UNIVERSITI PUTRA MALAYSIA

EXTRACTION AND CHARACTERISATION OF COLLAGEN AND GELATIN FROM RED TILAPIA (Oreochromis nilotica) SKIN

HARVINDER KAUR

FSTM 2006 25

EXTRACTION AND CHARACTERISATION OF COLLAGEN AND GELATIN FROM RED TILAPIA (Oreochromis nilotica) SKIN

HARVINDER KAUR

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA 2006

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EXTRACTION AND CHARACTERISATION OF COLLAGEN AND GELATIN FROM RED TILAPIA (Oreochromis nilotica) SKIN

By HARVINDER KAUR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of Science

December 2006

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DEDICATION

This thesis is dedicated to my son, Hanspal who has been the source of strength and inspiration for me. It was a real challenge to produce this thesis with him around. Nevertheless, it would not have been a success without him. Thank you and love you very much, my dear Hanspal.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science EXTRACTION AND CHARACTERISATION OF COLLAGEN AND GELATIN FROM RED TILAPIA (Oreochromis nilotica) SKIN By HARVINDER KAUR December 2006 Chairman: Faculty: Professor Jamilah Bakar, PhD Food Science and Technology

Collagen and gelatin were extracted by a series of washings with acid and alkali. Collagen extractions were carried out using pepsin, trypsin and papain. Effects of frozen storage of skins at -20C for up to 8 weeks on collagen characteristics were also studied at 2 weeks interval. The collagens obtained were evaluated for yield, protein, amino acid profile, molecular weight and mineral analysis. Gelatin was then extracted from the collagen and their properties vis visual appearance, odor, pH, bloom strength, viscosity, melting point and amino acid profile were determined. Papain-extracted collagen which showed reasonably high yield, protein and amino acid content was chosen for the study of effects of enzyme on gelatin extraction. Gelatin extraction with papain was carried out at several time and temperature variable i.e. 4 hr, 8 hr and 12 hr, and 26C, 45C and 65C respectively. The properties of both enzymatic and non-enzymatic extracted gelatin were then compared with properties of commercial mammalian gelatins. The collagen yield range from 31.6 63.6% (dry weight) with papain-extracted collagen showing the highest yield followed by pepsin-extracted collagen (32.5% dry weight) and trypsinextracted collagen (31.6% dry weight). The collagens have 15 30% protein and an

apparent molecular weight of 20,000 Da to 220,000 Da. In the mineral analyses by Energy Dispersive X-ray (EDX), carbon, oxygen, sodium and chlorine were detected. Glycine and proline were the major amino acids of collagen and gelatin at which constituting 25% and 20% of total amino acids respectively. Total amino acid content of trypsin and papain-extracted collagen were higher than pepsin-extracted collagen. The gelatins extracted had barely detectable odor, was snowy white and light-textured in appearance. Yield recorded was in the range 12 18% (dry weight, w/w) with enzymatic treatment giving higher yields and protein contents. Lower loss of residual enzyme activity at 26C and 45C for all extraction time indicate that the enzyme was stable at these conditions. Lower residual enzyme activity at 65C shows higher loss of papain activity. Enzyme treatments have little effect on the characteristics of the gelatin. Commercial mammalian gelatins were of yellow to brown color with higher protein (> 71.6%) and moisture content (> 9.7%). pH, viscosities and melting points of the commercial gelatin were also higher than fish gelatin. Total amino acid compositions of both fish gelatins were lower than the commercial gelatins. Threonine and tyrosine were not detected in commercial gelatins. Enzyme-extracted fish gelatin failed to gel whereas non-enzymatic fish gelatin showed higher bloom strength (250.9g) compared with commercial gelatins.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains PENGESTRAKAN DAN CIRI-CIRI KOLAGEN DAN GELATIN DARI KULIT TILAPIA MERAH (Oreochromis nilotica) Oleh HARVINDER KAUR Disember 2006 Pengerusi: Fakulti: Profesor Jamilah Bakar, PhD Sains dan Teknologi Makanan

Kolagen dan gelatin diekstrak melalui satu siri langkah pencucian dengan asid dan alkali. Pengekstrakan kolagen dijalankan dengan menggunakan enzim pepsin, tripsin dan papain. Kesan penyimpanan kulit ikan pada -20C (sejukbeku) selama 8 minggu pada ciri-ciri kolagen juga dijalankan, pada setiap selang 2 minggu. Kolagen yang diperolehi dianalisa untuk perolehan, protin, analisis asid amino, berat molekul dan analisis mineral. Gelatin kemudiannya diekstrak dari kolagen dan ciri-ciri seperti analisa warna secara visual, bau, pH, kekuatan gel, kelikatan, takat cair dan analisis asid amino ditentukan. Kolagen diekstrak dengan papain memberikan perolehan, protin dan komposisi asid amino yang agak tinggi, telah dipilih untuk kajian kesan enzim pada pengekstrakan gelatin. Pengekstrakan gelatin dengan papain dijalankan pada beberapa kombinasi masa dan suhu i.e. 4 j, 8 j, 12 j dan 26C, 45C dan 65C masing-masing. Ciri-ciri gelatin yang diekstrak dengan dan tanpa enzim kemudiannya dibandingkan dengan gelatin mamalia komersil. Perolehan kolagen adalah di antara 31.6 63.6.8% (berat kering), iaitu kolagen diekstrak dengan papain menunjukkan perolehan tertinggi diikuti kolagen diekstrak dengan pepsin (32.5% berat kering) dan kolagen diekstrak dengan tripsin (31.5% berat kering). Kolagen

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yang diperolehi mempunyai komposisi protin antara 15 30% dan berat molekul ketara antara 20,000 Da ke 200,000 Da. Dalam analisis mineral melalui Energy Dispersive X-Ray (EDX), karbon, oksigen, natrium dan klorin telah dapat dikenalpasti. Glisin dan prolin merupakan asid amino utama kolagen dan gelatin yang merangkumi 25% dan 20% daripada jumlah asid amino masing-masing. Jumlah asid amino gelatin diekstrak dengan tripsin dan papain adalah lebih tinggi daripada kolagen diekstrak pepsin. Gelatin yang diperolehi tidak mempunyai bau yang ketara, adalah putih salji dan bertekstur ringan. Perolehan gelatin adalah di antara 12 18% (berat kering) dengan pengekstrakan enzim menunjukkan perolehan dan kandungan protin yang lebih tinggi. Kehilangan aktiviti enzim residual yang lebih rendah pada suhu 26C dan 45C bagi setiap masa ekstraksi menunjukkan enzim tersebut adalah stabil pada keadaan tersebut. Aktiviti enzim residual yang rendah pada 65C menunjukkan kehilangan aktiviti papain yang lebih tinggi. Pengekstrakan dengan enzim tidak memberikan kesan ketara pada ciri-ciri gelatin. Gelatin mamalia komersil mempunyai warna kuning atau perang dan kandungan protin (> 71.6%) dan kelembapan (> 9.7%) yang lebih tinggi berbanding gelatin ikan. pH, kelikatan dan takat cair gelatin komersil juga adalah lebih tinggi. Jumlah komposisi asid amino kedua-dua gelatin ikan adalah lebih rendah berbanding dengan gelatin komersil. Treonin dan tirosin tidak dapat dikesan dalam gelatin komersil. Gelatin ikan yang diekstrak dengan enzim gagal menggel manakala gelatin ikan yang tidak diekstrak dengan enzim menunjukkan kekuatan gel (250.9g) yang lebih tinggi berbanding dengan gelatin komersil.

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ACKNOWLEDGEMENTS

Sincere gratitude to the Chairperson of the Supervisory Committee, Professor Dr. Jamilah Bakar for all her guidance, patience, encouragement and supervision throughout the project course. Her constant advice and support has given me the strength and capabilities to complete the thesis write up. All the valuable experience and knowledge gained throughout this study will always be remembered and appreciated. Many thanks also go to my co-supervisors, Professor Dr. Russly Abdul Rahman and Associate Professor Badlishah Sham Baharin for their valuable support and constructive advice.

Heartfelt thanks also go to all my friends: Nazri, Manichand, Azizah, Levina and Shanthi, for sharing their knowledge, for the cooperation and supportive spirit throughout my entire course of study.

Great appreciations also go to the dedicated technical staff En. Shoib, En. Halim, En. Azman, En. Rosli and Kak Jem for guiding me with my research work.

Deepest gratitude to my beloved parents for their ever-present support and guidance. Not to forget my husband, Karam who has given me the strength and encouragement to carry on with the thesis write up. Also to my wonderful son, Hanspal who has been the light and inspiration for me to make this project a success.

To those who are not mentioned here, the help and guidance offered is sincerely appreciated and always remembered.

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I certify that an Examination Committee met on the 27th December 2006 to conduct the final examination of Harvinder Kaur on her Master of Science thesis entitled Extraction of Collagen and Gelatin from Skin of Red Tilapia (Oreochromis nilotica) and Its Characteristics in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Nazamid Saari, PhD Professor Faculty of Graduate Studies Universiti Putra Malaysia (Chairman) Mohd Yazid bin Abdul Manap, PhD Professor Faculty of Graduate Studies Universiti Putra Malaysia (Internal Examiner) Suhaila Mohamed, PhD Professor Faculty of Graduate Studies Universiti Putra Malaysia (Internal Examiner) Salam Babji, PhD Professor Faculty of Graduate Studies Universiti Putra Malaysia (External Examiner)

HASANAH MOHD. GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows:

Jamilah Bakar, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairperson)

Russly Abd. Rahman, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member)

Badlishah Sham Baharin, PhD Associate Professor Faculty of Food Science and Technology Universisti Putra Malaysia (Member)

AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date: 14 JUNE 2007

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

HARVINDER KAUR Date: 11 APRIL 2007

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LIST OF TABLES

Table

Page

1 2a

Distinct features of collagen types Yield (%) and protein content (%) of collagen samples from red tilapia skins as affected by different enzymes Yield (%) and protein content (%) of collagen samples from red tilapia skins as affected by storage period Visual observation of red tilapia skin collagen as extracted by different enzymes and storage study Instrumental colour of collagen samples as affected by different enzyme extraction Instrumental colour of collagen samples as affected by different storage period Amino acids composition of collagen samples as affected by different enzyme extraction (mg/g sample) Amino acids composition of collagen samples as affected by storage period (mg/g sample) Yield (%) enzymatic and non-enzymatic gelatin samples As affected by extraction time and temperature Protein Content (%) enzymatic and non-enzymatic gelatin samples as affected by extraction time and temperature Hunter color values of enzymatic and non-enzymatic treated gelatin as affected by different extraction time and temperature Amino acid composition of gelatin at 26C extraction Amino acid composition of gelatin at 45C extraction Amino acid composition of gelatin at 65C extraction Residual enzyme activity of papain in gelatin samples at 30C incubation

17

82

2b

83

84

4a

86

4a

87

5a

89

5b

90

109

111

114 116 117 118

9 10 11 12

123

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Residual enzyme activity of papain in gelatin samples at 40C incubation Proximate composition of red tilapia skin gelatin and commercial mammalian gelatin Visual observation of red tilapia skin gelatin and commercial mammalian gelatin Instrumental color of red tilapia skin gelatin and commercial mammalian gelatin pH, viscosity and bloom strength of red tilapia skin gelatin and commercial mammalian gelatine Amino acid composition of tilapia skin gelatin and commercial mammalian gelatin Amino acid composition in several fish gelatins Melting point (C) of red tilapia skin gelatin and commercial mammalian gelatin at different maturation temperature

125

14

135

15

139

16

140

17

142

18

149 151

19 20

152

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LIST OF FIGURES

Figure

Page

1 2

Schematic cross-section of ox-hide skin A stereo pair showing a segment of collagen triple helix (wire model) A stereo pair showing a segment of collagen triple helix (sphere model) Gelatin world production in 2002 by % raw materials Gelatin world production in 2001 by % raw materials Gelatin production in 2002 by regions (%) Gelatin production in 2001 by regions (%) SDS-PAGE patterns of collagen from skins of red tilapia As affected by different enzyme extractions SDS-PAGE patterns of collagen from skins of red tilapia as affected by storage study Scanning Electron Micrograph (SEM) of trypsin extracted collagen fibrils from skins of red tilapia (Magnifications, 1000x) Scanning Electron Micrograph (SEM) of trypsin extracted collagen fibrils from skins of red tilapia (Magnifications, 5000x) Scanning Electron Micrograph (SEM) of papain extracted collagen fibrils from skins of red tilapia (Magnifications, 1000x) Scanning Electron Micrograph (SEM) of papain extracted collagen fibrils from skins of red tilapia (Magnifications, 5000x) Scanning Electron Micrograph (SEM) of pepsin extracted collagen fibrils from skins of red tilapia (Magnifications, 1000x) Scanning Electron Micrograph (SEM) of pepsin extracted collagen fibrils from skins of red tilapia (Magnifications, 5000x) Scanning Electron Micrograph (SEM) of collagen fibrils from shark and pig (Magnifications, 35,000x; bars 1)

7 22

23

4a 4b 5a 5b 6a

66 66 67 67

92

6b

93

7a

95

7b

95

8a

96

8b

96

9a

97

9b

97

10

98

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Experimental design of of gelatin extraction as affected by variable extraction time and temperature with and without papain addition Bloom strength curves of tilapia and commercial mammalian gelatin gels (6.67%

104

12

146

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LIST OF PLATES

Plate

Page

1a 1b 2

Pepsin, trypsin and papain extracted collagen Pepsin extracted collagen samples as subjected to storage study Tilapia skin and commercial mammalian gelatin gels

86 86 148

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LIST OF ABBREVIATIONS

Gelatin Manufactures of Europe Isoelectric Points Daltons Sodium Dodecyl SulphatePolyacrylamide Gel Electrophoresis Glycine Proline Hydroxyproline Nanometre Hydrochloric acid Molecular weight Energy Dispersive X-ray Scanning Electron Microscopy Sodium Hydroxide Sodium Chloride Enzyme-aided Gelatin Non-enzymatic Gelatine Centipoises Millipoises

GME pHI Da

SDS-PAGE Gly Pro Hyp nm HCl Mw EDX SEM NaOH NaCl EG NEG cP mP

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TABLE OF CONTENTS
Page DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENT APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS CHAPTER I II INTRODUCTION LITERATURE REVIEW Collagen Introduction Sources of collagen Mammalian sources Aquatic sources Fish collagen Other aquatic sources Invertebrate collagen Extraction procedures Acid extraction Enzymatic extraction Collagen Types Structure and properties of collagen Molecular structure of collagen Fibril structure of collagen Amino acid composition of collagen Reactivity and stability Stability to chemicals and enzymes Denaturation Applications of collagen Gelatin Introduction Sources of gelatin Mammalian gelatin Fish gelatin Types of gelatin 1 5 5 5 6 6 8 8 11 13 13 13 14 15 21 21 25 26 29 32 34 36 37 37 38 38 41 45 ii iii v vii viii x xi xiii xv xvi

Acid pre-treatment Alkaline pre-treatment Conversion of gelatin from collagen Washing treatments Extraction of gelatin Enzymatic extraction of gelatin Physico-chemical properties of gelatin Color and Clarity pH Solubility Viscosity Gel strength Melting temperatures Amino acid composition Gelatin market and applications Gelatin market Edible gelatin Technical and pharmaceutical uses

45 47 50 51 52 54 55 55 56 57 58 59 61 63 66 66 67 70

III

EFFECTS OF ENZYME EXTRACTION AND STORAGE PERIOD ON COLLAGEN PROPERTIES OF RED TILAPIA (Oreochromis nilotica) SKINS Introduction Materials and methodology Materials Chemicals Methods Storage study Analyses Yield of gelatin (% w/w) Visual observation and instrumental color Protein content Amino acids compositions Molecular weight determination Mineral content Electron microscopic observation Statistical Analyses Results and discussion Yield Protein content Visual observation and instrumental color Amino acids profile Molecular weight distribution Mineral analysis Scanning electron microscopy of collagen Conclusion

74 74 77 77 77 77 78 78 78 79 79 79 79 80 80 81 81 81 83 84 87 91 93 94 99

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ENZYMATIC EXTRACTION OF GELATIN FROM RED TILAPIA (Oreochromis nilotica) Introduction Materials and methodology Experimental design Materials Chemicals Methods Analyses Yield of gelatin (% w/w) Determination of color, crude protein and amino acid profile Determination of residual enzyme activity Statistical analyses Results and discussion Yield Protein determination Instrumental color Amino acid composition Residual enzyme activity Conclusion PHYSICO-CHEMICAL PROPERTIES OF GELATIN FROM RED TILAPIA SKINS COMPARED TO COMMERCIAL MAMMALIAN GELATIN Introduction Materials and methodology Materials Methods Physico-chemical Analyses Moisture content Ash content Fat content Determination of color, crude protein and amino acid profile Determination of pH Determination of viscosity Determination of melting point Determination of gel strength (BS 757 method) Statistical Analyses Results and discussion Proximate analyses moisture - ash - protein - fat Visual observation and instrumental color pH determination Viscosity determination

101 101 103 103 105 105 105 106 106 107 107 108 108 108 111 113 115 122 126

128 128 130 130 131 131 131 131 132 132 132 132 133 133 134 134 134 136 137 138 138 141 143

Gel strength Amino acids compositions Melting point determination Conclusion VI GENERAL CONCLUSIONS

144 148 151 154 156 159 167 181 182

REFERENCES APPENDICES BIODATA OF THE AUTHOR LIST OF PUBLICATIONS

CHAPTER I

INTRODUCTION

The use of hydrocolloids in numerous industries has been escalating. This is due to the tremendous development in production and its applications in various industries. The more well known and applied hydrocolloids in the food industry are alginates, carrageenans, agar, guar gum, Arabic gum, methylcellulose, carboxymethylcellulose and gelatins. The applications of these substances are extended to other industries such as textiles, pharmaceutical and cosmetics. These compounds have a vegetal origin though in some cases they could be obtained from animals.

Gelatin is the most versatile of the hydrocolloids in the modern food industry. In comparison to gelatin, carrageenan forms brittle, barely elastic gel. Pectin gels have no elastic properties at all and are not stable as a result. Alginates form clear, elastic gels, but their melting point is much higher than that of gelatin (Gelatin Manufacture Europe (GME), 1990). The use of starches and modified starches in food processing can lead to unpleasant textures due to the large quantities needed.

Gelatin, a protein derived from collagen is the major structural protein in connective tissue of animal skin and bone (Choi and Regenstein, 2000; Leuenberger, 1991; Segtnan et.al., 2003; Tosh et al., 2003; Cho et al., 2004; Ingvild et al., 2004; GomezGuillen et al., 2001; Gilsenan and Ross-Murphy, 2000; Gudmundsson and Hafsteinsson 1997; Grossman and Bergmann, 1992). It is an important constituent in a number of food and non-food products due to its multi-functional properties,

thermal stability, digestibility, solubility and its biological characteristics. In the food industry, it serves primarily as a gelling agent, but it is also used as a thickener, film former, stabilizer, emulsifier, adhesive agent, foaming agent, protective colloid and as a beverage fining agent (Johnston-Banks, 1990; Segtnan et al., 2003). The quality of gelatin for a particular application therefore depends largely on its rheological properties that are desirable for that application (Stainsby, 1987; GomezGuillen et al., 2002). Gelatin sales in year 2001 were US $1.2 billion and world average annual sales growth was estimated at 2-3% (GME). Statistics showed that total world production by percent raw material in 2001 was 269,400 metric tons and this increased to 272,500 metric tons in year 2002 (GME).

Commercially, gelatin is made from skins and skeletons of bovine and porcine. Mammalian gelatin has been intensively studied (Ward and Courts, 1977; Gilsenan and Ross-Murphy, 2000; Cho et al., 2004). For many socio-cultural reasons, alternative sources are increasingly demanded. Among such reasons are religious proscription of Judaism and Islam. Diseases such as bovine spongiform encephalopathy (BSE) and foot-and-mouth disease (FMD) crisis have also caused restrictions on collagen trade (Fernandez-Diaz, 2003; Cho et al., 2004; Ogawa et al., 2004). Interest in investigating possible means of making more effective use of underutilized resources and industrial wastes is not a new ambition in the food industry. The quantity of industrial waste produced is increasing by year for example the waste from fish processing after filleting can account for as much as 75% of the total catch weight (Shahidi, 1994). About 30% of such waste consists of skin and bone with high collagen content. This waste is an excellent raw material for the preparation of high protein foods, besides helping to eliminate harmful

environmental aspects. Therefore, gelatin from marine sources has been looked upon as possible alternatives to mammalian gelatins. Several patents (Grossman and Bergmann, 1992; Holzer, 1996) as well as several published methods (GomezGuillen and Montero, 2001; Gudmundsson and Hafsteinsson, 1997; Nagai and Suzuki, 2000) for fish gelatin production have been reported. The pure dehydrated gelatin is a partially crystalline polymer, depending on the storage and thermal history of the macromolecule (Slade and Levine, 1987). Gelatin consists of different amounts of 18 amino acids, where glycine, proline and hydroxyproline are the most abundant. Commercially, two main types of gelatin are used: Type A and Type B gelatins (Veis, 1964; Ward and Courts, 1977; Segtnan et al., 2003). Type A gelatins result from acid process and Type B gelatin results from alkaline process. Dry commercial gelatins for the food industry usually contain about 88% protein, 10% water and 1 2% salts (GME, 1990).

Collagen, the precursor of gelatin is a fibrous protein and the most abundant protein in animals i.e. approximately 1/3 of the total protein. About 10% of mammalian muscle protein is collagen, but the amount in fish is generally much less (Foegeding et al., 1996). In general, collagens and gelatins from fish skins have lower imino acids (proline and hydroxyproline) content than collagens and gelatins from mammalian sources (Pikkarainen, 1986, P. Johns, 1977, Luenberger, 1991). Current findings showed that fish gelatins have lower melting point and lower gel strength than mammalian gelatins (Leunberger, 1991). However, gelatins from cold water fish (cod, megrim and hake) have different properties than warm water fish (tilapia and tuna) which show properties more similar to mammalian gelatins.