PROPERTIES OF EDIBLE FILMS PREPARED FROM FISH SKIN GELATIN ADDED WITH DIFFERENT FATTY ACIDS AND THEIR SUCROSE ESTERS by NOR AMALINI BINTI AHMAD Thesis submitted in fulfillment of the requirements for the degree of Master of Science in Food Technology DECEMBER 2015
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PROPERTIES OF EDIBLE FILMS PREPARED FROM
FISH SKIN GELATIN ADDED WITH DIFFERENT
FATTY ACIDS AND THEIR SUCROSE ESTERS
by
NOR AMALINI BINTI AHMAD
Thesis submitted in fulfillment of the requirements for the
degree of Master of Science in Food Technology
DECEMBER 2015
ii
ACKNOWLEDGEMENT
Alhamdulillah. Thank God for all the blessings.
This thesis is specially dedicated to my late father, Ahmad Busran. I hope I make
him proud.
I would like to thank my supervisor, Prof. Dr. Norziah Mohd Hani for all the
chances, guide, support and knowledge I have gained through this study.
I am grateful to Ministry of Higher Education, Malaysia for giving me the financial
assistance through MyMaster, and also to Universiti Sains Malaysia for its Graduate
Assistant Scheme. I am also thankful to all lecturers who gave comments, knowledge
and courage during postgraduate seminars. Also, to lab assistants and administration
staffs, I strongly appreciate all the help.
Much love to my mother, Jariah Ismail, who always be my best friend and biggest
supporter. To my brothers and sisters, and all my family members, thank you for
being so understanding and supportive. No words to describe our beautiful family.
Sincere thanks to all my labmates, friends and those who involved in helping me to
complete this study. All the experience I gained through this journey, they are all
precious and worth.
I am so small here.
NOR AMALINI AHMAD
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TABLE OF CONTENTS
Page
Acknowledgement ii
Table of Contents iii
List of Tables viii
List of Figures ix
List of Plates xiii
List of Appendices xiv
List of Abbreviations xv
List of Symbols xvii
Abstrak xix
Abstract xxii
CHAPTER 1 : INTRODUCTION
1.1 Background
1.2 Research objectives
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CHAPTER 2 : LITERATURE REVIEW
2.1 Gelatin
2.1.1 Properties of gelatin
2.1.2 Prospect of gelatin production
2.2 Introduction to edible films
2.3 Preparation methods of edible films
2.3.1 Dry process
2.3.2 Wet process
2.4 Types of edible films
2.4.1 Polysaccharide based films
2.4.2 Protein based films
2.4.3 Lipid based films
2.4.4 Composite films
2.4.5 Active films
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2.5 Properties of edible films
2.5.1 Protective barriers
2.5.2 Mechanical supports
2.5.3 Carriers of functional ingredients
2.6 Modifications of edible films
2.6.1 Plasticization
2.6.2 Chemical methods
2.6.3 Enzymatic methods
2.6.4 Radiation
2.6.5 Incorporation of hydrophobic materials
2.6.5.1 Fatty acids
2.6.5.2 Fatty acid sucrose esters
2.6.5.3 The actions of lipids in emulsion films
2.7 Applications of edible films
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CHAPTER 3 : EXTRACTION AND CHARACTERIZATION OF
GELATIN FROM TILAPIA FISH SKINS
3.1 Introduction
3.2 Materials and methods
3.2.1 Materials
3.2.2 Gelatin extraction
3.2.3 Characterization of gelatin samples
3.2.3.1 Yield of extracted fish gelatin obtained
3.2.3.2 Determination of protein, moisture and ash
4.2.2 Preparation of glycerol plasticized gelatin films with
addition of fatty acids
4.3 Determination of physical properties of gelatin films
4.3.1 Film thickness measurement
4.3.2 Film solubility measurement
4.3.3 Opacity measurement
4.4 Determination of water vapour permeability of gelatin films
4.5 Determination of mechanical properties of gelatin films
4.6 Determination of moisture sorption isotherm
4.7 Scanning Electron Microscopy (SEM) study
4.8 Attenuated total reflectance-Fourier transform infrared (ATR-FTIR)
spectroscopy analysis
4.9 Statistical analysis
4.10 Results and discussion
4.10.1 Thickness and appearance
4.10.2 Film solubility in water
4.10.3 Opacity
4.10.4 Water vapour permeability (WVP)
4.10.5 Mechanical properties
4.10.6 Isotherm curves
4.10.7 Morphology of modified films
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4.10.8 FTIR profile
4.11 Conclusion
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CHAPTER 5 : PHYSICO-MECHANICAL AND MORPHOLOGICAL
PROPERTIES OF GELATIN BASED FILMS ADDED
WITH PALMITIC AND STEARIC FATTY ACID
SUCROSE ESTERS
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials
5.2.2 Preparation of glycerol plasticized gelatin films with
addition of fatty acid sucrose esters
5.3 Determination of physical properties of gelatin films
5.3.1 Film thickness measurement
5.3.2 Film solubility measurement
5.3.3 Opacity measurement
5.4 Determination of water vapour permeability of gelatin films
5.5 Determination of mechanical properties of gelatin films
5.6 Determination of moisture sorption isotherm
5.7 Scanning Electron Microscopy (SEM) study
5.8 Attenuated total reflectance-Fourier transform infrared (ATR-FTIR)
spectroscopy analysis
5.9 Statistical analysis
5.10 Results and discussion
5.10.1 Thickness and appearance
5.10.2 Film solubility in water
5.10.3 Opacity
5.10.4 Water vapour permeability (WVP)
5.10.5 Mechanical properties
5.10.6 Isotherm curves
5.10.7 Morphology of modified films
5.10.8 FTIR profile
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5.10.9 Comparative study: Properties of gelatin films added with
fatty acids and their sucrose esters
5.10.9.1 Film solubility in water
5.10.9.2 Opacity
5.10.9.3 Water vapour permeability (WVP)
5.10.9.4 Mechanical properties
5.11 Conclusion
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CHAPTER 6: CONCLUSION AND RECOMMENDATION
6.1 Conclusion
6.2 Recommendation
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REFERENCES
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APPENDICES
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LIST OF TABLES
Page
Table 3.1 Protein, moisture and ash contents of gelatin samples 49
Table 4.1 Formulations of gelatin films with and without addition of fatty acids, palmitic acid and stearic acid
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Table 4.2a Thickness, solubility and opacity of gelatin films added with palmitic acid at different levels of substitution for glycerol
71
Table 4.2b Thickness, solubility and opacity of gelatin films added with stearic acid at different levels of substitution for glycerol
72
Table 4.3a Tensile strength (TS), Young’s modulus (E) and elongation at break (EAB) of gelatin films added with palmitic acid at different levels of substitution for glycerol
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Table 4.3b Tensile strength (TS), Young’s modulus (E) and elongation at break (EAB) of gelatin films added with stearic acid at different levels of substitution for glycerol
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Table 5.1 Formulations of gelatin films with and without addition of fatty acid sucrose esters, palmitic acid sucrose ester and stearic acid sucrose ester
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Table 5.2a Thickness, solubility and opacity of gelatin films added with palmitic acid sucrose ester at different levels of substitution for glycerol
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Table 5.2b Thickness, solubility and opacity of gelatin films added with stearic acid sucrose ester at different levels of substitution for glycerol
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Table 5.3a Tensile strength (TS), Young’s modulus (E) and elongation at break (EAB) of gelatin films added with palmitic acid sucrose ester at different levels of substitution for glycerol
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Table 5.3b Tensile strength (TS), Young’s modulus (E) and elongation at break (EAB) of gelatin films added with stearic acid sucrose ester at different levels of substitution for glycerol
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LIST OF FIGURES
Page
Figure 1.1 Flow chart of overall research methodology 7
Figure 2.1 Mechanism of transport of molecules through films 22
Figure 2.2 Chemical structures of polyols 26
Figure 2.3 Structure of saturated fatty acid 31
Figure 2.4 Structure of fatty acid sucrose ester 33
Figure 2.5 Types of lipid droplets in emulsions (A) Uniform spherical droplets in a closely-packed system (B) Non-uniform droplets in a closely-packed system (C) Polyhedral droplets in a closely-packed system
36
Figure 2.6 Physical states of medium or long chain fatty acids at different temperatures and pH values
38
Figure 3.1 Extracted fish gelatin derived from Tilapia skins 47
Figure 3.2 Molecular weight analysis of extracted fish gelatin (EFG), commercial halal bovine gelatin (CHBG) and commercial beef skin gelatin (CBG)
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Figure 3.3 Bloom strength of extracted fish gelatin (EFG), commercial beef skin gelatin (CBG) and commercial halal bovine gelatin (CHBG)
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Figure 3.4 Viscosity of extracted fish gelatin (EFG), commercial beef skin gelatin (CBG) and commercial halal bovine gelatin (CHBG)
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Figure 3.5 FTIR spectra of extracted fish gelatin (EFG), commercial beef skin gelatin (CBG) and commercial halal bovine gelatin (CHBG)
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Figure 4.1 Solubility of gelatin films plasticized with different ratios of glycerol to palmitic acid (PA) or stearic acid (SA) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 4.2 Opacity values of gelatin films plasticized with different ratios of glycerol to palmitic acid (PA) or stearic acid (SA) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 4.3 Water vapour permeability of gelatin films: unplasticized (CTRL); plasticized with glycerol at 5 % (5G), 10 % (10G) and 15 % (15G); and plasticized (5P, 10P and 15P) at different glycerol and palmitic acid ratios, 75:25 (PA25), 50:50 (PA50), 25:75 (PA75) and 0:100 (PA100)
79
Figure 4.4 Water vapour permeability of gelatin films: unplasticized (CTRL); plasticized with glycerol at 5 % (5G), 10 % (10G) and 15 % (15G); and plasticized (5P, 10P and 15P) at different glycerol and stearic acid ratios, 75:25 (SA25), 50:50 (SA50), 25:75 (SA75) and 0:100 (SA100)
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Figure 4.5 Water vapour permeability of gelatin films plasticized with different ratios of glycerol to palmitic acid (PA) or stearic acid (SA) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 4.6 Alignment of the film components in gelatin films 88
Figure 4.7 Tensile strength; A, B and C at 5, 10 and 15 % plasticizer concentrations, respectively, and Young’s modulus; D, E and F at 5, 10 and 15 % plasticizer concentrations, respectively, of gelatin films plasticized with different ratios of glycerol to palmitic acid (PA) or stearic acid (SA)
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Figure 4.8 Elongation at break of gelatin films plasticized with different ratios of glycerol to palmitic acid (PA) or stearic acid (SA) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 4.9 Moisture sorption isotherm curves of gelatin films: plasticized with glycerol only at 15 % (15G); and plasticized with a mixture of 25:75 glycerol to PA (15P-PA75) or glycerol to SA (15P-SA75) at 15 % plasticizer level
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Figure 4.10 Scanning Electron Micrographs (1500x) of film surfaces (a) plasticized with glycerol at 15 % (15G) (b) plasticized with glycerol and palmitic acid at 15 % plasticizer level (15P-PA75) (c) plasticized with glycerol and stearic acid at 15 % plasticizer level (15P-SA75); and their cross-sections (d) 15G (e) 15P-PA75 (f) 15P-SA75
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Figure 4.11 FTIR spectra of gelatin films: plasticized with glycerol only at 15 % (15G); and plasticized with a mixture of 25:75 glycerol to PA (15P-PA75) or glycerol to SA (15P-SA75) at 15 % plasticizer level
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Figure 5.1 Solubility of gelatin films plasticized with different ratios of glycerol to palmitic acid sucrose ester (PASE) or stearic acid sucrose ester (SASE) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 5.2 Opacity values of gelatin films plasticized with different ratios of glycerol to palmitic acid sucrose ester (PASE) or stearic acid sucrose ester (SASE) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 5.3 Water vapour permeability of gelatin films: unplasticized (CTRL); plasticized with glycerol at 5 % (5G), 10 % (10G) and 15 % (15G); and plasticized (5P, 10P and 15P) at different glycerol and palmitic acid sucrose ester ratios, 75:25 (PASE25), 50:50 (PASE50), 25:75 (PASE75) and 0:100 (PASE100)
112
Figure 5.4 Water vapour permeability of gelatin films: unplasticized (CTRL); plasticized with glycerol at 5 % (5G), 10 % (10G) and 15 % (15G); and plasticized (5P, 10P and 15P) at different glycerol and stearic acid sucrose ester ratios, 75:25 (SASE25), 50:50 (SASE50), 25:75 (SASE75) and 0:100 (SASE100)
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Figure 5.5 Water vapour permeability of gelatin films plasticized with different ratios of glycerol to palmitic acid sucrose ester (PASE) or stearic acid sucrose ester (SASE) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 5.6 Tensile strength; A, B and C at 5, 10 and 15 % plasticizer concentrations, respectively, and Young’s modulus; D, E and F at 5, 10 and 15 % plasticizer concentrations, respectively, of gelatin films plasticized with different ratios of glycerol to palmitic acid sucrose ester (PASE) or stearic acid sucrose ester (SASE)
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Figure 5.7 Elongation at break of gelatin films plasticized with different ratios of glycerol to palmitic acid sucrose ester (PASE) or stearic acid sucrose ester (SASE) at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C)
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Figure 5.8 Moisture sorption isotherm curves of gelatin films: plasticized with glycerol only at 15 % (15G); and plasticized with a mixture of 25:75 glycerol to PASE (15P-PASE75) or glycerol to SASE (15P-SASE75) at 15 % plasticizer level
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Figure 5.9 Scanning Electron Micrographs (1500x) of film surfaces (a) plasticized with glycerol at 15 % (15G) (b) plasticized with glycerol and palmitic acid sucrose ester at 15 % plasticizer level (15P-PASE75) (c) plasticized with glycerol and stearic acid sucrose ester at 15 % plasticizer level (15P-SASE75); and their cross-sections (d) 15G (e) 15P-PASE75 (f) 15P-SASE75
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Figure 5.10 FTIR spectra of gelatin films: plasticized with glycerol only at 15 % (15G); and plasticized with a mixture of 25:75 glycerol to PASE (15P-PASE75) or glycerol to SASE (15P-SASE75) at 15 % plasticizer level
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xiii
LIST OF PLATES
Page
Plate 4.1 Photographs of gelatin films: unplasticized (CTRL); plasticized with glycerol only at 5 % (5G), 10 % (10G) and 15 % (15G); and plasticized with a 50:50 ratio of glycerol to palmitic acid (PA) or stearic acid (SA) at plasticizer concentrations, 5 % (5P-PA50 or 5P-SA50), at 10 % (10P-PA50 or 10P-SA50) and at 15 % (15P-PA50 or 15P-SA50)
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Plate 5.1 Photographs of gelatin films: unplasticized (CTRL); plasticized with glycerol only at 5 % (5G), 10 % (10G) and 15 % (15G); and plasticized with a 50:50 ratio of glycerol to palmitic acid sucrose ester (PASE) or stearic acid sucrose ester (SASE) at plasticizer concentrations, 5 % (5P-PASE50 or 5P-SASE50), at 10 % (10P-PASE50 or 10P-SASE50) and at 15 % (15P-PASE50 or 15P-SASE50)
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LIST OF APPENDICES
Page
Appendix A Solubility of gelatin films plasticized with different ratios of glycerol to fatty acids or their sucrose esters at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C). PA-Palmitic acid; SA-Stearic acid; PASE-Palmitic acid sucrose ester; SASE-Stearic acid sucrose ester
161
Appendix B Opacity values of gelatin films plasticized with different ratios of glycerol to fatty acids or their sucrose esters at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C). PA-Palmitic acid; SA-Stearic acid; PASE-Palmitic acid sucrose ester; SASE-Stearic acid sucrose ester
162
Appendix C Water vapour permeability (WVP) of gelatin films plasticized with different ratios of glycerol to fatty acids or their sucrose esters at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C). PA-Palmitic acid; SA-Stearic acid; PASE-Palmitic acid sucrose ester; SASE-Stearic acid sucrose ester
163
Appendix D Tensile strength of gelatin films plasticized with different ratios of glycerol to fatty acids or their sucrose esters at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C). PA-Palmitic acid; SA-Stearic acid; PASE-Palmitic acid sucrose ester; SASE-Stearic acid sucrose ester
164
Appendix E Young’s modulus of gelatin films plasticized with different ratios of glycerol to fatty acids or their sucrose esters at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C). PA-Palmitic acid; SA-Stearic acid; PASE-Palmitic acid sucrose ester; SASE-Stearic acid sucrose ester
165
Appendix F Elongation at break of gelatin films plasticized with different ratios of glycerol to fatty acids or their sucrose esters at plasticizer concentrations of 5 % (A), 10 % (B) and 15 % (C). PA-Palmitic acid; SA-Stearic acid; PASE-Palmitic acid sucrose ester; SASE-Stearic acid sucrose ester
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LIST OF ABBREVIATIONS
ANOVA Analysis of variance
AOAC Association of Official Analytical Chemist
ASTM American Society for Testing and Materials
ATR-FTIR Attenuated total reflectance-Fourier transform infrared
BSE Bovine Spongiform Encephalopathy
C Carbon
CBG Commercial beef skin gelatin
CHBG Commercial halal bovine gelatin
CTRL Control
E Young’s modulus
EAB Elongation at break
EFG Extracted fish gelatin
FA Fatty acid
FASE Fatty acid sucrose ester
FTIR Fourier transform infrared
G Glycerol
GMIA Gelatin Manufacturers Institute of America
GRAS Generally Recognized as Safe
HLB Hydrophilic-lipophilic balance
P Plasticizer (Percent)
PA Palmitic acid
PASE Palmitic acid sucrose ester
SA Stearic acid
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SASE Stearic acid sucrose ester
SD Standard deviation
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEM Scanning Electron Microscopy
SPSS Statistical Package for the Social Sciences
TS Tensile strength
WVP Water vapour permeability
WVTR Water vapour transmission rate
xvii
LIST OF SYMBOLS
% Percent
oC Degree celcius
mg Milligram
g Gram
kg Kilogram
mm Milimetre
cm Centimetre
m Metre
s Second
min Minute
h Hour
μl Microlitre
ml Millilitre
V Volt
kV Kilovolt
mol Mole
M Molar
N Newton
Pa Pascals
MPa Megapascals
mPa.s Milipascals seconds
kDa Kilodalton
rpm Revolutions per minute
xviii
w/w Weight/ weight
w/v Weight/ volume
v/v Volume/ volume
O/ W Oil in water
vs. Versus
α Alpha
β Beta
γ Gamma
aw Water activity
pI Isoelectric point
Tg Glass transition temperature
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SIFAT-SIFAT FILEM BOLEH MAKAN YANG DIHASILKAN DARIPADA
GELATIN KULIT IKAN DITAMBAH DENGAN PELBAGAI ASID LEMAK
DAN DERIVATIF ESTER SUKROSANYA
ABSTRAK
Di antara biopolimer-biopolimer yang digunakan untuk menghasilkan
filem-filem boleh makan, gelatin telah mendapat banyak perhatian kerana
kebolehannya yang baik dalam membentuk filem. Walau bagaimanapun, filem-filem
gelatin sangat hidrofilik justeru modifikasi adalah diperlukan untuk mengurangkan
kelemahan ini. Dalam kajian ini, gelatin ikan telah diekstrak daripada kulit-kulit ikan
Tilapia dan sifat-sifat fiziko-kimianya ditentukan. Hasil gelatin ikan yang diekstrak
(EFG) ialah sebanyak 20.2 % dengan kandungan protein iaitu 91.5 %, kelembapan
7.0 %, dan abu 1.3 %. Kekuatan bloom EFG ialah 241.1 g, dianggap tinggi dan juga
menunjukkan kehadiran protein yang memiliki berat molekul yang tinggi seperti
rantaian-rantaian α dan β dalam corak protein elektroforesis. Spektrum FTIR bagi
EFG menunjukkan kumpulan-kumpulan amida (A, B, I, II dan III) dalam lingkungan
nombor-nombor gelombang yang sama seperti ditunjukkan oleh gelatin-gelatin
komersial daripada sumber lembu. Gelatin ikan yang diekstrak (EFG) kemudian
digunakan untuk menghasilkan filem-filem boleh makan berasaskan protein
diplastikkan dengan gliserol. Gliserol telah ditambah pada kepekatan, 5, 10 dan 15 %
berdasarkan berat gelatin. Penggunaan EFG sebagai matrik biopolimer berjaya
menghasilkan filem-filem gelatin yang bebas terbentuk dan stabil. Penambahan
gliserol sebagai bahan pemplastik telah mengurangkan kekuatan tensil (TS) dan
modulus Young (E) tetapi meningkatkan pemanjangan pada titik putus (EAB) filem-
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filem gelatin berbanding filem yang tidak diplastikkan. Walau bagaimanapun,
dengan penambahan kandungan gliserol dari 5 ke 15 %, filem-filem yang
diplastikkan juga menunjukkan penambahan kebolehtelapan air (WVP) dan
kelegapan tetapi pengurangan kelarutan. Justeru bagi menambah baik sifat-sifat
filem terutamanya sifat-sifat penyekat air, modifikasi ke atas filem-filem gelatin
yang diplastikkan telah dilakukan dengan penambahan asid palmitik (PA) atau asid
stearik (SA), atau ester-ester sukrosanya; ester sukrosa asid palmitik (PASE) atau
ester sukrosa asid stearik (SASE) sebagai pengganti bagi gliserol pada pelbagai