3silage, fish 9±10, 56, 57, 118, 441±2,

486, 490, 491silent cutter/mixer 203single cell oils (SCOs) 268±9, 270size exclusion chromatography 128, 383skin 4, 6, 7, 8, 12, 109deskinning using marine enzymes

386

enzymatic extraction of collagen129±31, 132

fatty acids 27using 188

skipjack tuna 260slurry ice (binary ice) 177smooth muscle 69±70sodium chloride 324bio-gas production 477±8

soil conditioner 509soil enrichment 364soil nutrients 364, 506sole 291solid waste treatment 468±71solubilisationcalcium solubilisation using fish bone

peptide 333±5isoelectric precipitation and

solubilisation 75±84, 152±61marine enzymes 381

solubilitychitin 341±3N-alkylation for improved solubility

348±9chitosan 341±3of fish mince and pH 200FPH 234, 235proteins 251

solubles, fish 438, 440sonolysis 352sorbitol 218sorting 16, 49±52, 61, 175±6see also on-board handling

soy protein hydrolysate 324speciality ingredients 66speciesfatty acid composition 28±9and functional properties 11±16fish mince/surimi 211±12

and storage stability of fish mince/surimi 217

taste active compounds specific to 324traceability of by-products 134±5, 136

sphagnum peat 498, 499Spirulina 423±4sprats 178spray-dried flavour products 322±4spreading (wastes) 489, 491squalamine 451±2, 456stabilisationcarotenoid pigments 419collagen and gelatin 287

Staphylococcus aureus 359starches 97

530 Index

starfish 294stick water 438, 440stomachs 185storageby-product fractions 15±16stability of fish mince/surimi 217±19comparison between fish mince andsurimi 218±19

factors affecting 217±18mince from surimi by-products 219

storage modulus 253±4striated muscle 69±70strictaketal 402string lettuce 423±4stroma proteins (connective tissue) 70sturgeon caviar 29, 181±2sugars 97, 218sulphited phlorotannins 400±1super-absorbent hydrogel (SAH) 82supercritical methanol method 476±7supplements, food see food supplementssurface/interfacial properties 236±7surimi 8, 58, 96, 196±228by-products 204±5functional properties 210±16machinery for preparation 200±4manufacturing 198±200conventional method 198±9new technology 199±200

nutritional characteristics 216±17protein recovery from surimi

processing water 84±7, 145±6,149±52, 154±5, 205±6

alkaline conditions 162characteristics of by-products 207±8water-insoluble proteins 209±10

storage stability 217±19comparison with fish mince 218±19

utilisation 219±23swim bladders 185±6synthetic colorants 420±2

tablets 297tarako 183taste active compounds 324taste active free amino acids (TAFAA)

318tea extract 34temperatureanaerobic digestion 468composting and 492, 493vermicomposting 505, 506

storage/processing and fish mince/surimi 213

wash water 214terpenoid phenols 407±8tert-butylhydroquinone (TBHQ) 397tetraprenyltoluquinols 401±2thermal gravimetric (TG) analysis 255thermophilic temperature range 468thrombosis 454, 455tilapia 291, 295lipase from stomach and intestine of

378time, storage/processing 213tissue engineering 452±3titanium dioxide 101±3tocopherols 402tongues 92±3using 179±81

total suspended solids (TSS) 464traceability 134±5, 136, 190transesterification 114, 465±7transglutaminases (TGases) 379±80,

386±7TGase modification and gelatin 298±9

triacylglycerols (TAG) 22, 26, 31, 73, 74,265±6, 268

reducing bodily concentrations of 272see also fatty acids; fish oils; lipids;

marine oilsTrichoderma harzianum 360trimethylamine oxide (TMAO) 95trimethylamine oxide (TMAO)

demethylase 209trimmings (cut-offs) 4, 5, 6, 7, 8, 186trinitrobenzenesulphonic (TBNS) acid

method 127tropomyosin 70troponin 70trout 80±2trypsin 14, 121, 376, 387tryptophan 149tumour cell inhibition 356tuna 260tuna intestinal enzymes (TICE) 331±2tuna pyloric caeca crude proteinase

(TPCCP) 122, 332tusk 5, 6, 7

ulcer healing 356±7ultrafiltration 299, 382ultraviolet (UV) radiation 425Umamizyme 124, 126underutilised fish parts 179±89belly flap/trimmings 186fins and skins 188frames 187±8

Index 531

heads, cheeks and tongues 179±81liver 186±7maws 185±6milt 185roe 181±5stomachs 185

underutilised species 144, 145, 171±9,189±90

by-catch, by-products and discards171±3

key drivers for using 173±4upgrading of 177±9using 174±9

United Kingdom 4unsaturated fatty acids 23±4, 403upflow anaerobic filter reactor 469upflow anaerobic sludge blanket (UASB)

reactor 468, 469, 477

V cut 201vacuum packaging 33±4Valorga digester 470value-added foods 66vegetable oil 419vermicomposting 502, 503±7process parameters 504±6seafood wastes 506±7

viscera 4, 5, 6, 7, 12, 109fatty acids 27gutting 16, 50±1sorting 52

viscoelastic properties 253±4viscosity 290±1vitamin A 417vitamin E 29±30, 32volatile compounds 320von Post classification system 499

warm-water fish 281, 288, 291warmed-over flavour (WOF) 363washing 99, 150and functional properties of surimi and

fish mince 213effect of wash water and washingconditions 214

storage stability of fish mince/surimi217±18

surimi manufacture 198±9waste 108alternatives for uses 488±90, 491characteristics of seafood wastes 487

from primary processing 91±2see also by-products

waste disposal 489, 491composting see compostingproblem of 450±1regulations 174, 426, 436±7, 460±1at sea 173±4, 460±1, 486, 487

wastewatercharacteristics 463, 464treatmentanaerobic digesters 468, 469chitin and chitosan 364

wateraqueous extraction of seafood flavour

305content and composting 492, 493

vermicomposting 505content of seafood wastes 462±3dewatering 151, 199properties in aquatic foods 68±9protein±water interactions 70±3purification using chitosan 364used in surimi/fish mince preparation

214water-binders 163water gel desserts 295±6water-holding capacity 13, 68±9water-insoluble proteins 209±10water jet deboning 207water-soluble chitin (WSC) 354water-soluble protein 205±6wax esters 265±6weight control 355wet reduction process 438whale blubber oils 265±6, 272white fish frames, fish flavour from

313±15white muscle 70, 73whiteness of fish mince 97, 99±103hydrogen peroxide 99±101, 102titanium dioxide 101±3

windrows 495±6PAWS 497±501

wound healing 354±5

xanthan gum 102±3xanthophylls xxii, xxiii

yeast 324

zeaxanthin 416, 417

532 Index

Documents

Maximising the Value of Marine By-products