UNIVERSITI PUTRA MALAYSIA

SAMANA SHABANIKAKROODI

IB 2014 8

CHARACTERIZATION OF OIL FROM LIVER AND VISCERAL FATS OF PATIN (Pangasianodon hypophthalmus SAUVAGE) AND ITS USE IN

HAND CREAM PREPARATION

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CHARACTERIZATION OF OIL FROM LIVER AND VISCERAL FATS OF

PATIN (Pangasianodon hypophthalmus SAUVAGE) AND ITS USE IN HAND

CREAM PREPARATION

By

SAMANA SHABANIKAKROODI

Thesis submitted to the School of Graduates Studies, Universiti Putra Malaysia, in

Fulfillment of the Requirements for the Degree of Doctor of Philosophy

June 2014

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of

the requirement for the degree of Doctor of Philosophy.

CHARACTERIZATION OF OIL FROM LIVER AND VISCERAL FATS OF

PATIN (Pangasianodon hypophthalmus SAUVAGE) AND ITS USE IN HAND

CREAM PREPARATION

By

SAMANA SHABANIKAKROODI

June 2014

Chairman: Annie Christianus, PhD

Institute: Institute of Bioscience

The visceral fat and liver of patin, Pangasianodon hypophthalmus, are considered as

waste from fish processing. Converting these byproducts into fish oil not only provide a

good source of health promoting fatty acids but also can be used for the production of

value added products.

The proximate and fatty acid compositions of the visceral storage fat and liver of patin

were determined. Fat content of the visceral fatty tissue of female and male patin were

77.64 and 73.23%, respectively, whereas fat content of the liver of female and male

were 11.71 and 9.59%, respectively. The total amounts of monounsaturated and

polyunsaturated fatty acids of the liver of female and male catfish were 51.03 and 50.55

%, respectively, whereas in fatty tissue of female and male were 50.46 and 50.31%,

respectively.

Refining was done on the extracted oil from byproducts of patin to produce a more pure

and stable product. The effect of refining procedure on the fatty acid composition of oil

from various refining steps and the quality of the final product were investigated. Fatty

acids found in crude and refined patin oil were C12:0, C14:0, C14:1, C16:0, C16:1,

C18:0, C18:1, C18:2, C18:3, C18:4, C20:0, C20:1, C20:4, C20:5, and C22:6. The

amount of saturated fatty acids of refined oil were significantly (P < 0.05) higher than

that of crude oil. The total amounts of monounsaturated fatty acids of oil from different

refining steps were not significantly (P > 0.05) different, whereas the total amount of

polyunsaturated fatty acids of refined oil was significantly (P < 0.05) lower than that of

crude oil. The eicosapentaenoic acid, docosahexaenoic acid, and n-3 to n-6 ratio of

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refined oil were 0.83, 0.79%, and 1.01, respectively. Analysis of the physical and

chemical properties of refined oil indicated the suitability of patin oil to be used in the

formulation of hand cream.

In the present study, the efficacy and stability of various hand cream formulations using

0, 1.0, 2.5 and 5.0% of fish oil were investigated and compared. The appearance, odor,

texture, and pH of formulations containing 1 and 2.5% of fish oil remained stable in

very good qualities during the storage, whereas the related values to the appearance and

texture of formulation containing 5.0% of fish oil at the end of 6 months of storage at

45°C were significantly (P < 0.05) lower than other formulations and previous

evaluations of this formulation. Weight loss and microbial contamination were not

observed in all formulations under study. Efficacy of preservative system was proved

through preservative challenge tests. Efficacy test indicated that the formulations

containing fish oil were preferred by volunteers rather than the one without the fish oil.

Addition of fish oil significantly (P < 0.05) increased the moisturizing effects of hand

creams. The formulation containing 2.5% of fish oil obtained the best related values in

all investigated items including skin texture, moisture, smoothness, brightness, and well

being perception.

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Abstrak tesis dikemukakan kepada Senat Universiti Putra Malaysia untuk memenuhi

keperluan untuk ijazah Doktor Falsafah

PENCIRIAN MINYAK DARI HATI DAN LEMAK VISCERA

PATIN (Pangasianodon hypophthalmus SAUVAGE) DAN PENGGUNAANNYA

DALAM PENYEDIAAN KRIM TANGAN

Oleh

SAMANA SHABANIKAKROODI

Jun 2014

Pengerusi: Annie Christianus, PhD

Institut: Institut Biosains

Simpanan lemak viscera dan hati patin, Pangasianodon hypophthalmus merupakan sisa

dari pemprosesan ikan. Penukaran bahan sampingan ini kepada minyak ikan bukan

sahaja sebagai sumber asid lemak yang baik untuk kesihatan tetapi boleh digunakan

untuk penghasilan produk yang boleh ditambah nilai.

Komposisi proksimat dan asid lemak simpanan lemak viscera dan hati patin telah

ditentukan. Kandungan lemak tisu lemak viscera patin betina dan jantan adalah 77.64

dan 73.23%, masing-masingnya. Manakala kandungan lemak hati betina dan jantan

adalah 11.71 dan 9.59%, setiap satunya. Jumlah kandungan asid lemak mono tak tepu

dan poli tak tepu hati betina dan jantan adalah 51.03 dan 50.55 %, setiap satunya,

manakala tisu lemak betina dan jantan adalah 50.46 dan 50.31%, masing-masingnya.

Penulenan dijalankan ke atas minyak yang diekstrak dari bahan sampingan patin untuk

menghasilkan produk yang lebih tulen dan stabil. Kesan prosedur penulenan ke atas

komposisi asid lemak minyak dari berbagai peringkat penulenan dan kualiti produk

yang terhasil dikaji. Asid lemak yang terdapat dalam minyak patin tanpa dan dengan

penulenan adalah C12:0, C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3,

C18:4, C20:0, C20:1, C20:4, C20:5, dan C22:6. Kandungan asid lemak tepu dalam

minyak yang telah ditulenkan ketara lebih tinggi (P < 0.05) benbanding minyak

mentah. Jumlah kandungan asid lemak mono tak tepu dari minyak yang terhasil dari

peringkat penulenan yang berbeza tidak menunjukkan perbezaan yang ketara (P > 0.05),

manakala jumlah kandungan asid lemak poli tak tepu minyak yang ditulenkan ketara

lebih rendah (P < 0.05) berbanding dengan minyak mentah. Asid eikosapentaenoik,

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dokosahexaenoik, dan nisbah n-3 kepada n-6 minyak yang ditulenkan adalah 0.83,

0.79%, dan 1.01, masing-masingnya. Analisis ciri fizikal dan kimia minyak yang

ditulenkan menunjukkan kesesuaian minyak patin untuk digunakan dalam formulasi

krim tangan.

Dalam kajian ini, keberkesanan dan kestabilan beberapa formulasi krim tangan

menggunakan 0, 1.0, 2.5 dan 5.0% minyak ikan dikaji dan dibandingkan. Rupabentuk,

bau, tekstur, dan pH formulasi yang mengandungi 1 dan 2.5% minyak ikan ternyata

stabil dengan kualiti yang baik semasa tempoh penyimpanan, manakala nilai yang

berkaitan dengan rupabentuk dan tekstur formulasi yang mengandungi 5% minyak ikan

pada akhir tempoh 6 bulan simpanan pada 45°C ketara menurun (P < 0.05) berbanding

dengan yang formulasi yang lain dan dari formulasi yang telah dinilai sebelumnya.

Tidak terdapat sebarang kehilangan berat dan pencemaran mikrobial diperhatikan dalam

kajian ini. Keberkesanan sistem pengawetan terbukti melalui kajian cabaran

pengawetan. Kajian keberkesanan menunjukkan bahawa formulasi yang mengandungi

minyak ikan menjadi pilihan sukarelawan berbanding dengan yang tidak meengandungi

minyak ikan. Penambahan minyak ikan meningkatkan kesan pelembapan krim tangan

dengan ketaranya (P < 0.05). Formulasi yang mengandungi 2.5% minyak ikan mendapat

nilai terbaik diantara semua bahan yang dikaji termasuk tekstur kulit, lembapan,

kelembutan, kecerahan, dan persepsi keselesaan.

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ACKNOWLEDGEMENTS

I would like to express the deepest appreciation to my supervisory committee chairman,

Dr. Annie Christianus, who has the attitude of a devoted genius. She continuously and

convincingly conveyed a spirit of adventure in regard to this research. Without her

guidance and persistent help this dissertation would not have been possible.

I would like to express my very great appreciation to Prof. Dr. Tan Chin Ping for his

indispensable and constructive comments during the planning and development of this

thesis. He generously has given his precious knowledge, wisdom, and time which have

been very much appreciated.

I would like to express my sincere gratitude in memory of Prof. Dr. Yaakob b. Che Man

for his kind co operations. He will be sorely missed by those who knew him.

I would like to express my deep gratitude to Dr. Fariborz Ehteshami for his enthusiastic

encouragement and useful critiques of this thesis. My grateful thanks are also extended

to all those who provided me the possibility to complete this thesis.

Furthermore, I would like to acknowledge with much appreciation the crucial role of the

staff of Mero Cosmetic Factory in Iran, who gave me the permission to use all required

equipments and the necessary materials to prepare and evaluate various hand cream

formulations. I would also like to thank the staff of the Maad laboratory in Iran for their

valuable technical supports during fish oil analysis and refining.

I wish to express my heartfelt thanks to my parents for their endless love, kindness,

encouragement and invaluable support they have shown during all my life and

especially during the past four years that has taken me to finalize this thesis. I would

also like to express my gratitude to my sister, Setareh, who has been greatly tolerant and

supportive during my study.

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I certify that a Thesis Examination Committee has met on 26 June 2014 to conduct the

final examination of Samana Shabanikakroodi on her thesis entitled “Characterization of

Oil from Liver and Visceral Fats of Patin (Pangasianodon hypophthalmus SAUVAGE)

and its Use in Hand Cream Preparation” in accordance with the Universities and

University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia

[P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded

the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Hassan bin Hj Mohd Daud, PhD

Associate Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Chairman)

Che Roos bin Saad, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Internal Examiner)

Hishamuddin bin Omar, PhD

Senior lecturer

Faculty of Science

Universiti Putra Malaysia

(Internal Examiner)

Paul Kam Shing Shin, PhD

Associate Professor

City University of Hong Kong

China

(External Examiner)

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 21 July 2014

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The

members of the Supervisory Committee were as follows:

Annie Christianus, PhD

Senior lecturer

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Tan Chin Ping, PhD Professor

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Member)

Yaakob bin Che Man, PhD

Professor

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Member)

_______________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate studies

Universiti Putra Malaysia

Date:

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DECLARATION

Declaration by graduate student

I hereby confirm that:

This thesis is my original work;

Quotations, illustrations and citations have been duly referenced;

This thesis has not been submitted previously or concurrently for any other

degree at any other institutions;

Intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

Written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and Innovation) before thesis is published (in the

form of written, printed or in electronic form) including books, journals,

modules, proceedings, popular writings, seminar papers, manuscripts, posters,

reports, lecture notes, learning modules or any other materials as stated in the

Universiti Putra Malaysia (Research) Rules 2012;

There is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: _______________________ Date: __________________

Name and Matric No.: _________________________________________

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Declaration by Members of Supervisory Committee

This is to confirm that:

The research conducted and the writing of this thesis was under our supervision;

Supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: _______________________

Name of Chairman of Supervisory

Committee: _______________________

Signature: _______________________

Name of Member of Supervisory

Committee: _______________________

Signature: _______________________

Name of Member of Supervisory

Committee: _______________________

Signature: _______________________

Name of Member of Supervisory

Committee: _______________________

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vi

APPROVAL vii

DECLARATION ix

LIST OF TABLES xiv

LIST OF FIGURES xix

LIST OF APPENDICES xxiii

LIST OF ABBREVIATIONS

xxiv

CHAPTER

1. INTRODUCTION 1

1.1 Problem statement and justification 2

1.2 Specific objectives

3

2. LITERATURE REVIEW 5

2.1 Fish oil 5

2.1.1 Patin catfish 8

2.1.2 Principal components of fish oil 7

2.1.3 Factors influencing the fatty acid composition of fish oil 8

2.1.4 Common practice of fish oil extraction 10

2.1.4.1 Low temperature solvent extraction 10

2.1.4.2 Wet and steam rendering method 10

2.1.4.3 Enzymatic extraction 11

2.1.4.4 Supercritical fluid extraction 12

2.1.4.5 Urea complexation 12

2.1.5 Fish oil refining processes and their influences 13

2.1.6 Fish oil applications 14

2.1.6.1 In medicine and pharmaceutics 14

2.1.6.2 In food industry 16

2.1.6.3 In cosmetics 16

2.1.6.4 Other applications 17

2.1.7 Principals of the effect of fatty acids on skin 17

2.1.8 The antimicrobial efficacy of oil 18

2.1.8.1 Historical background 18

2.1.8.2 Antimicrobial efficacy of fatty acids 18

2.2 Hand cream 21

2.2.1 Skin morphology 22

2.2.2 Principals of hand care products 22

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2.2.2.1 Hand cleansing products 22

2.2.2.2 Hand tonics 22

2.2.2.3 Hand moisturizing and nourishing products 23

2.2.3 Development of hand cream based on natural product 23

2.2.4 Quantitative and qualitative evaluation of hand cream 24

2.2.5 Safety of hand cream 26

2.2.5.1 Skin irritation 27

2.2.5.2 Skin sensitization

27

3. MATERIALS AND METHODS 28

3.1 Fatty acid composition of fatty tissue, liver, and combination of both 28

3.1.1 Sample preparation 28

3.1.2 Fat extraction 28

3.1.2.1 Fat extraction by the method of Bligh and Dyer 28

3.1.2.2 Fat extraction by the method of Sathivel et al 29

3.1.3 Esterification of fatty acids 30

3.1.4 Fatty acid analysis by Gas Chromatography 30

3.2 Proximate analysis 31

3.2.1 Lipid content 31

3.2.2 Moisture content 31

3.2.3 Protein content 31

3.3 Oil refining 32

3.3.1 Procedure of oil refining 32

3.3.1.1 Degumming 33

3.3.1.2 Neutralizing 33

3.3.1.3 Bleaching 34

3.3.1.4 Deodorizing 35

3.3.2 Evaluating the refined fish oil specifications 36

3.3.2.1 Peroxide value 36

3.3.2.2 Free fatty acid 37

3.3.2.3 Iodine value 37

3.3.2.4 Saponification value 38

3.3.2.5 Melting point 38

3.3.2.6 Specific gravity 39

3.3.2.7 Refractive index 39

3.4 Challenging the antimicrobial efficacy of fish oil 39

3.4.1 Microbial strains and culture condition 39

3.4.2 Disk diffusion test 40

3.4.3 Agar well diffusion test 42

3.4.4 Broth dilution test 43

3.5 Hand cream preparation 44

3.5.1 Procedure of hand cream production 44

3.5.2 Testing the stability of hand creams 46

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3.5.2.1 Sampling set up 46

3.5.2.2 Appearance, odor, and texture 47

3.5.2.3 pH 47

3.5.2.4 Weight loss 48

3.5.2.5 Microbiological limit tests 48

3.5.2.5.1 Total aerobic microbial count 48

3.5.2.5.2 Total combined molds and yeasts count 49

3.5.2.5.3 Test for Pseudomonas aeruginosa 49

3.5.2.5.4 Test for Staphylococcus aureus 49

3.5.2.5.5 Test for Escherichia coli 49

3.5.2.5.6 Test for Candida albicans 50

3.5.2.6 Preservative challenge tests 50

3.5.2.6.1 Bacteria 50

3.5.2.6.2 Yeasts 50

3.5.2.6.3 Molds 51

3.5.3 Skin irritation test 51

3.5.3.1 The in vivo dermal irritation test 51

3.5.3.2 Clinical observations of skin reactions 53

3.5.4 Testing the efficacy of hand creams 55

3.6 Statistical analysis

56

4. RESULTS AND DISCUSSION 57

4.1. Proximate composition of liver and fatty tissue 57

4.2. Fatty acid profile of liver and fatty tissue 58

4.3. Yield of oil from each refining step 62

4.4. Fatty acid compositions of oil from each refining step 63

4.5. Specifications of the refined patin catfish oil 67

4.6 Antimicrobial efficacy of crude and refined patin catfish oil 70

4.7. Stability of the hand creams 75

4.8. Skin irritation 101

4.9. Efficacy of the hand creams

105

5. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS FOR

FUTURE RESEARCH

121

REFERENCES 123

APPENDICES 139

BIODATA OF STUDENT 159

LIST OF PUBLICATIONS 160

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LIST OF TABLES

Table

Page

2.1 Factors influencing the fatty acid composition of fish oil

9

2.2 Various refining steps and their effects

14

2.3 Antibacterial effect of Eicosapentaenoic Acid (EPA)

19

2.4 Effect of the structure of various fatty acids against Bacillus larvae

20

3.1 Components in each formulation under study

46

3.2 Grading of erythematic reactions

54

3.3 Grading of edema formation

55

4.1 The total length and weight of female and male P. hypophthalmus and

average weight of the liver and fatty tissue

57

4.2 Protein, fat, and moisture content of fatty tissue and liver of male and V

female P. hypophthalmus

58

4.3 Fatty acid composition of fatty tissue and liver of male and female P.

hypophthalmus

60

4.4 The total n-3 fatty acids, n-6 fatty acids, and n-3/ n-6 ratios of fatty

tissue and liver of male and female P. hypophthalmus

62

4.5 Quantity of oil recovered from each refining step

63

4.6 Fatty acid composition of oil from each refining step

65

4.7 Total fatty acid content of patin and three commercial fish oils

66

4.8 The total n-3 fatty acids, n-6 fatty acids, and n-3/ n-6 ratios of P.

hypophthalmus oil from each refining step

67

4.9 Melting points of various fatty acids

68

4.10 The physical and chemical properties of refined patin catfish oil

70

4.11 The amounts of EPA and DHA of crude and refined patin catfish oil,

Sardinella longiceps, and Sardinella fimbriata

71

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4.12 The results of total aerobic microbial count of various formulations

under study during storage at 4°C

79

4.13 The results of total aerobic microbial count of various formulations

under study during storage at 25°C

79

4.14 The results of total aerobic microbial count of various formulations

under study during storage at 37°C

80

4.15 The results of total aerobic microbial count of various formulations

under study during storage at 45°C

80

4.16 The results of total combined molds and yeasts count of various

formulations under study during storage at 4°C

81

4.17 The results of total combined molds and yeasts count of various

formulations under study during storage at 25°C

81

4.18 The results of total combined molds and yeasts count of various

formulations under study during storage at 37°C

82

4.19 The results of total combined molds and yeasts count of various

formulations under study during storage at 45°C

82

4.20 The results of Pseudomonas aeruginosa count of various formulations

under study during storage at 4°C

83

4.21 The results of Pseudomonas aeruginosa count of various formulations

under study during storage at 25°C

83

4.22 The results of Pseudomonas aeruginosa count of various formulations

under study during storage at 37°C

84

4.23 The results of Pseudomonas aeruginosa count of various formulations

under study during storage at 45°C

84

4.24 The results of Staphylococcus aureus count of various formulations

under study during storage at 4°C

85

4.25 The results of Staphylococcus aureus count of various formulations

under study during storage at 25°C

85

4.26 The results of Staphylococcus aureus count of various formulations

under study during storage at 37°C

86

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4.27 The results of Staphylococcus aureus count of various formulations

under study during storage at 45°C

86

4.28 The results of Escherichia coli count of various formulations under

study during storage at 4°C

87

4.29 The results of Escherichia coli count of various formulations under

study during storage at 25°C

87

4.30 The results of Escherichia coli count of various formulations under

study during storage at 37°C

88

4.31 The results of Escherichia coli count of various formulations under

study during storage at 45°C

88

4.32 The results of Candida albicans count of various formulations under

study during storage at 4°C

89

4.33 The results of Candida albicans count of various formulations under

study during storage at 25°C

89

4.34 The results of Candida albicans count of various formulations under

study (F1: without fish oil, F2: 1.00% fish oil, F3: 2.50% fish oil, and

F4: 5.00% fish oil) during storage at 37°C

90

4.35 The results of Candida albicans count of various formulations under

study during storage at 45°C

90

4.36 The results of Pseudomonas aeruginosa challenge test on various

formulations under study after 6 months of hand cream storage at 4°C

91

4.37 The results of Pseudomonas aeruginosa challenge test on various

formulations under study after 6 months of hand cream storage at

25°C

92

4.38 The results of Pseudomonas aeruginosa challenge test on various

formulations under study after 6 months of hand cream storage at

37°C

92

4.39 The results of Pseudomonas aeruginosa challenge test on various

formulations under study after 6 months of hand cream storage at

45°C

93

4.40 The results of Escherichia coli challenge test on various formulations

under study after 6 months of hand cream storage at 4°C

93

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4.41 The results of Escherichia coli challenge test on various formulations

under study after 6 months of hand cream storage at 25°C

94

4.42 The results of Escherichia coli challenge test on various formulations

under study after 6 months of hand cream storage at 37°C

94

4.43 The results of Escherichia coli challenge test on various formulations

under study after 6 months of hand cream storage at 45°C

95

4.44 The results of Staphylococcus aureus challenge test on various

formulations under study after 6 months of hand cream storage at 4°C

95

4.45 The results of Staphylococcus aureus challenge test on various

formulations under study after 6 months of hand cream storage at

25°C

96

4.46 The results of Staphylococcus aureus challenge test on various

formulations under study after 6 months of hand cream storage at

37°C

96

4.47 The results of Staphylococcus aureus challenge test on various

formulations under study after 6 months of hand cream storage at

45°C

97

4.48 The results of Candida albicans challenge test on various

formulations under study after 6 months of hand cream storage at 4°C

97

4.49 The results of Candida albicans challenge test on various

formulations under study after 6 months of hand cream storage at

25°C

98

4.50 The results of Candida albicans challenge test on various

formulations under study after 6 months of hand cream storage at

37°C

98

4.51 The results of Candida albicans challenge test on various

formulations under study after 6 months of hand cream storage at

45°C

99

4.52 The results of Aspergillus niger challenge test on various formulations

under study after 6 months of hand cream storage at 4°C

99

4.53 The results of Aspergillus niger challenge test on various formulations

under study after 6 months of hand cream storage at 25°C

100

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4.54 The results of Aspergillus niger challenge test on various formulations

under study after 6 months of hand cream storage at 37°C

100

4.55 The results of Aspergillus niger challenge test on various formulations

under study after 6 months of hand cream storage at 45°C

101

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LIST OF FIGURES

Figure

Page

1.1 Patin catfish on the measuring ruler

1

1.2 Gonad of male (a) and female (b) patin catfish

4

2.1 Patin (Pangasianodon hypophthalmus)

5

2.2 Production cycle of P. hypophthalmus

6

2.3 Global production statistics of P. hypophthalmus

7

2.4 Schematic representation of a: eicosapentaenoic acid (EPA, 20:5 n-3)

and b: docosahexaenoic acid (DHA, 22:6 n-3)

8

2.5 Industrial method for the production of fish oil and fishmeal

11

2.6 Schematic figure of supercritical fluid extraction and fractionation of

fish oil fatty acid ethyl esters

12

3.1 The liver (a) and visceral storage fat (b) of patin

28

3.2 Heating the mixture of liver and fatty tissue (a) and sample after

centrifugation (b)

29

3.3 Gas Chromatography (GC Varian, model 3400)

30

3.4 Oil sample after addition of aqueous citric acid solution

33

3.5 Heating the sample after addition of sodium hydroxide (a), sample after

addition of demineralized water (b), remaining soap in the beaker (c),

and sample after centrifugation (d)

34

3.6 Sample before the addition of activated earth (a), after the addition of

activated earth (b), and after centrifugation (c)

35

3.7 Laboratory deodorization unit

36

3.8 Acetone, suspension of refined and crude patin oil in acetone from left

to right (a), and stocks of five different microbs which were used in

assaying the antimicrobial efficacy of crude and refined patin catfish

41

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oil (b)

3.9 Microbial strain was swabbed on Mueller Hinton Agar plates

42

3.10 One well was cut in each plate

43

3.11 Refined patin catfish oil (left) and crude oil (right)

44

3.12 Water phase (left) and oil phase (right) at 75°C (a), mixing the two

phase (b), addition of perfumes to the emulsion

45

3.13 Hand cream in its special container

47

3.14 Evaluating the pH value of various hand cream samples

48

3.15 Three mice were used for irritation test of each hand cream

formulation

52

3.16 Fur was removed from the dorsal area of the mouse

52

3.17 Hand cream was applied on the skin

53

3.18 Non-irritating tapes around the bodies of mice

53

4.1 Biosynthesis of polyunsaturated fatty acids

61

4.2 Sn-1, sn-2, and sn-3 positions in the structure of triacylglycerol

64

4.3 Disk diffusion susceptibility assay testing the effect of aceton against

Pseudomonas aeruginosa (before (a-1) and after (a-2) incubation),

Staphylococcus aureus (before (b-1) and after (b-2) incubation),

Escherichia coli (before (c-1) and after (c-2) incubation), Candida

albicans (before (d-1) and after (d-2) incubation)

72

4.4 Disk diffusion susceptibility assay testing the effect of crude patin oil

against Staphylococcus aureus (before (a-1) and after (a-2) incubation),

Escherichia coli (before (b-1) and after (b-2) incubation), Candida

albicans (before (c-1) and after (c-2) incubation), Aspergillus niger

(before (d-1) and after (d-2) incubation)

73

4.5 Disk diffusion susceptibility assay testing the effect of refined patin oil

against Pseudomonas aeruginosa (before (a-1) and after (a-2)

incubation), Staphylococcus aureus (before (b-1) and after (b-2)

74

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incubation), Escherichia coli (before (c-1) and after (c-2) incubation),

Candida albicans (before (d-1) and after (d-2) incubation), Aspergillus

niger (before (e-1) and after (e-2) incubation)

4.6 Agar well diffusion test assaying the effect of crude patin oil against

Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli,

Candida albicans, Aspergillus niger

75

4.7 Liquid paraffin (a) and refined patin catfish oil (b)

76

4.8 The values related to the appearance (a), odor (b), and texture (c) of the

hand cream formulations under study (F1: without fish oil, F2: 1.00%

fish oil, F3: 2.50% fish oil, and F4: 5.00% fish oil). The symbol (*)

indicates statistically significant difference between treatments (P <

0.05)

77

4.9 The in vivo dermal irritation test did not show skin irritation of mouse

after four hours of hand cream application

102

4.10 Erythematic reactions and edema were not observed after one hour of

hand cream application

103

4.11 Erythematic reactions and edema were not observed after 24 hours of

hand cream application

103

4.12 Erythematic reactions and edema were not observed after 48 hours of

hand cream application

104

4.13 Erythematic reactions and edema were not observed after 72 hours of

hand cream application

104

4.14 The values related to the effect of the various hand creams on skin

texture after 30 days of application

105

4.15 The values related to the effect of the various hand creams on skin

moisture after 30 days of application

106

4.16 The values related to the effect of the various hand creams on skin

smoothness after 30 days of application

107

4.17 The values related to the effect of the various hand creams on skin

brightness after 30 days of application

108

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4.18 The values related to the well being perception of the effect of the

various hand creams on skin after 30 days of application

109

4.19 The values related to the effect of the various hand creams on skin

texture one week after the end of the treatment

110

4.20 The values related to the effect of the various hand creams on skin

moisture one week after the end of the treatment

111

4.21 The values related to the effect of the various hand creams on skin

smoothness one week after the end of the treatment

112

4.22 The values related to the effect of the various hand creams on skin

brightness one week after the end of the treatment

113

4.23 The values related to the well being perception of the effect of the

various hand creams on skin one week after the end of the treatment

114

4.24 The picture of both hands before application of hand cream (F3)

containing fish oil (a) and the left hand of a volunteer after

application of F3 as compared to her right hand without application

of hand cream (b)

115

4.25 The statistical significant difference (P < 0.05) of the mean values

related to the skin brightness (a), moisture (b), smoothness (c),

texture (d), and well being perception (e) of the hand cream

formulations under study after 30 days of application

117

4.26 The statistical significant difference (P < 0.05) of the mean values

related to the skin brightness (a), moisture (b), smoothness (c),

texture (d), and well being perception (e), of the hand cream

formulations under study one week after the end of the treatment

119

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LIST OF APPENDICES

Appendix

Page

A: Hand cream’s stability questionnaire

139

B: Hand cream’s efficacy questionnaire (Group one)

147

C: Hand cream’s efficacy questionnaire (Group two)

151

D: Hand cream’s efficacy questionnaire (Group three)

155

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LIST OF ABBREVIATIONS

AA Arachidonic Acid

AOCS American Oil Chemists’ Society

ATCC American Type Culture Collection

BHA Butylated Hydroxy Anisole

CFU Colony Forming Units

DHA Docosahexaenoic Acid

EFA Essential Fatty acids

EPA Eicosopentaenoic Acid

F1 Hand cream Formulation without fish oil

F2 Hand cream Formulation containing 1.00% fish oil

F3 Hand cream Formulation containing 2.50% fish oil

F4 Hand cream Formulation containing 5.00% fish oil

FAME Fatty Acid Methyl Esters

FAO Food and Agriculture Organization of the United Nations

FFA Free Fatty Acids

GC Gas Chromatography

MUFA Monounsaturated Fatty Acid

PUFA Polyunsaturated Fatty Acid

SFA Saturated Fatty Acid

USFA Unsaturated Fatty Acid

U.S. FDA U.S. Food and Drug Administration

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CHAPTER 1

INTRODUCTION

Biotechnologies are those technologies in which living organisms or parts of them

are used to produce large quantities of products which are useful for human

(Barbarisi, 2011). According to this definition of biotechnology and to fulfill a study

in the field of aquatic biotechnology, extraction and refining of oil from byproducts

of patin (Pangasianodon hypophthalmus) was considered to be applied in a

formulation of a cosmetic hand cream. Production of hand cream using fish oil

extracted from by-products can add value to these wastes and provide health

promoting advantages for human skin.

According to Department Of Fisheries Malaysia (2010) patin is the second largest

cultured fish species in Malaysia. Patin was chosen for this study because it is easier

to harvest large amount of by-products from patin since they are being sold at big

size (> 2.8 kg/fish). However, African catfish (Clarias gariepinus) is the first largest

cultured fish species but they are being sold in smaller sizes of around < 0.2 kg/fish

which means too small amount of by-products per fish which is not very suitable for

this study. The big size of patin catfish is obvious on the measuring ruler in Figure

1.1.

Figure 1.1. Patin catfish on the measuring ruler

Ovary

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In the present study, application of fish oil in a formulation of hand cream was

considered because application of fish oil is beneficial for human skin (Heard et al.,

2003; Puglia et al., 2005; and Zulfakar et al., 2010) and Mero Cosmetic (Tehran,

Iran) is interested on production of hand cream using fish oil and provided all the

materials and equipments which made this study possible.

1.1 Problem statement and justification

- The visceral storage fat and liver of patin are discarded. Disposal of these

wastes incurs cost and environmental pollution. Therefore, finding a usage

for them can decrease environmental pollution caused by fish waste.

- Because of well-known health benefits of fish oil there is an increased

interest in fish oil consumption, while fish resources are restricted. Therefore,

finding new sources are important for on growing human population. Fish

byproducts can be considered as valuable available sources.

- The visceral storage fat and liver accumulate fat. Therefore, they are potential

sources of extractable fish oil. Evaluating the fatty acid composition of fish

oil extracted from by-products of patin can provide useful information and

emphasize their values for food, pharmaceutical, and cosmetic industries

which are eager to use fish oil in their products.

- Fish oil extracted from fish wastes is economically important as it can be

obtain in very cheap price which is favorable for industries and final

consumers.

- Crude fish oil is a mixture of several compounds which decrease the quality

and shelf life of oil. Therefore, it is suitable to be refined. Unfortunately, a

few researches have been conducted to provide data on the effect of refining

on the valuable fatty acid composition of fish oil. Thus, this study considered

to evaluate the fatty acid composition of refined patin catfish oil after

extraction, degumming, neutralizing, bleaching, and deodorizing to provide a

better understanding of their changes. However, refining can affect the fatty

acid composition of fish oil but it is still is one of the most common methods

for removing and decreasing impurities (such as free fatty acids, lipid

oxidation products, odor, and colorant compound) from fish oil (Young,

1982). Results of this research are important for oil industry interested in

refined patin catfish oil production and scholars who are interested in

modification of refining processes.

- There are a few examples of available cosmetic skin care products containing

fish oil in the market but it is too hard to find a scholar study presenting data

on formula and procedure suitable for production of these kinds of products.

Therefore, this study can provide vital information on choosing hand cream

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ingredients, their suitable amounts, and processes for formulation of hand

cream which can contain fish oil and can be stable and effective.

- Generally, small amounts of fish oil are used in cosmetic products as

compared to topical therapeutic products (Abamba, 1993). It is important to

provide data which indicate the efficacy of these small amounts. Therefore,

in this study comparison of hand cream without the fish oil was conducted to

the hand cream containing fish oil.

- Unfortunately, there is a lack of data for determining the suitable and

functional amounts of fish oil in a cosmetic skin care product and specifically

in hand cream. This study attempted to find the suitable amount of fish oil

which does not adversely affect the stability of hand cream during storage

and to provide convincing evidence of effectiveness of that amount.

1.2 Specific objectives

The objectives of this study were divided into two main categories:

1. Patin catfish oil

1.1 Extracting and evaluating the fatty acid composition of liver and fatty tissue of

female and male and combination of both

(At the place that patin is filleted and during filleting processes, because of big size

of patin catfish it is practical and easy to separate female and male as well as their

byproducts. Obvious differences in the gonad of female and male are shown in

Figure 1.2. Moreover, without scientific data it is hard to predict whether the males

and females have similar fatty acid composition or not. Moreover, for cosmetic and

fish oil industries, providing information on male and female byproducts separately

and combination of them is important to provide a better understanding of fatty acid

composition of final fish oil product and its variation based on row materials which

are used).

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a b

Figure 1.2. Gonad of male (a) and female (b) patin catfish

1.2 Degumming, neutralizing, bleaching, and deodorizing the patin catfish oil and

evaluating the fatty acid composition of oil at each step

1.3 Evaluating the physical and chemical properties of refined oil

1.4 Evaluating the antibacterial efficacy of crude and refined patin catfish oil through

disk diffusion test, agar well diffusion test, and broth dilution test against

Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739),

Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231), and

Aspergillus niger (ATCC 16404)

2. Hand cream

2.1 Formulating and producing hand cream using various amounts of refined patin

catfish oil

2.2 Evaluating the appearance, odor, texture, pH, weight loss, preservative efficacy,

bacterial, fungal, and molds growth of various formulations of hand cream during six

months of storage at 4, 25, 37, and 45°C

2.3 Evaluating various hand cream formulations through the in vivo dermal irritation

test and clinical observations of skin reactions of mice

2.4 Evaluating various hand cream formulations through human study

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