65272819 Enzymes in Food Technology

5/28/2018 65272819 Enzymes in Food Technology

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Enzymes in Food

Technology

ROBERT J. WHITEHURST

BARRY A. LAW,

Editors

Sheffield Academic Press

CRC Press


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Enzymes

in

Food Technology


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Sheffield

Food Technology

Series Editors: B.A. Law and A.J. Taylor

Aseries

which

presents

thecurrent stateof the art of

chosen

sectorsof the

food and

beverage

industry. Written atprofessional and reference level, it

is

directed

at

food scientists

and

technologists, ingredients suppliers, pack-

aging technologists, qu ality assurance personnel, analytica l chem ists and

microbiologists. Each volumein the series provides an accessible source of

information on thescienceand technology of aparticular area.

itles

in the series:

Chemistry

and

Technology

of

Soft

Drinks and

Fruit Juices

Edited

by

P.R. Ashurst

NaturalToxicantsinFood

Edited

by

D.H. Watson

TechnologyofBottled

Water

Edited by D .A.G. Senior and P.R. As hu rst

Environmental Contaminants in

Food

Edited

by

C.F. Moffat

and

K.J.W hit tle

Handbook

of Beverage

Packaging

Edited by G.A. Giles

Technology of

Cheesemaking

Edited by B.A. Law

Mechanisation

andAutomationin

DairyTechnology

Edited byA.Y. Tamime and B.A. Law

Enzymes

inFoodTechnology

Edited by R.J. Whitehurst and B.A. Law


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Edited by

ROBERT J. WHITEHURST

Kerry SPP

Cambridge,

U K

and

BARRY A. LAW

Food Science Australia

Victoria

Australia

Sheffield

Academic

P r e s s

CRC ress

Enzymes in Food Technology


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First published

2002

Copyright

2002Sheffield Academic Press

Published by

Sheffield

Academic Press

Ltd

Mansion House, 19Kingfield Road

Sheffield

S1 1

9AS,

UK

ISBN 184127223X

Published

in the

U.S.A.

an d

Canada

(only)b y

CRCPress

LLC

2000

CorporateBlvd., N.W.

Boca Raton,

FL33431,

U.S.A.

Orders from the U.S.A. and Canada (only) to CRC Press LLC

U.S.A. and Canada only:

ISBN

0-8493-9782-0

All rights reserved. No part of this publication may bereproduced, stored in a retrieval system

or transm itted in any form or by any means, electronic, m echanical, pho tocop ying or otherwise,

without

the

prior permission

of the

copyright owner.

This book con tains info rm atio n obtained from authentic

and

highlyregarded

sources.

Reprinted

material

is quoted with permission, and sources are indicated. Reasonable efforts have been

made to publish reliable data and in form ation, but the author and the pub lisher cannot assume

responsibility

for the

validity

of all

materials

or for the

consequences

of

their use.

Trademark Notice:Product

or

corporate names

may be

trademarks

or

registered trademarks,

and are used

only

fo ridentification an d explanation,without intent to infringe.

Printed

on

acid-free paper

in

Great Britain

by

Antony

Rowe Ltd., Chippenham , W iltshire

British Library

Cataloguing-in-Publication Data:

A catalogue record

fo r

this book

is

availablefrom

the

British Library

LibraryofCongressCataloging-in-PublicationData:

A

catalog record

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available

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Congress


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Preface

Enzymesmay bedescribed as 'functional catalytic proteins'. I dohope,how-

ever, that this volume will re-introduce them

as

useful

and

targeted work-

forces. Like the more familiar human workforces, they have preferred work-

ing conditions, may be trained (cultured) to carry out very specific tasks, and

theycannot function when their food (substrate) runs out.

The purpose of this volume is to provide both a basic grounding, for those

not

experienced in the use of enzymes, and a state-of-the-art account of

today's enzyme technology as applied to food and drink. Authors have been

selected not only for their practical, working knowledge of enzymes but also

for their infectious enthusiasm for thesubject.

Enzymes are introduced first according to their nomenclature and then by

their nature and mode of action. Chapters go on to describe the basic theory

and

practical applications of exogenous enzymes in food and drink tech-

nology, and how enzymes improve raw materials and influence and

modify

th e biochemical

and

physical events that

we

describe

as

'food processing'.

Finally,

methods

of

culturing

and

manufacturing enzymes

in

commercial

quantities

are

described, together with

the

role that genetic engineering

has to

play intheir further development.

Indigenous enzymes

in

food

raw

materials have long played

a

role

in

food

production. Today, however, enzymologists, working together with food

development technologists and wi th a view to market requirements, have

helped and improved upon nature to bring us varieties of food and drink that

were unheard of a relatively short time ago. Examples of these are wines

which mature earlier and have enhanced aroma and colour stability, 'naked'

but

undamaged citrus fruit (devoid of its pith or peel), bread that resists

staling, and enzyme-modified cheeses. Furthermore, production yields and

purities of intermediate raw materials have been improved, and by-products,

previously thought of as waste, may now be utilised.

M y thanks to all the contributors to this book for sharing their practical

approach to the subject. I hope that thereader finds the volumeasrewarding

as Ifound itspreparation.

R. J.Wh itehurst

B.

A. Law


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Contributors

Ms

Rose-M arie Canal-Llauberes

M rs

Cornelia

Drost-Lustenberger

DrPierre Fauquembergue

M r

Mari

J. M . C. van

Gestel

Dr

Catherine Grassin

Professor

Barry A. Law

M rPer

M unk Nielsen

M r

Hans

Sejr

Olsen

M r

Maarten

van

Oort

M r

RichiePiggott

M sJoanQi Si

M rDiederik J. M.Schmedding

M r

Reinhold

Urlaub

Novozymes, Cite Mondiale, 23 Parvis

desChartrons,F-33074Bordeaux Cedex,

France

Novozymes Switzerland

AG,

Neumatt ,

4243 Ditting en, Switzerland

DSM Food Specialties, 20 rue duBallon,

59041Lille Cedex, France

PB 28, Quest International, PO Box 2,

1400 CABuss,The Netherlands

DSM

Food

Specialties,

20 rue du

Ballon,

59041 Lille Cedex, France

Food

Science Australia, Sneydes Road,

Private

Bag 16,

Werribee, Victoria

3030,

Australia

Novozymes A/S, Laurentsvej 55, DK-

2880Bagsvaerd, D enmark

Novozymes A/S,CLaurentsvej

55, DK-

2880Bagsvaerd, Denm ark

Novozymes, Bil tseveste 12, 3432 AR

Nieuwegein,The Netherlands

Quest International, 5115 Sedge Blvd.,

Hoffman

Estates, Illinois 60192, USA

Novozymes SA, 79 Avenue Frangois

Arajo,

92017 Nanterre Cedex, France

PB 28,

Quest International,

PO Box 2,

1400 CABuss,The Netherlands

Rohm Enzyme Gmbh, Kirschenallee 45,

D-64293 Darmstadt, Germany


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Contents

1 The nature of enzymes and their action in foods 1

B A R R Y

A. LAW

1 . 1

Introduction

1.2 Enzym es

in

food 2

1.2.1 Enzym e nomenc lature 2

1.2.2 Enzym e kinetics 3

1.2.3 Enz ym e (in)s tability

6

1.2.4 Composition andactivityofcommercial enzym e preparations 8

1.3 Sources and range of enz ym es for food technology 9

1.4 Food enzyme legislation 14

1.5

Modificationof food enzyme activityb yprotein engineering 16

1.6 Summary

and

conclusions

18

References 18

Enzymes

for

bread, pasta

and

noodle products

19

JOANQI SI and CORNELIA

DROST-LUSTENBERGER

2.1 Introduction 19

2.2

Bread

19

2.2.1

Fungal a/p/za-amylases 20

2.2.2 Amylases toextend shelf life 21

2.2.3 Xylanases/pentosanases/hemicellulases

28

2.2.4 Lipase 31

2.2.5 Oxidases 33

2.2.6 Synergisticeffects ofenzymes 34

2.2.7 Enzymes

fo r

frozen dough

and

part-baked bead

39

2.3 Enz ym es for Asian noodles and non- duru m pasta 46

2.3.1 Red ucin g speckiness 46

2.3.2 Increas ing brightne ss and colour

stability

46

2.3.3 Imp rov ing texture 48

2.3.4 Mechanisms

for the effect of

lipase

51

Acknowledgement

54

References

54

Enzymesinbrewing

57

DIEDERIK

J. M.

SCHMEDDING

andM A R I J. M. C.

vanGESTEL

3. 1

Introduction

3 . 1 . 1 Historyofbrewing

3.2 Rawmaterials


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CONTENTS

3.2.1 Malt

and

adjuncts

58

3.2.2

Hops 59

3.2.3 Yeast

59

3.2.4 Water

59

3.2.5 Exogeno us enzy me s as processingaids 59

3.3 Theprocesseso fmalt inga ndbrewing 60

3.3.1

The

maltingprocess

61

3.3.2 M alt specification 61

3.3.3 Thebrewingprocess 62

3.4 Enzymesin thebrewingprocess 64

3.4.1 Enzymes inmalting 64

3.4.2 Enzymes in mashing 66

3.4.3 Enzymes inadjunct cooking 70

3.4.4 Enzymes

in

lautering/mash

filtration 70

3.4.5 Enzymes

in

fermentation

71

3.4.6 Enz ym es in ma turatio n 73

3.4.7 Ch ill proofing enzym es

73

3.4.8 Futu re developm ents 74

Acknowledgements

75

Further reading

75

Enzymes

in

wine production

76

MAARTEN vanOORTand ROSE-MARIE

CANAL-LLAUBERES

4.1

Introduction

76

4.2

Legalaspects

of the use of

enzymes

in

winemaking

76

4.2.1 Recom mendations from international organisations 77

4.3 Enzym es in winem aking: history and

definitions

77

4.4 Enzyme

properties

an dcom position 78

4.5

Applications

of

enzymes

79

4.5.1 Enzymes

fo r

pressing

and

maceration

80

4.6

Enz ym e applications

fo r

white

an d

pink wine grape varieties

81

4.7

Enz ym e applications

for red

wine

grape

varieties

83

4.8 Enzyme

applications

formust andpresswines: clarification enzymes 83

4.9 Enzym e applications fo ryoung wines: maturationand filtrationenzymes 84

4.10 Enz ym e preparations fo r aroma liberation 85

4.11 Enzym es for colour extraction 87

4.12

Other enzymes used inwinemaking 87

4.12.1

Urease 87

4.12.2

Lysozyme

88

4.13 N ew developments 88

4.13.1 Corkcleaning 88

4.13.2 Colour stabilisation

88

4.13.3 He alth issues 89

4.14 Conclusions

89

References 89


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CONTENTS

X I

Enzymes

in the

manufacture

of

dairy products

90

B A R R Y A . LAW

Introduction

90

Milk-clotting enzymes 90

5.2.1 The nature and identity of rennets and coagu lants 91

5.2.2 Maincharacteristicsof rennets and coagulants from differ ent sources 92

5.2.3 Production

of

rennets

and

coagulants

95

5.2.4 Form ulation and standardisation of renn ets and coagulan ts 96

5.3

Lactoperoxidase

96

5.4

Cheese

ripening enzymes 97

5.4.

i

Types

of

enzymes available commercially

97

5.4.2 Enzy me addition technology 99

5.4.3 Enzy me-m odified cheese (EM C) technolog y

101

5.5 Lysozym e 103

5.6

Transglutaminase

104

5.7

Lipase

104

5.7.1

Lipolysed mi lk

fat

(LMF)

104

5.7.2 Lipase-catalysed

intra- an d

intermo lecular modification

of

milk

fat 105

5.8 Lactase 105

5.8.1 Com mercial dairy products

o f

lactase technology

107

References

107

Enzymic modification

of

food protein

109

PERM U N K NIELSENandH A N SSEJROLSEN

6.1 Introduction 109

6.2 Industrialproteases 109

6.3

Protein hydrolysis

in

enzyme applications

110

6.3.1 Control of hydrolysis reaction 11 1

6.3.2 Cal culati on of the degree of hydr olysis of proteins using the

pH-stat technique 1 12

6.3.3 Calc ulation of thedegreeof hydro lysis of proteins us ing the

osmometry technique

114

6.3.4 Some kinetic aspects ofprotein hydrolysis 114

6.3.5

The

effect

of

proteolysis

116

6.4 The bitterness problem 1 17

6.4.1

Other

off-flavour

problems related tobitterness 118

6.5

Protein hydrolysis

fo r

food processing

119

6.5.1

Inactivationofenzyme activitya ndd ownstream processing 120

6.6 Functional protein hydrolysates 122

6.7 Low allergenic peptidesfor baby food form ulae 124

6.8

M eat extracts 25

6.9

Bone cleaning

127

6.9.1

Hydrolysisofmeat protein in relationt obone clean ing 127

6.10 Enz ym atic tenderisat ionofmeat 129

6.11 Modif icat ion

of

wheat gluten

130

6.12 Use of

mem branes

in

protein hydr olysis

processes 131

6.12.1 Fun ctiona l protein hydrolysa tes 131

6.12.2 Lowmolecular weight hydrolysates 132

6.12.3 The

mem brane reactor

133


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xii

CONTENTS

6.13 Flavour enhancers 134

6.13.1 Production

of

flavour enhancers

134

6.13.2

Enzym atically hydrolysed vegetable proteins( e -HV P ) 135

6.13.3

H ydrolysed a nim al proteins (e-H V P) 135

6.14 Yeast extracts

13 6

6.15 Fish

processes 137

6.16 Protein cross-linking

in

food processing

138

6.16.1

Applications

of

transglutaminase

139

References 140

7 Enzymes in fruit and vegetable juice extraction 144

REINHOLD URLAUB

7.1

Introduction

14 4

7.2 The legal situation 145

7.3 Definitions and characteristics 146

7.4

Pectins

147

7.4.1 Sm ooth-region pectinases 148

7.4.2 Hairy -regio n pectinases 149

7.5 Cellulose and hem icellulose 150

7.6

Starch

151

7.7 Protein 153

7.8 Applicationoftechnical enzym e products 154

7.9 Pome

fruit

processing 155

7.9.1 Enzym atic treatmentof the mash 157

7.9.2 Pom ace extr actio n 158

7.9.3 M aceration 159

7.9.4 Pomace ma ceratio n 160

7.9.5 Juice treatm ent 161

7.9.6 Traditiona l clarification and filtration 165

7.9.7

Ultrafiltration

of

apple juice

166

7.9.8 Clou dy apple juic e

or

apple juice concentrate

167

7.10 Grapes

168

7.10.1 Processing of Concord grapes 168

7.10.2 Manufacturing white grape juiceandgrape juic e concentrate 169

7.11

Berries

170

7.12

Stonefruit 172

7.13 Citrus fruit 172

7.13.1 WESOS

173

7.13.2 Citrus juice concentrates

175

7.13.3 Clear

an d

semi-cloudy citrus concentrates

176

7.13.4

Extraction of citrus oil 177

7.13.5 Cloudifier

178

7.14

Tropical

fruit

179

7.15 Vegetables

181

7.16 M embrane cleaning 182

References 182


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CONTENTS

xiii

8 Enzymes infruit

processing

184

CATHERINE GRASSIN and PIERRE FAUQUEMBERGUE

8.1

Cit rus peeling

184

8.2 Citr us peel processing 186

8.3 Fruit firming 187

8.3.1 Strawberries 194

8.3.2 Tomato

19 6

References

199

9

Enzymes

in

starch modification

200

H A N SSEJR OLSEN

9.1 Introduction 20 0

9.2 Processing an denzymology 20 1

9.2.1 Starch liquefaction

201

9.2.2 tf/pte-Amylases 204

9.2.3 Saccharification of liquefied starch 210

9.2.4 Tailor-made glucose syru ps

210

9.2.5

Use of

syrups

211

9.2.6 Production of maltose syrups

2

13

9.2.7

DX, DE and

reducing value

213

9.2.8 High conve rsion syrup 214

9.2.9 Production

of

high

dextrose

syrups

215

9.2.i 0 Am yloglucosidase/pullulanus combination in the productiono f

high dextrose syrup

216

9.2.11 Co ntinu ous saccharification 217

9.2.12 Continuous saccharification

in a

membrane reactor

218

9.3 En zy me s as processing aids in the

purification

of saccharified wh eat starch 218

9.4 Glucose isomerisation 219

9.4.1 The isomerisation reaction 219

9.4.2 Isomerisation cond itions 220

9.4.3 Isomerisation temperature 220

9.4.4 Isomerisation

pH 220

9.4.5 Isomerisation glucose (dextrose-DX) and fructose conc entration 221

9.4.6 The immobilised enz ym e system 221

9.4.7 Process lay-out

222

9.4.8 Enzy me decay 224

9.4.9 Con trolling isomerisation costs 224

9.5 Use of high-fructose corn syrup s (M FCS) 225

9.6 Cyclodextrins 226

9.7 The

future

227

9.8 Conclusion 227

References

228

10 Commercial enzym e production andgenetic modification of

sourceorganisms

229

RICHIE

PIGGOTT

10.1 Briefhistory 22 9

10.2 Sources of comm ercial enzym es 229


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xiv CONTENTS

10.2.1 Anim al-derived enzym es 230

10.2.2 Plant-derived enzymes

230

10.3 Microbial enzyme fermentation 232

10.4 Preservation

of

industrial microorganisms

233

10.4.1 Freezing 234

10.4.2 Freeze-drying 234

10.4.3 Subcul turing 234

10.5 Inocu lum developm ent

234

10.6 Submerged fermentation 235

10.7 Separation

of

broth

236

10.7.1 Filtration withfilter aid 236

10.7.2 Microfiltration

237

10.7.3 Cen trifug ation 237

10.8 Concentration

237

10.9 Drying 23 8

10.10 Enz yme formu lation 239

10.10.1

Powder blending

239

10.10.2 Liq uid blending 240

10.11 Surface culture

(Koji)

fermentation 240

10.12 Intracellular enz ym e production

241

10.13 Geneticso fproducer organisms 24 2

Acknowledgements 244

References 244

Index 245


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1

The

nature

of enzymes

andtheir actionin foods

Barry A. Law

1 .1

Introduction

Enzymes are proteins that are produced by all living organisms. They speed

up chemical reactions selectively as part of essential life processes such as

digestion, respiration, metabolism andtissu e maintenance.Inother words, they

are highly specificbiologicalcatalysts. The enzym es work under more or less

mild

conditions (they have to, in order to operate in living cells and

life-

sustainingenvironm ents), mak ing them ideal catalyststo use infood technology,

in which

the

manufacturer wants

to modify

food

raw

materials selectively

withoutdestroying essential nutrients. The historical uses of enzymes to make

beer, wine ,

cheese

and bread are elegant examples of the indu strial explo itation

of

the

power

and

selectivity

of

enzymes. Early food enzyme technologists were

clever craftsmen,buttheydid notrealise that thisisw hat they w ere doing,and

that

they could haveadoptedsuch

a

splendid

job

title.

To understand modern food enzyme technology, it is important to realise

that these early enzymatic processes were not only fermentations, but also

complex

and

coordinated enzyme-mediatedprocesses.

The

enzymes w ere then,

and remain, essential for the provision of fermentation substrates (beer and

bread), the development of flavour and aroma (wine) or the creation of the

very structure

of the

product (cheese).

The

following chapters

of

this volume

will describe and discuss how enzymes do all of these things in these and

other

foods.

The

picture will emerge

of a

technology that began

as a

craft

and has become a sophisticated high-technology, high value-added industrial

sector. Enz ym e production

and

application

in the

food m anufacturing industry

isbased

on a

profound understanding

of the

role

o f

enzymes

in

traditional foods,

from which technologists have improved the basic processes to supply bigger

markets with safer

and

higher quality products. This understanding, together

with

improvements in enzyme sourcing and production technology, has also

yielded novel enzym etechnologies

to

create

new

foods

and

food ingredients,

as

willalso

be

revealed

by the

authors. This opening discussion will introduce

the

reader

to the

basic properties

of

enzymes,

the

methods used

to

quantify them

for technological use, their non-ideal behaviou rinfood systems,and therange

of food technologies

in

whichthey

are

used.


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2

ENZYMES

IN FOOD

TECHNOLOGY

1.2

Enzymes

in

food

1.2.1

Enzyme nomenclature

Any particular enzyme onlycatalyses reactionsbetween onetype(or a narrow

range) of chemical compound (its substrate). This defines its 'specificity' and

provides

thebasisof its

c lassification

and

name. Often,

the

trivial name

of the

enzyme, derived from the truncated substrate name with 'ase' added, identifies

thesubstrateorsubstrate

range

betterforfood

technologists

than

does

itssystem-

atic name or its International U nion of Bioche mistry E nzy m e Com mission (IU B

or EC)number [1].Forexample, thetrivial nameforlipid-hydrolysing enzymes

is lipase, but the

official

name and number is triacylglycerol acylhydrolase

(IUB/EC

3.1.1.3). The

latter

is

precisely descriptive

of

what such enzymes

do, and the

term/number

is

vital

fo r

unam biguous comm unication between

biochemistsand product chemists.As far as food technologists areconcerned

though, lipases act on lipids and break them down to their component

fatty

acidsandglycerol.Thisis the important informationto beused in food product

and process terms, and the trivial names of all food enzymes

will

be used

throughout this chapter. Thus, proteinases (usually shortened

to

protease) chop

up

proteins by breaking the am ide bonds that join their com ponent amino acids

into

the

protein polypeptide chain. Carbohydrates wo uld logically

be

broken

down

by

carbohydrases, though this

is not a

commonly used term, largely

because carbohydrate building blocks are very diverse, as are the polymeric

structures formed

from

them (the starches, cellulose, pectins). This dive rsity

willbecomeclearer

to the

reader

as

this volume unfolds.

For

now, suffice

it

to say

that more useful general terms

fo r

mem bers

of

this class

of

enzyme

are

derived from individual

substrates.

Thus

lactase

breaks down lactose, maltase

breaks down maltose, pectinase b reaks dow n pectin and cellulase breaks down

cellulose. There

is no

'starchase'

because

there

are too

many structural isomers

of starch on w hic h to base the ob vious

trivial

nam e for starch degrading enzymes.

Themost ubiquitous

'starchase'

iscalledalpha-amylase

because

itchopsup the

amylose component of starchat the most common glucose-to-glucose bondin

the amylose polymer (a lpha - l 4 ) .

Although

th em ajorityofenzym es (over 90%) used currentlyincomm ercial

scale food technology are hyd rolytic substrate degraders like those m ention ed

above, some enz ym es used to improve and

modify

food m aterials catalyse sy n-

thetic

reactions

and

substrate interconversions. Most

are

q uite easy

to identify

with

their biochemical

function

because their nam es describe

the

m ain substrate

and the reaction; for example, glucose oxidase oxidises glucose to gluconic

acid, using up oxygen. However, this name does not

tell

us much about the

technological

functions

it performs, namely reducing glucose content of egg

whites to reduce Maillard browning, and scavenging oxygen in packaging

technology. There

is

nothing

to be

done about

this,

other

than for the

food

technologist to beawareof thetechnological

significance

ofglucose oxidation


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E N Z Y M E SA N DTHEIR ACTIONINFOODS 3

by

a

nice ge ntle enzym e. S imilarly,

the

names glucose isomerase

or

lipoxy genase

tell

all

about

the

biochemistry involved,

but we

just have

to

remember

th e

technology.

The

isomer

of

glucose produced

by

glucose (strictly, 'xylose ' )

isomerase is fructose which, mole for mole, is sweeter than glucose so the

enzy me is used to make the sweetener, high fructose corn sy rup (section 1.3).

The reactions catalysed by lipoxygenases used in bread imp rovem ent are qu ite

complex

[2] and not

altogether understood

by

research biochemists. Fortunately,

food technolog ists understand em pirically that the enzy m e catalyses reactions

between lipids

in flour and

oxygen. Directly

and

indirectly these reactions

bleach the flour and make nice white bread, and also modify wheat proteins

so that they help in forming good crumb structure and high loaf volumes

(chapter 2).

Some enzymesareeasiertoidentify with their food applications than others,

butmanyofthese connections betw een nom enclatu reandtechnological

function

will

become

familiar

to the reader thro ugh out this

volume.

1 .2 .2 Enzyme kinetics

The u nde rlying me chanism s of enzym e action and the interactions of enzym es

with

the ir physical and chemical en vironm ent can be described mathem atically

with

reasonab le precision. However, most

of the

equations, constants

and

ground

rules (enz ym e kinetics) have been worked

out for

idealised situations

in

which

single

enzymes act on simple, single substrates under predictable conditions

found

within living cells.

The

reader

who

wants

to

understand these parameters

in mathematical terms can find an excellent summary and reference list by

Fullbrook

[3] in

'Industrial Enzymology' [4].

In the

same work, Fullbrook

[5 ]

also goes on to explain some of the pitfalls of applying 'classical' enzyme

kineticstonon-ideal industrial processes.

Food techno logistsnotonly needtoknow which enzym es degrade, synthesise

or interconvert which food material substrates, but also need to have a means

of working out how much of the chosen enzyme to use under any particular

cond itions to achieve an economical rate and

efficiency

of material conversion.

Qualitative and quantitative enzyme kinetics show us that enzymes behave

quite predictably in simple ideal systems such as those used to classify and

characterise enzyme preparations in research and QA laboratories. They work

atpeak rates at particular pH values, temperatures and substrate concentrations

according

to

well-established rules (figure 1.1).

At

fixed

substrate concentrations, enzymic reaction rates depend

on

enzyme

concentration up to a maximum, dependent on the turnover

efficiency

of the

particular

enzy m e preparation. The 'activity curv es' of the type shown diagra-

matically in figure 1.1 are used to derive the numerical values of these param-

eters, and they are vital basic inp uts into deciding wh ich enzym e prep aration(s)

should

be

used

in

w hic h food m odification process [3].


19/270

E N Z Y M E SINFOODTECHNOLOGY

3 5 7

pH

10 20 30 40

Temperature (C)

Substrate

concentration

Enzyme

concentration

Figure

1 .1

Effect of pH, temperature, enzyme concentration and substrate concentration on the

initial

rate

of

enzyme catalysed reactions

in

solution.

The temperature and pH 'profiles' derived

from

simple test conditions are

usually

applicable

to

complex food environments because they

are

dependent

on the

molecular

properties of the

enzyme protein itself, rather than

on

those

of the substrate. Enzyme proteins are precisely folded polypeptide chains, held

together byre latively weak molecu lar forces. The folded structure determines

the

integrity

of the

cataly tic site (th e 'active

site',figure

1 .2)

within the

enzyme,

and this is easily disrupted by energy changes in the enzym e's environm ent

(temperaturebeingaprime example). This phenomenoniscalled 'denaturation'


20/270

Figure 1 .2

Th e

three-dim ensiona l folded polype-ptide chain

of an

enzyme protein,

illustrating th e

interactions between active site

functional

groups

(A and B)

an d

substrate

(C )

wh ich lower

th e

energy needed

to

break

th e

chem ical bond

in the

substrate

an d

thus

createt he

cataly t ic

activity of the

enzyme.


21/270

6 ENZYMESIN

FOOD TECHNOLOGY

and it can be reversible or irreversible dependent on the severity of structural

deformation

and

damage.

However, even small changes in intramolecular forces in the enzyme , such as

those caused

by

small temperature changes,

or

pH-dependent charge differenc es

on the amino acids making up the primary polypeptide chain structures, can

also

cause conformational changes in the structure that

fall

short of denatu-

ration.

Changes

of

this magnitude

are

manifested

in the

typical temperature

and pH/activity curves in figure 1.1 and illustrate how

precise

the spatial

juxtaposition

of

active site

functional

groups

must be to

reach

th e maximum

rate

of

catalysis. Thus,

as

reaction temperature

is

increased, classical chemical

kinetics dictate th at the reaction

willspeed

up, but beyond a certain temperature

(characteristic

of any

particular enzyme )

the

disruption

to the

enzy me protein

folded

structure reduces

its

catalytic

efficiency andactivity

tails

o ff

again.

In

the

case

of pH, the bell-shaped curve is the manifestationof anoptimal

spatial structureof theenzy me protein w hich formsat acertain pH, whenthe

relative ionisations

within the

structure combine

to

orientate

the

active site

to

give maximum substrate binding, bond modification,and release of product.

Either

side

of the

optimum

pH for

activity, changes

in

charge

and

hydrogen

bondingefficiency

cane ither distortth eactive site through changes in thethree-

dimensional fold

of the

protein polypeptide chain

(figure

1.2),

or

reduce bond

dipoles

in

active site

functional

groups, reducing their capacity

to

lower

th e

activation energy

fo rconversionof thesubstrate(s).

The understanding of the relationships between the amino acid sequences,

three-dimensional structures

and

catalytic

efficiencies

of

food enzymes

is now

sufficiently

comprehensive

to

allow molecular enzymologists

to

change

th e

structures of food enzy m es to improve their technological p rocessing properties

such

as heat resistance, p H o ptim um , resistance to catalytic poisons/inhibitors

and even substrate preference. This is called 'protein engineering', and is

reviewed

by

Goodenough

[6] and Law [7]

specifically

in

relation

to the

advanta-

geous

modification of

enzymes

fo r

food ingredients synth esis. Other exam ples

appear laterin section 1.5.

1.2 .3 Enzyme (in)stability

Enzymes have

a finite

working

life, or

half-life,

due to

inherent physical insta-

bility,

the action of antagonists/inhibitors, and 'poisoning' bycontaminants in

the

reaction

m ixture.

In

foods

and

food

technology,

physical instability

can be

induced by the pH and temperature effects outlined above, but also by relatively

mild forces such

as

surface tension

in

foams

and

emulsions.

M ost enzy me inhibitors would not be present in foods because they are gen-

erally poisonous (e.g. heavy metals and organometallic compounds), but many

enzyme antagonists and catalytic poisons are common in foods and food raw

materials(e.g. respectively, proteolytic enzy m esandfree radicals

from

oxidised


22/270

ENZYMES

AN DTHEIR ACTIONIN FOODS

7

unsaturated fattyacids). Enzyme instability measured

for

purifiedproteins

in

aqueous

bufferscan

give guidance

to

technologists

as to the

comparative stability

of

rival enzyme preparations under real processing conditions, but absolute

stabilitycanonlybedetermined inreal food system s.The firstdecision tomake

is

whether

the

process will benefit

from

stable

or

unstable enzymes.

Highly

stable enzymes

are

normally used

in

processes that take

a

long tim e

to

complete, such

as

malting

and

Koji fermentations,

or

where

the

enzyme

is

part

of a

diagnostic kit,

and has to

survive drying

and

long

storage

before use,

withoutlosing its standardised activity at m anu factu re. On the other hand , some

enzy m es used in food m anu fac ture are only required to be active for a short

time

to

scavenge oxygen,

to

precipitate milk proteins

or to

assist ripening

for

example,

and

their long-term

persistence

in the

food

is

either irrelevant,

or

even

detrimental. Specific examples

of

these applied scenarios

and the

criteria used

to picktheright enzym efor the jobw ill emergein thefollow ing chapters. Some

ofthespecialfactors thatinfluencethestabilityandhalf-life ofenzymesinfood

system s and food processing are listed in table 1.1.

There are no hard and fast rules to guide food technologists through this

largely

unexplored areaofapplied enzy m e science becausetheresearch com mu -

nityhas not systematically studied theeffectsofphase structures and interfaces

in

food

on all of the

factors used

to

predict enzyme activity. However, based

on table

1 .1 ,

it m ay be

possible

to

avoid some

pitfalls, or

even exploit some

Table 1 .1 Special factors that

influence

the

stability

ofenzymes infood

Condition

Effect on

stability Un derlying cause

Phase concen tration

of

enzyme

Presence

of other

non-enzymeproteins

Presenceofother

enzymeproteins

Stabilises

Stabilises

Destabilises

Presenceof impurities Destabilises

Increasedenergyp er uni t volum e required

to

denature enzym e

Lowers proportion of denaturing energyorm olecular

source available

t o

denature enzyme protein

If

accompanying

enzymes

or

proteinases, they

may

degrade

th e

added en zym e protein

Catalyticpoisoning

oflipasesbyfreeradical-induced

chemical

damageto theenzyme

(especiallylipases)

of anyenzymeby

heavy m etal atoms blocking

th e

activesite

of

metal requiring enzymes

by

chelating

agentssuchascitric acidorpolyphosphates

Phase interfaces Destabilises Denaturationbysurface tension forces


23/270

8 ENZYMES INFOOD TECHNO LOGY

opportunities. For example, a supplier might recommend an enzyme addition

rate of x

units

per

kilogram

of

food product,

based on the

activity measured

against the substrate in solution. The food manufacturer calculates the cost of

th e

enzymatic conversion

based on

this

figure,

adjusts

it to

compensate

for

differences in pH and

temperature,

and

decides

how

much

th e

enzymatic stage

will

cost. This decision process is

based

on the assumption that the enzyme

will

be acting in a homogeneous environ me nt sim ilar to that und er wh ich

the

producer assayed it . However, most foods are heterogeneous, consisting

of discontinuous solid

composites

of fat, protein, carbohydrates and water, or

emulsions

and

foams with distinct phase boundaries. Such structures

in

food

tend to cause any added substance to be concentrated by

affinity,

osmosis or

differential

solubility into one particular component or phase of the food. It is

therefore worth seeking from the sup plier data on the solubility, hydrop hobicity

and resistance to surface tension de naturation of the enzym e, so that the costing

figures can be

adjusted

to '.v

units

per kg

b ulk protein,

fat or

carbohydrate',

or

even 'per u ni t volum e of water in an em uls ion '. This mi gh t

produce

a cost figure

substantially

more or less than that based on assum ptions of ho mog eneity.

1.2 .4 Com position and activity of comm ercial enzyme preparations

Most commercial enzyme preparations contain

not

only

the

specific enzyme

whose activity is printed on the label, but also other enzymes that happen to

beproduced by the same source material/organism [8]. Theenzy me user mu st

always

be

aware

of

this factor

in the

enzyme product specification

to

avoid

side effects in complex foods (e.g. starch breakdown by an enzyme preparation

bought

for its

proteinase f unc tion ). Even

if the

application

is for an

isolated food

component suchaswhey protein,aheterogeneous enz ym e preparation can pass

unexpected

and

unwanted enzyme activities

on to the

customer

who

buys

the

enzyme-modified

food ingredient unless steps

are

taken

to

inactivate

it

after

processing.

Thus, enzyme/substrate interrelationships are more complex and less pre-

dictable

in

food reaction environments because food materials

are not

pure

chemicals, but complex structures and/or poorly defined m ixtu res of potential

(possibly competing) substrates andinhibitors.

The

amount

of

enzyme required

to

convert

a

given amount

of

substrate

is

measured

in

'Enzyme Units ' .

In

simple systems

th e

original International

Union of

Biochemistry Unit

(U) was the

'amount

of

enzym e that

will

catalyse

the transformation of one micromole of substrate per minute under defined

conditions' .The SIunitforenzym e activityis the 'katal'. definedas the' amou nt

of

enzyme that

will

cause

the

transformation

of one

m i l limole

of

substrate

per

second under specified conditions'.

Unfortunately

for food technologists, even a substrate that is definable in

food technology terms (e.g.

fungal

protein, vegetable protein extracts,

animal


24/270

E N Z Y M E SA N DTHE IR ACTIONINFOODS 9

muscle,com

starch, milk fat)

is

heterogeneou s

as far as an

enzyme

is

concerned,

and the

above unit definitions

are oflittleuse to

them

inprocess

design.

For

example,

corn starch consists o f glucose polym ers ofinfinitely-variab lemolecu-

lar weigh t with huge batch-to-batch difference s, rendering impossible any fixed

standard enzy medoseto achieve an agreed specification for a hydrolysed starch

ingredient

product. Indeed, itwouldbe impossibletodefineanenzyme unitin

relation

to

such

a

substrate simp lybecause

a

m illimole

ormicromoleof a

mixed

molecular

weight polymer cannot be defined. These factors mean that there is

no

simple, consistent

way to

define

or

pre-determine

how

much enzyme

to add

to how much raw material in food processing.

In

practice, enzyme suppliers provide essential information for their cus-

tomers bydefining units intermsof the

technological

functionof the enzyme.

This not

only

allows the user to define and control the enzymatic process on

an industrial scale, but sets out thebasis fo r relatingthecostof the enzymeto

the

value

of the

product produced,

and

allows comparison

of the

productivity

of enzymes from

different

suppliers. A good example of such a pragmatic

and

direct approach to quantifying enzymes for sale anddosing is the use

of

'international milk clotting units' (IMCU)fo r

qu antifying

m ilk-coagulating

enzym es ( chy m osin, microbial renn ets) for sale and use in cheesemaking. The

enzymes

are all acid proteinases that degrade caseins, but casein breakdown

itselfis not

usefully

relatedto the technological

function

ofdestabilising c asein

micelles andcausing m ilktoclot.The IM CU is ameasureof theabilityof acid

proteinases, sold

ascheese

m ilk coagulants,

to

clot

a

standard amount

o f

m ilk

unde r standard conditions in a standard time relative to an internationa l enzy m e

reference standard [8]. Other function-based unitsof food processing enzyme

activity

are based on such parameters as viscosity (reduction or increase),

conductivity and

colourbinding.

1.3

Sources

and

range

of

enzymes

for

food

technology

The

traditional sources

of

food technology enzymes have been

the

tissues

of

plants and animals (table 1.2). Although these are still widely used in food

manufacture, there are many influences driving food enzyme production and

use towards microbial alternatives, including genetically-modified derivatives

of these organisms (GMOs).

There are many examples of the use of carbohydrate-degrading enzymes

in

food manufacture, particularly

in

baking, beer brewing

and

fruit

juice pro-

duction,

and these will be covered in detail in chapters 2, 3 and 7 of this

volume.How ever, their economical produc tionfrom efficient microorganisms

in

industrial-scale fermenters means that

the

utilisation

of the

enzymes such

as

amylase and pectinase present in the traditional raw m aterials (w heat, barley,

citrus

fruits,flour) isconfinednow totheir action

in

situ,andtheyare notwidely

extracted for

exoge nous use.


25/270

10

ENZYMES

INFOOD

TECHNOLOGY

Table 1.2 Enzymes widelysourced from animals and plants used in food manufacturing technology

Enzyme

alpha-

Amylase

beta-Amylase

Papain

Bromelain

Ficin

Trypsin

Source

Cereal

seeds

e.g. wheat, barley

Sweet potato

Latex of unripe

papaya

fruit

Pineapplejuice

and

stem

Fig fruit

latex

Bovine/porcine

pancreas

Action

in

food

Starch hydrolysis

to

oligosaccharides

Starch hydrolysis

to maltose

Food

and

beverage

proteinhydrolysis

Muscle

and connective

tissue

protein hydro lysis

As

bromelain

Food

protein hydrolysis

Food

applications

Breadmaking

brewing

(malt ing)

Production

of

high

malt

syrups

Meattenderisation

chill

haze prevention inbeer

Meat tenderisation

Asbromelain an dpapainbu t

not

widely used

due to

cost

Production

of

hydrolysates

for

food

flavouring

(mos t ly

Chymosin

(rennet)

Pepsin

Lysozyme

Calf abomasum

Bovine

abomasum

Lipase/esterase

Gullet

of

goat

and

lamb: calf abomasum:

pi g

pancreas

Lipoxygenase Soy bean

Hen

eg g

white

Lactoperoxidase

Cheese

whey:

bovine colostrum

hydrolysis

As

chymosin

+

more general casein

hydrolysis in cheese

Triglyceride

( f a t )

hvdrolvsis

Oxidation of

unsaturated

fatty

acids

in flour

Hydrolysis

of

bacterial

cellwall

polysaccharides

Oxidation of

thiocyanate

ion

to bactericidal

hypothiocyanate

replaced

now by

microbial

proteinases)

Coagulation

of milk in

cheesemaking

Usually

present

with

chymosin as pan of

' rennet '

Flavour enhancement

i n

cheese products:

fa t

function modification

by

interesterification

Bread dou gh impro veme nt

Prevention of late blowing

defects incheeseby

spore-forming bacteria

Cold sterilisation

of milk

Certain

plantandanimal

pro teinases,

on theotherhand, rem ain inwidespread

usethrough

their

well-established

effectiveness

insomefundamental processes

in cheese

production and meat

processing (chapters

5 and 6). Of

particular

note are papain (and th e related proteinases bromelain and ficin) in meat


26/270

E N Z Y M E S

A ND

THEIR ACTION

IN

FOODS

1 i

tenderisation, and chymosin (withalittle pepsinfo rgoodmeasure) in themilk

coagulation stage of cheesemaking. Ch ym osin extracted

from

calf

offal

has been

displaced in some countries by the same enzy me produced by ferm entation of

yeasts

and

fungi containing cloned chymosin genes (chapter

5), but it is

still

the preferred

choiceof

many traditional cheesemakers, despite

the

supply

and

purityadvantagesof the fermentation-produced product.

Bovine

and porcine trypsin are still used for producing food protein

hydrolysates as food

flavour

ingredients, but there are so many good microbial

alternatives

to these classical serine proteinases now on the market (table 1.3),

especially those with less tendency to make bitter products, that trypsin is no

longer

as

important

to

food manufacturers.

Like proteinases, the food grade animal lipases that have been the m ainstay of

the dairy flavours industry in the past (chapter 5) are gradually being replaced

by equ ivalent enzy me s of microbial origin. This is especially thecase in the

newer technologies such

as fat function

modification

by

enzymatic interester-

ification

[10] in which complex and interdependent enzyme reactors put new

demands

on

long-term enzym e stability, catalyticefficiency, compatibility with

multi-step

processes

and resistance to poisoning by im purities in com m odity

food fats.

However, some 'traditional' app licatio ns remain, particularly in the

production of

piquant-flavoured

Italian cheese varieties (chapter 5).

Soy and

wheat lipoxygenase

is an

important enzyme

in bread

baking

(chapter 2) and has traditionally been used in the

form

of the endogenous

wheat flour enzyme, supplemented with soy flour. The latter not only adds

to the lipoxygenase activity in the dough, but

also

supplies lipid substrates to

both

enzyme sources toimprovethe texture, 'workability' andcolour ofbread

dough, through (probably)

sulfhydryl

group oxidation

in

wheat proteins,

and

oxidativebleachingofplant pigme nts.

Lysozyme and lactoperoxidase are both animal enzymes extracted

from

natural

sources (table 1.2),

but

they

differ

in

their application

from

most anim al

andplant enzymes in that they are both an timicrob ial, and can be used to control

different typesofspoilageincheeseandmilk (chapter 5).

M ore and m ore enzym es for food technology are now

derivedfrom

specially

selected

or

genetically modified microorganisms grown

in

industrialscalefer-

menters (chapter 10) and table 1.3 lists a range of examples and app lications.

The

number

and

range

of

these exam ples

of

m icrob ially sourced alternatives

reflect

the logistical and comm ercial advantage s of us ing microbial ferm entation

rather than animalorplant extraction to produce food enzymes. Logistics are

based

on

both political geography

and

transpo rtation costs; clearly

it is

desirable

for an enzyme producer and user to have a reliable, predictable source of

an

enzym e that is pivotal for a m anu factu ring process. T his rule holds good

for quantity, quality and price, and the fermentation alternative delivers on

all counts.

It can be

produced anywhere

in the

world, irrespective

of

climate

and

agro-economics,

th e

enzyme yield

is

predictable

from th e

fermentation


27/270

Table 1 .3 Enzymes derived

from

microorganisms

an d

used

in

food

manufacturing

technology

Enz.yme

alpha

-Amylase

alpha-

Acetoluctute

decarboxylase

Amyloglucosidase

Aminopeptidase

Catalase

Cellulase

Chymosin

Cyclodextrin

glucanotransferase

beta-Galactosidase

(lactase)

Source

Axpergillux spp.

Bacillus spp.*

Micmhacterium

irnperiale

Bacillussubtilis*

Axpergillux niger

Rhizopux spp.

Lcictococcus lactix

Axpergillux

spp.

Rhizopux oryzae

Aspergillux

niger*

Micrococcux

lute us

Axpergillux niger

Trichodernw spp.

Axpergillux awtunori*

Kluyvemmycex

lactix*

Bacillus spp.*

Axpergillux spp.

Kluyvennnvcex spp.

Action

in

food

Wheat starch hydrolysis

Converts acetolactate

to

acetoin

Hydrolyses starch dextrins to

glucose (saccharitication)

Releases free amino acids

from

N- t e r m i n u s

of proteins and peptides

Breaks down hydrogen peroxide

to water and oxygen

Hydrolyses

cellulose

Hydrolyses kappa-casein

Synthesisecyclodextrins

from liquified starch

Hydrolyses mi lk lactose

to glucose and galactose

Application in food technology

Dough softening, increased bread volume, aid production of

sugars for yeast fermentation

Reduction of wine

maturation time

b y

c i rcumvent ing need

for secondary fermentation of diacetyl to acetoin

On e stage of high fructose corn syrup production;

production

of

M i t e beers

De-bittering

protein hydrolysates accelerating

cheese

maturat ion

Oxygen removal technology, combined

with glucose oxidase

Fruit liquifaction injuice production

Coagulation of milk fo rcheeseinaking

Cyclodextrins

a re

food-grade micro-encap sulants

fo r

colours,

f lavours and vitamins

Sweetening

milk an d

whey; products

fo r

lactose-intolerant

individuals;

reduction of crystallisation in icecream containing

beta

-Glucanase

Glucose isomerase

Aspergilltix

spp.

Bacillus

xuhtilix*

Actinplani's

missouriensis

Bacillus coaguans

Streptomyces

lividanx*

Streptomycesrubiginosus

Hydrolyses beta-glucans

i n beer mashes

Converts glucose

to

fructose

whey; improvingfunctionality

of

whey protein concentrates;

manufactu re of

lactulose

Fil t rat ion

aids, haze prevention

in

beer production

Production of high fructose corn syrup

(beverage sweetener)


28/270

Glucose oxidase

Hemicellulase and

xylanase

Lipase andesterase

Pectinase

(polygalacturonase)

Pectinesterase

Pentosanase

Pullulanase

Protease

(proteinase)

Aspergillus niger*

Penicillium chrysogenum

Aspergillus

spp.*

Bacillussubtilis*

Trichoderma reesei*

Aspergillus spp.*

Candida spp.

Rhizomucor miehei

Penicillium

roqueforti

Rhizopus spp.

Bacillus

subtilis*

Aspergillus

spp.

Penicilliumfuniculosum

Aspergillus

spp.*

Humicola insolens

Trichoderma

reesei

Bacillusspp.*

Klebsiella

spp.*

Aspergillus

spp.*

Rhizomucor miehei

Cryphonectria

parasitica

Penicillium

citrinum

Rhizopus niveus

Bacillus

spp.*

Oxidises glucose to gluconic acid

Hydrolyses hemicelluloses

(insoluble non-starch

polysaccharides in flour)

Hydrolyses triglycerides

to

fatty

acids andglycerol;

hydrolyses alkyl

esters

to

fatty

acids and alcohol

Hydrolyses pectin

Removes methyl groups

from

galacoseunits

i n

pectin

Hydrolyses

pentosans

(soluble

non-starch polysaccharides

inwheat flours)

Hydrolyses 1 - 6bonds that form

'branches'in starch structure

Hydrolysis of

kappa-casein;

hydrolysis of

animal

an d

vegetable food

proteins;

hydrolysis of wheat glutens

Oxygen removal

from

food packaging;

removal of glucose

from

egg white to prevent browning

Bread improvement through improved crum b structure

Flavour enhancem ent

in

cheese products;

fat function modificationby interesterification;

synthesis

of flavour

esters

Clarification of fruitjuices by depectinisation

With pectinase in depectinisation technology

Part ofbreaddough improvem ent technology

Starch

saccharification (improvesefficiency)

Milkcoagulation

fo r

cheesemaking;

hydrolysate production for soups and savoury foods;

bread doug h improvement

*These enzymesarecommercia l ly

available

from GM O versionsof thesource microorganism .


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ENZYMES

IN

FOOD TECHNOLOG Y

parameters,and the purity is guaranteed by both the ferme ntation specification

andthe

downstream processing technology. Also,

the

ferm entation source com-

pletely avoids problems

posed

by the threat of the spread of diseases in the plan t

and animal population.

Nothing illustrates these points better than

the use of

food-grade microor-

ganisms to make chymosin, the

cheese

m ilk coagulant. T his topic is dealt

with

in

detail in

chapter

5, so a

summary will suffice here. Chymosin

is an

acid

proteinase that

is

traditionally

a

by-product

of the

milk

and

veal industry.

It

is

extracted as

'rennet'

from the calf abomasum after slaughter, and ideally

has a

hig h ratio

of

chymosin

to

pepsin,

the

adult bovine acid proteinase. Even

before the spread of bovine spongiform encephalopathy (BS E) and its hu m an

form

CJD,

the

supply

of

calf

offal

from

the

meat industry

was

unpredictable,

and

insufficient

to match the demands of the global

cheese

industry. Now the

supply situation

is

even worse,

and

unlikely

to

recover

in the

forseeable future.

Fortunately the shortfall can be made up throug h the sup ply and use of m icrobial

alternatives produced by the growing food enzyme industry.

These

alternatives

range from

fungal

acid proteinases (mainly

from

Rhizomucor miehei) to calf

chym osin produced by gene cloning technology so that the identical calf en zy m e

can be

produced

by

ferm enting food yeast

or

mould under strictly controlled

conditions.

The

following chapters

of

this volume describe

and

discuss many more

examplesof theapplicationof

microbial food enzymes

in the

commodity

sectors

of the food manufacturing and processing industry. Indeed, th eav ailability

and use of

microbial enzymes

is so

widespread that

a

considerable body

of

national

legislation has emerged and matured to ensure environmental and

consum er safety. This is trueofenzymes derived

from

both GM and non-GM

microorganisms.

1 .4 Foodenzyme legislation

The

following

is a

summary only

to

complete this overview

of the

special

considerations attached to the use of enzymes in food technology. In food

enzy m e legislation, most enzymes

are

regarded

as

processingaids because they

are added during processing for technical

reasons,

and have no function in

the

food itself. Additives have

a definite function in the

food product, such

as preservation,

antioxidation,

colouring, flavouring or

stabilisation.

There

are

grey areas,

of

course, most no tably

in thecaseof egg

w hite lysozyme

in

cheese,

where

i t

continues

to

inh ibit gas-forming bacteria

up to the

time

of

consumption.

However, the majority of food enz ym e applications are as processing aids, and

theyare

often denatured

by

heat during processing.

Food

enzymes are regulated in the UK and European Union (EU) member

states by permitted list.For an enzyme to be on the

list

itmust pass stringent


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ENZYM ES AND THEIR ACTION INFOODS 15

testing to prove absence of toxins, allergens, heav y metals, pathogen ic microor-

ganisms and other hazardous contaminants, as specified by the WHO/FAO Joint

Expert Comm ittee forFoodAdditives (JECFA). Thu s, although enzym es are not

by

definition food additives, they

are

treated

as

such

for

scrutiny before going

into

the

Permitted List. Thereafter they 'become' processing aids

and

need

not

belabelled(unless the source organism is a GM O see below). US legislation

andregulation

of

food enzymes

is

based

on

safety

in

use, whatever

the

source,

andrelies heavily on the concept of 'G enerally R egarded as Safe' (G RA S) status

for additives, ingredients and their sources.

UK and EUFoodLaw is

harmo nised

for

additives

by

Directives

and

Regula-

tionswh ich must become StatuteLaw inM ember States,but not forprocessing

aids. This can lead to differences,not in the rigour of

safety

regulation,but in

the need to label foods. US and EU Law is not harmonised at all andenzyme

applications infood m ustbehandledon a case-by-case basis.

Although there

is a

Permitted List

of

food enzymes that specifies origin,

application field and attests to safety, this does not perm it an existing enzym e

preparation on the list to be used if it has been sourced from a new organism,

or by a new

production process,

or if it is to be

used

in a

novel food process.

In

the UK , all

such intended

new

sources

and

processesmust

be

referred

to the

Advisory Committee on Novel Foods and Processes (ACNFP); the European

Commission (EC) operates an equivalent system of approval. ACNFP was

formerly an advisory body to the Ministry of Agriculture, Fisheries andFood

(M AFF) that was consulted by the industry on a vo lunta ry basis, but its increas-

ingrole

in

regulating GM Os

and

their p roducts

has led to it

becoming

a

statutory

body within the new Food Standards Agency (FSA). Clearance from ACNFP

is

necessary before

any new

food enzy m e/enzy m atic process

(GM or

non-GM )

can be broug ht to market w ithin generalFoodLaw.

ACNFP operates a system of decision trees that lead to sets of informa-

tion/testing requirements which the w ould-be producer or user of the enz ym e

must provide/carry out before ACNFP will consider the case fo rcomm ercial

application to food. This ACNFP system is the basis of new EU legislation

placing

the

same stringent requirements

on

proposed

new

uses

of

enzymes

in

EU Member States [Regulation (EC) No.258/97 onNovelFoods andNovel

FoodIngredients]. It also incorporates specific information systems, checks and

rulestoregulatethe introductionofenzymesfrom GM Os into foods.

For the purposes of EU Food Law, a GMO is 'an organism, capable of

replicating or transferring genetic material, in which the genetic material has

been altered in a waythat does nothappen naturallyb ymating and/or natural

recombination'.

This

is a

very comprehensive definition,

and it is

important

to

note that there

are now

many methods available

for

constructing GMOs that

either do not use any DNA 'foreign' to the host organism, or use methods

that produce

a final

construct that contains

no

unnatural mutations.

As

such,

these are inherentlyas safe astheir wild-typeequivalentsbut must, under EU


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16

E N Z Y M E S

IN

FOOD TECHNOLOGY

regulations, be regarded asGMOs in the same way as those constructs that

contain 'foreign' genes (e.g. maize

and soy

resistant

to

weedkiller

and

rot-proof

tomatoes). Such organisms and enzymes produced

from

them would have to

be

referred

to

ACNFP

(or

equivalent),

but

would

not

necessarily have

toappear

on the food label. In the U S, the type of genetic m odifications described above

would not result in GM O classification,andwould only require normal proof

of

safety

in

use.

All

G M O-derived food enzy me s that pass scrutiny by ACNFP and equivalen t

must

also pass th estringent safety testsand regulations requiredbyJECFA.

1.5

Modification

of

food

enzyme

activity

byprotein

engineering

Just as basic and applied research in enzymology has brought about GMO

technology

to

improve

the

range, reliability

and

purity

of

food enzymes,

it has

also made available

the

technology

of

enzy me protein engineering.

The

princi-

ples involved in protein eng ineering as they spe cifically relate to food e nzymes

are

comprehensively discussed

by

Goodenough

[6] and

need

not be

repeated

here. Suffice

it to say

that protein engineering

is a

knowledge-based method

of

changing

th e

forces

and

interactions

within

the

three-dimensional structure

of an

enzyme protein

bydirected

changes

to its

amino acid sequence through

manipulationof the gene controlling its production in the source org anism . The

aim is to alter the 'technological properties' of the enzy m e to make it more or less

stable,

change

its

optimum operating range,

or

change

its

substrate converting

range (spec ificity).

Because this technology

can

alter

th e

fundamental properties

of naturally

occurring enz ym e proteins, regulators and en zym e producers alike are not yet

considering food applications through w hich consume rs wo uld eat or be exposed

tothese novel materials.Assafety assuranceand surveillance methods become

more sophisticated,

th e

benefits

of the

technology

may be fully

realised,

but for

the present, engineered enzymes are only use in non-food applications and in

one

specific food ingredient technology invo lving

tightly-immobilised

enzymes

in

a

bioreactor. This

is the

well-established process

for

seque ntially converting

corn (maize) starch

to

dex trins, glucose,

and finally

fructose,

to

produce

a

sweet

syrup

(high -fructose corn syrup, HFCS)

that

is

used

extensively

in

carbonated

soft

drinks [11 ,12 ] .

The key

enzyme

in

this process

is

xylose isomerase (also known

as

glu-

cose isomerase within

th e

industry).

It is

immobilised

and

used

in a

packed

column reactor as the final processing stage in which the glucose released by

starch saccharification

is

isomerised

to

fructose.

The

conditions under

which

the

native microbial xylose

isomerase is

used

in the

complete process

are set

by a compromise between the optimum reaction temperature fo renzymat ic

isomerisation,

and the

rate

of

enzym e denaturation

as aresultofdestabilisation


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ENZYM ES AND THEIR ACTION IN FOODS i 7

of

the folded (quarternary) structure of the enzyme protein. The active site of

the enz ym e is formed through a molecular association of protein monom ers

held together

by

interracial hydrogen bonding

and

salt bridges

(figure

1,3).

The

process-limiting inactivation

of

xylose

isomerase

involves destabilisation

of

this

molecular association through the chemical glyc osylation of lysine epsilon-

amino groups that mediate

th e

molecular association.

The

glycosylation happens

quite quickly

because

the enzyme reactor temperature is relatively high, and

starch-derived glucose is present at high concentrations to react with the am ino

groups. Redesign engineering strategies, based

on

computer graphics-aided

protein engineering, identified a particular lysine residue (253) at the interface of

threeof the glucose isomerase monomers (A, B and D;

figure

1.3) which , wh en

substituted

with

arginine throug h site-directed mutagenesis, reduced

th e

rate

of

am ino group glycosylation, maintained the spatial orientation of position 253 in

the

quarternary structure,

and

created

a new

stabilising hydrogen bond between

the mo nom ers [13].

T he

practical result

of the

engineering

exercisehas

been

the

creation of a new catalytically-active xyloseisomerasewhich is not only more

stablein

solution

at6070C,but

less susceptible

to

glycosylation

by

glucose

at

the

elevated operating temperature. Th e 'new ' enzym e has a volum etric half-life

at

60C

of 155 0h over a 10 week test period, whereas the equivalentvalue for

wild-type

x ylose isomerase

is 607 h.

Figure

1 .3 The

effects

of replacing

lysine

residue 253 in the amino acid sequence of a monomero f

Actinoplanes missouriensis

xylose isomerase

by an arginine

residue. Bold lines represent

the

carbon

skeleton

of lysine 253 (dashed) in the

wild-type enzyme,

and the

replacement arginine (solid)

in the

mutated

enzyme.

The

other amino acids

a nd

their sequence numbers

are

m erely representations

of

their

approximate

positions near

the

monomer boundaries

of

protein monomers

A, C and D .

Hy drogen bond

interactions

ar e

indicated

by

dotted linesbetween

th e

monomer boundary interfaces. Reproduced

with

permission ofSocietyofChemicalIndustry.

ImageNot Available


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18

ENZYMESINFOOD TECHNOLOGY

1.6 Summary

andconclusions

The food manufacturing and ingredients industry makes widespread use of

enzym es in both traditionalsectorssuch as baking, brewing and cheesemaking,

butalso in new

areas

such as fat mo dification and sweetener technology. A

degree

of

care

and in ge nuity is oftenneeded to adapt these fragile biological catalysts

to industrialprocesses,but acom binationofbasic biochemical knowledge and

modern biotechnology

is

opening

up new

areas

of

application, especially

fo r

enzymes of microbial origin, and animal enzymes produced in microbes by

genetic engineering technology.

References

1.

Dix on, M .. Webb,E.G.

Enzymes.

London: Longman;1979.

2. Wong,W.S.Food

Enzymes. Structure and Function.

New York: Chapm an & H all:1995.

3.

Fullbrook,

P.D.

Practical applied kinetics.

In:

Godfrey,

T. ,

West,

S.

(eds).

Industrial Enzym ology.

2

nd

edn.

Basingstoke: Macmillan

Pressand New

York: Stockton

Press:

1996:483501.

4.

Godfrey,

T.,

West.

S.(eds)


2

nd

edn.

Basingstoke: M acm illan Press

and

New

York: Stockton

Press;1996.

5.

Fullbrook.

P.D.

P ractical

limits

and prospects (k ine tic s) . In: God frey, T., West, S. (eds).

Industrial

Enzymology. 2

nd

edn. Basingstoke: Mac millan

Pressand New

York: Stockton

Press;1996: 503540.

6. Goodenough, P.W.

Food

enzymes and the new technology. In: Tucker.G.A..Woods, L.F.J. (e ds) .

Enzymes in

Food

Processing.

2

nd

edn. Glasgow: B lackie Academ ic

and

Professional:

1995:41113.

7. Law, B.A.

Manipulation

of

enzymes

fo r industrial

application: Protein

and

environmental

engineering.

In: G odfrey, T.. West, S. (eds).


2

nd

edn. Basingstoke :

M acmillan

Pressand New

York: Stockton Press;

1996:385-393.

8. Godfrey, T, W est. S. Introduc tion to

industrial

enzym ology. In: Godfrey, T., West, S. (eds).

Industrial

Enzymology.

2

nd

edn.

Basingstoke: M acmillan

Press

and N ew York: Stockton Press:

1996: 18.

9. Intern ation al Dairy Federation. Provisional IDF Standard 157 A.1997.

10 . Pandey,A.,Benjamin,S. ,Soccol,C.R.,Nigam.P. .Krieger.N .,

Soccol.

V.T.The

realm

oflipasesin

biotechnology.

Biotechnol. Appl. Biochem.

1999:29: 119131.

11.

Che n, W.-P. Glucose isomerase.

A

review.

Process Biochem.

1980;

June/July:

30-35.

12. Chen, W.-P. Glucose isomerase.

A

review.

Process Biochem. 1980;August/September:3641.

13 .

Q uax. W.J., M rabet, N.T., Luiten , R.G .M ., Sh wer huiz en, P.W., Stanssens.

P. .Lasters.I.Stabilisation

of

xylose isomerase

by

lysine residue engineering.

Bio/Technology.

1991:

8:

783742.


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Enzymes

for

bread, pasta

and

noodle

products

Joan

Qi Si andCorneliaDrost-Lustenberger

2.1 Introduction

Bread, noodlesandpastaareamongthemost basic, comm onandlow-cost foods

and their manufactureis

often

dependent on the traditions of the countries or

regions in which they are consumed. However, ancient as bread, noodle and

pasta-making are, theyare also closely related to biotechnology.

This chapter reviews

the

effects

ofdifferent

types

of

enzymes

and the

syner-

gistic

effects

when they are combined w ith each other during the bread-m aking

process.

It

willalso give examples

of

enzyme

use in

Asian noodles

and

pasta.

2.2

Bread

Con sum ers h ave certain quality criteria

fo r

bread, inclu ding appearance,

fresh-

ness, taste, flavour, variety, and a consistent q uality. It is a great challenge for

the

baking industry

to

meet these criteria,

for

several reasons. Firstly,

the

main

ingredient

of the

breadflourvaries

due to

wheat variety, weather during

the

growing season,andmilling technology. Although millers attempttoblend

wheatfrom

different sources to produce flour w ith a good and consistent baking

quality, itoften

proves difficult

to

satisfy both high-quality

and

low-cost stan-

dards at the same time. Secondly, because bread preferences

differ,

the baking

industry

uses ingredients with

different

qualities

and

em ploys different baking

procedures.Forinstance, English sandw ich bread, w ithits finecrum b structure

and

very soft texture,

is not

popular with

the

French,

who

want baguettes

with

crispy crust, large holes,

and

good crumb chewiness. Thirdly, consumer

preferences are

shifting

towards healthier products. It is sometimes possible

to make new bread varieties by simply adjusting the formulation or baking

procedure. However,

in

other

cases,

bakers

m ay

have

to

develop

new

techniques.

Therefore, both millers and bakers need ingredients or process aids such as

chemical oxidants, emulsifiers

and

enzymes

to

standardise

the

quality

of the

products and

diversify

the product range.

For

decades enzym es such

as

malt

and

microbial alpha-amylases have been

used

fo r

bread making.

Due to the

changes

in the

baking industry

and the

demand for more varied and natural products, enzymes have gained more and

mo re impo rtance in bread form ulations. Throu gh new and rapid developments in

biotechnology,a

number

of new

enz ym es have recently been m ade available

to


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20

E N Z Y M E S

IN

FOOD TECHNOLOGY

the baking industry. One exam ple is pure xylanase, w ith single activity instead of

traditional hem icellulase preparations, which improves the doug h mach inab ility.

Alipasehas a

gluten

strengthening

effect that results

inmorestable

dough

and

improved crumb structure similar

to

DATEM

orSSL/CSL and a

maltogenic

alpha-amylase thathas auniqueanti-staling effect.

2.2.7

Fungal

alpha

-amylases

Wheat and

thus wheat

flour

contains

endogenous and

indigenous enzymes,

mainly amylases. However, the level of amylase activity varies from one type

of wheat to another. The am oun t ofalpha-amylases in most sound, ungerm inated

wheat

or rye flours is

negligible

[1].

Therefore,

most

bread

flours

must

be

supplemented withalpha-amylases, added in the form of maltflour or

fungal

enzymes.

Many

methodsare

available

for the

determination

ofamylase

activities

as

reviewed by K ruger and Lineback [2]. The baking industry and millers use other

methods such as

falling

number (FN) and Brabender amylograph to determine

the

amylase content andcorrelate tobread making quality. Although FN is

excellent fo r measuring th e activityof cereal am ylases in clu ding those inmalt

flour,

it

is not

suitable

fo r

measuring

the

activity

of

fungal

alpha-

amylases

.

Fungal

alpha-amylasesare

generallyless

thermostable;

they

are

inactivated

at

temperatures near

65C.

Therefore,

fungal

alpha

-amylases cannot

be

detected

by the standard FN method, which is conducted at 100C (AACC method

56-8IB, 1972). Perten

and co-workers

reported

a

modified

FN

method

for

measuring

the fungal alpha

-amylases activity

in flour

[3].

Fungal alpha-amylases act on the damaged starch content, which can vary

depending

on

wheat variety

and

m illingconditions.Generally,

flour

made from

hard wheat contains more damaged starch than the

soft

wheat. The

alpha-

amylases widely used

in the

baking industry

can

hydrolyse amylose

and

amylopectin

to

release soluble intermediate-size dextrins

of

DP2DP12 [4].

The

alpha-amylases provide fermentable sugar, which results

in an

increased

volume, better crust colour, and improved flavour. Due to hydrolysis of the

damaged starch,

a

suitable dosage

of

alpha-amylases results

in a

desirable

dough softening. However, extensive degradation of the damaged starch due

to an overdose ofalpha-amylases com monly leads to sticky dough.

Figure

2.1

illustrates

the

effect

of a

fungal amylase

on bread

quality

in

terms of bread volum e, crumb structure and dough characteristics. The volum e

andcru mb structure improve w ith increasing dosage of

fungal

alpha-amylases.

Although

a

high

dosagecanprovidea

larger volume

increase,the

dough w ould

be too sticky to work w ith. The o ptim um dosage is thu s

defined

as the dosage

with maximum reachable volume without a sticky dough. For the examples

in figure

2.1,

the

optimum dosage

fo r

both

flours is

around

15

FAU/kg

flour

(FAU

=

fungal alpha-amylase units) .


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Documents

65272819 Enzymes in Food Technology