Salmon Skin Gelatin Extracted from Trypsin-Aided Process
for Film Formation with Improved Water Resistance
by
Hui Yin Fan
Department of Food Science and Agricultural Chemistry
Macdonald Campus, McGill University
Quebec, Canada
2018
A thesis submitted to McGill University in partial fulfillment of the requirement of the
degree of Doctor of Philosophy in Food Science and Agricultural Chemistry
©Hui Yin Fan, 2018
ABSTRACT
Protein-based biodegradable films from mammalian gelatins are not totally accepted due to
religion beliefs and rising concerns regarding animal diseases. As an alternative, fish gelatin from
fish processing wastes has gained interest. Fish gelatin has good film-forming properties, and its
utilization adds value to abundant fish wastes that are either discarded or underutilized. To date,
fish skin gelatin has been chemically extracted, with few recent studies on enzymatic approaches
to improve extraction yields. However, the high hygroscopicity of gelatin needs to be overcome
as the film’s water barrier properties are poor. Thus, this work aims at investigating the recovery
of gelatin from fish skins and to form film with improved water resistance.
The investigation began by comparing the yield of Atlantic salmon (Salmo salar) fish skin gelatin
extracted using three different pretreatments. The findings showed that the highest yield (53.05 ±
4.38%) was obtained from trypsin-aided pre-treatments, as compared to pretreatments with saline,
and saline in combination with alkaline. Good quality gelatin was obtained by the very low-level
trypsin supplementation process, but in low yield. The predominance of high molecular weight
chains in gelatin results in the formation of films with improved physical and structural properties.
Therefore, an optimization study was performed on the trypsin-aided extraction process using a
Plackett-Burman (PB) design and followed by a Box-Behnken design (BBD) of response surface
methodology (RSM) to obtain higher yield gelatins with high molecular weight protein chains.
Two-fold higher yields of gelatin with high molecular weight protein chains were successfully
achieved under the optimum conditions, determined as follow: trypsin concentration at 1.49 U/g;
extraction temperature at 45 °C; and extraction time at 6 h 15 min. To evaluate the film forming
capability of the extracted gelatins, the optimized trypsin-aided extraction process was used to
obtain salmon skin gelatin for film formation using wet process. Since the properties of the films
are influenced by the film constituents, different gelatin and glycerol concentrations were
investigated. Characterization of the resultant films showed an increased in mechanical strength
as gelatin concentration increased, while an increased in elasticity increased with an increased in
glycerol concentration due to the plasticization effect. From this study, a good tensile strength and
elasticity of salmon skin gelatin film was identified at 5% protein and 30% glycerol; however, the
film was highly soluble in water (89.07%).
II
The need for improving the water resistance of salmon skin gelatin film was deemed necessary.
To achieve this, the addition of 5% of corn zein resulted in a significant decrease in water solubility
(65.5%) since zein is a hydrophobic protein. Canola oil was used as replacement to glycerol as
plasticizer. The water solubility of the gelatin-zein composite film was reduced further (as low as
47.4%) after plasticization with canola oil. However, the mechanical properties of the films were
weakened owing to the disruption of the polymer chains organization. The interaction between the
films’ constituents was confirmed through infrared and morphological studies.
The last stage of this study investigated the use of glutaraldehyde as cross-linking agent to improve
the mechanical properties and water resistance of gelatin-zein composite films. A central
composite design (CCD) of RSM was used to optimize the concentration in zein and
glutaraldehyde. The films exhibited improved water resistance (water solubility of 38.83%, water
vapour permeability of 0.276 g mm h-1 cm-2 Pa-1), while mechanical strength was retained with a
slight reduction in elasticity for the optimized film comprised of 3% zein and 0.02%
glutaraldehyde. The addition of glutaraldehyde formed crosslinks in the gelatin-zein composite
film successfully, as shown in infrared and morphological studies. The work presented here,
therefore, provides a promising platform for the development of fish skin gelatin films with
improved water resistance.
III
RÉSUMÉ
Les films biodégradables à base de protéines de mammifères (gélatine) ne sont pas totalement
acceptés en raison de croyances religieuses et de préoccupations grandissantes au sujet des
maladies animales. Comme alternative, la gélatine de poisson provenant des résidus de
transformation a gagné en intérêt. La gélatine de poisson présente de bonnes propriétés filmogènes
et son utilisation ajoute de la valeur aux résidus qui sont jetés, ou sous-utilisées. La majorité des
études portant sur l’extraction de gélatine de peau de poisson ont été faite avec des procédés
chimiques. Récemment, quelques études furent portées sur les approches enzymatiques pour
accroître le rendement de l’extraction. Toutefois, les propriétés barrières de ces films à l’eau sont
pauvres dues à l’hygroscopicité élevée de la gélatine. Ainsi, cette thèse focus sur la préparation de
films de gélatine ayant des propriétés barrières améliorées.
En premier lieu, l’extraction de la gélatine provenant de la peau du saumon atlantique (Salmo salar)
à l’aide de trois différents prétraitements a été comparée. Les résultats ont montré que le rendement
le plus élevé (53,05 ± 4,38 %) provenait des traitements pré assistée par la trypsine, par
comparaison aux prétraitements avec une solution saline et une solution saline en combinaison
avec les solutions alcalines. De la gélatine de bonne qualité a été obtenue par le procédé de
supplémentation de trypsine à très basse concentration, mais avec un faible rendement. La
prédominance des chaînes de masse moléculaire élevée dans de la gélatine se traduit par la
formation de films avec des propriétés physiques et structurelles améliorées. Par conséquence, une
étude d’optimisation a été réalisée sur le processus d’extraction assistée par la trypsine en utilisant
un design Plackett-Burman (PB) suivi d’un design Box-Behnken (BBD) de méthodologie de
surface de réponse (RSM) pour obtenir des rendements en gélatine plus élevés tout en conservant
des chaînes de protéines de poids moléculaire élevé. Après optimisation, les rendements de
gélatine furent doublés. Les conditions optimales étaient les suivantes: concentration de trypsine à
1,49 U/g, température d’extraction de 45 °C, et temps d’extraction de 6 h 15 min. Après extraction,
des films de gélatine furent synthétisés par voie humide. Différents ratios de gélatine et de glycérol
(plastifiant) furent utilisés pour cette étude. Certaines propriétés mécaniques ont augmentées avec
l’augmentation de la concentration en protéines, alors que certaines autres propriétés telle que
l’élasticité a augmenté avec l’augmentation de la concentration en glycérol. De cette étude, une
IV
bonne résistance à la traction et l’élasticité furent observées pour les films contenant 5 % de
protéines et 30 % de glycérol. Cependant, ces films étaient très solubles dans l’eau (89,07%).
L’amélioration de la résistance à l’eau des films a été jugée nécessaire. L’addition de 5 % de la
zéine de maïs a entraîné une diminution significative de la solubilité dans l’eau (65,5 %), puisque
zein est une protéine hydrophobe. L’huile de canola a été utilisée comme substitut au glycérol, ce
qui a permis une réduction de la solubilité dans l’eau à 47,4 %. Toutefois, les propriétés
mécaniques des films ont été affaiblies en raison de la perturbation de l’organisation des chaînes
de polymère. L’interaction entre les constituants des films a été confirmée par des études
morphologiques et infrarouges.
La dernière étape de cette étude fut l’utilisation du glutaraldéhyde comme agent de réticulation
pour améliorer les propriétés mécaniques et la résistance à l’eau des films composites (gélatinezein). Un motif central composite (CCD) de RSM a été utilisé afin d’optimiser la concentration de
zein et glutaraldéhyde. Les films ayant une composition de 3 % en zéine et 0,02 % en
glutaraldéhyde présentaient une étanchéité améliorée (hydro-solubilité de 38,83 %, perméabilité
à la vapeur d’eau de 0,276 g mm h-1 cm-2 Pa-1). Les études morphologiques et infrarouges ont
montrées que la réticulation avait eu lieu avec l’ajout de glutaraldéhyde. Cette thèse fournit une
plate-forme prometteuse pour le développement de films de gélatine provenant de la peau de
poisson ayant une étanchéité améliorée.
V
CONTRIBUTION OF AUTHORS
This thesis consists of nine chapters and is presented in manuscript format.
Chapter I is a general introduction and provides a brief literature review of the research work,
rationale and objectives of the present study.
Chapter II provides a detailed review of the literature on the properties of fish skin gelatin for
film formation, methods used for extracting fish gelatin, processes employed for forming fish
gelatin films, strategies available for improving the properties of fish gelatin films, and assessment
of fish gelatin film properties.
Chapter III to VIII are presented in the form of manuscripts, either have or to be submitted for
publication. The connecting statements provide the rational linking between the different chapters.
Chapter III consists of the investigation of different trypsin-aided process conditions on the yield
and molecular weight distribution of gelatins from fish skins. Chapter IV reports the optimization
study of the trypsin-aided extraction process conditions for the recovery of higher yield of salmon
skin gelatins with high molecular weight protein chains. Chapter V provides the characterization
of films prepared using salmon skin gelatin extracted by the optimized trypsin-aided process.
Chapter VI discusses the synthesis and characterization of salmon skin gelatin film with the
addition of corn zein and canola oil in replacement of glycerol as plasticizer for improving the
water resistance. Chapter VII reports the optimization study of salmon skin gelatin film
formulation consisted of zein and glutaraldehyde as crosslinker for improving the mechanical
properties and water resistance. Chapter VIII provides the in-depth characterization of the
optimized glutaraldehyde-crosslinked gelatin-zein composite films.
Chapter IX provides general conclusions, contributions to knowledge and recommendations for
possible future work in this field.
Hui Yin Fan, the candidate, was responsible for designing and performing the experiments,
interpreted the results, and prepared the manuscript and the thesis. Professor Dr. Benjamin K.
Simpson, the supervisor of the candidate, guided experiments of this work, provided direction and
laboratory and research facilities for the experiments, and reviewed the manuscript and thesis
VI
before submission. Associate Professor Dr. Marie-Josée Dumont, co-supervisor of the candidate,
co-supervised the experiments, provided assistance including direction of this work, laboratory
facilities, editing and reviewing the manuscript and thesis for submission. Daniel Duquette, a
master student under Dr. Dumont, conducted a portion of the dynamic mechanical analysis and
was listed as a co-author on manuscript 6 (Chapter VIII).
VII
PUBLICATIONS
1. Fan, H.Y., Dumont, M. J., & Simpson, B. K. (2017). Extraction of gelatin from salmon (Salmo
salar) fish skin using trypsin-aided process: optimization by Plackett–Burman and response
surface methodological approaches. Journal of Food Science and Technology, 54(12), 40004008.
2. Fan, H.Y., Duquette, D., Dumont, M.J. & Simpson, B.K. (2018). Salmon skin gelatin-corn zein
composite films produced via crosslinking with glutaraldehyde: optimization using response
surface
methodology
and
characterization.
International
Journal
of
Biological
Macromolecules, 120, 263-273.
3. Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Trypsin supplementation process for the
extraction of gelatin from different fish skins. To be submitted.
4. Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Characterization of films prepared using
salmon skin gelatin extracted by a trypsin-aided process. To be submitted.
5. Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Synthesis and characterization of salmon
skin gelatin-corn zein composite films plasticized with canola oil. To be submitted.
6. Fan, H.Y., Duquette, D., Dumont, M.J. & Simpson, B.K. (2018). Characterization of salmon
skin gelatin-corn zein composite films crosslinked with glutaraldehyde. To be submitted.
VIII
ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Benjamin K. Simpson for his support of my Ph.D study,
for his patience, mentorship, and immense knowledge. My sincere thanks go to Dr Marie-Josée
Dumont, for her continuous support, encouragement and critical comments on my work which
incented me to widen my research from different perspectives.
I would also like to thank to all the Professors and the staff in the Department of Food Science and
Agricultural Chemistry and the Department of Bioresources Engineering, McGill University, for
their advice and encouragement, especially Dr. Salwa Karboune, Dr. Valérie Orsat, Mr. Yvan
Gariépy and Ms. Leslie Ann LaDuke. My sincere thanks also go to whom have helped directly
and indirectly to the completion of this work.
I am thankful to all my fellow labmates, especially Nana, Zhang Yi, He ShuDong, Xiao Ran, Chen
Chen, Tian Lei, for the stimulating discussions, for the weekends we were working together, for
the fun, friendship and encouragement we have had throughout my studies.
Last but not the least, I am infinitely grateful to my parents, my family members, my siblings and
my friend for their unconditional support, encouragement, and love. Without them, this wouldn’t
have been possible.
IX
TABLE OF CONTENT
ABSTRACT .................................................................................................................................. II
RÉSUMÉ ..................................................................................................................................... IV
CONTRIBUTION OF AUTHORS ........................................................................................... VI
PUBLICATIONS ..................................................................................................................... VIII
ACKNOWLEDGEMENTS ....................................................................................................... IX
TABLE OF CONTENTS ............................................................................................................. X
LIST OF FIGURES ................................................................................................................... XV
LIST OF TABLES .................................................................................................................. XVII
LIST OF ABBREVIATIONS ................................................................................................. XIX
CHAPTER I. General Introduction .............................................................................................1
CHAPTER II. Literature Review .................................................................................................6
2.1 Fish skin gelatin for film formation ...........................................................................................7
2.1.1 Amino acid composition ...................................................................................................8
2.1.2 Molecular weight distribution ...........................................................................................8
2.2 Extraction of fish skin gelatin ....................................................................................................9
2.2.1 Acid pre-treatment ..........................................................................................................10
2.2.2 Alkaline pre-treatment ....................................................................................................10
2.2.3 Other pre-treatments .......................................................................................................11
2.3 Fish gelatin film formation ......................................................................................................11
2.3.1 Wet process .....................................................................................................................12
2.3.2 Dry process .....................................................................................................................12
2.4 Fish gelatin film modification ..................................................................................................13
2.4.1 Blending ..........................................................................................................................13
2.4.2 Lamination ......................................................................................................................13
2.4.3 Cross-linking ...................................................................................................................14
2.4.4 Nanoparticle reinforcement ............................................................................................15
2.5 Assessment of fish gelatin film properties ...............................................................................19
2.5.1 Barrier properties ............................................................................................................19
2.5.1.1 Gas barrier properties .............................................................................................19
2.5.1.2 Light barrier properties ..........................................................................................20
2.5.1.3 Water barrier properties .........................................................................................20
2.5.2 Mechanical properties .....................................................................................................25
2.5.3 Thermal properties ..........................................................................................................32
2.5.4 Structural properties ........................................................................................................36
2.5.5 Morphological properties ................................................................................................39
2.6 Conclusion ...............................................................................................................................40
X
CONNECTING STATEMENT 1 ...............................................................................................42
CHAPTER III. Trypsin supplementation process for the extraction of gelatin from different
fish skins........................................................................................................................................43
3.1 Abstract ....................................................................................................................................44
3.2 Introduction ..............................................................................................................................45
3.3 Materials and methods .............................................................................................................47
3.3.1 Materials .........................................................................................................................47
3.3.2 Fish skins handling .........................................................................................................47
3.3.3 Studies of gelatin extraction methods .............................................................................48
3.3.4 Extraction method with saline solution pre-treatment ....................................................48
3.3.5 Extraction method with saline and alkaline solutions pre-treatment ..............................48
3.3.6 Extraction method with trypsin solution pre-treatment ..................................................49
3.3.7 Gelatin extraction from fish skins using trypsin-aided process ......................................49
3.3.8 Hydroxyproline content ..................................................................................................49
3.3.9 Protein electrophoresis profile analysis ..........................................................................50
3.4 Results and discussion .............................................................................................................51
3.4.1 Effect of the different extraction methods on the hydroxyproline content and yield of
gelatin..............................................................................................................................51
3.4.2 Effects of trypsin concentrations, incubation times and extraction temperatures on the
yield of gelatin ...............................................................................................................52
3.4.3 Effects of trypsin concentrations, incubation times and extraction temperatures on the
protein electrophoretic patterns of gelatin .....................................................................55
3.5 Conclusion ..............................................................................................................................58
CONNECTING STATEMENT 2 ...............................................................................................59
CHAPTER IV. Extraction of gelatin from salmon (Salmo salar) fish skin using trypsin-aided
process: optimization by Plackett-Burman and response surface methodological approaches
........................................................................................................................................................60
4.1 Abstract ....................................................................................................................................61
4.2 Introduction ..............................................................................................................................62
4.3 Materials and methods .............................................................................................................63
4.3.1 Chemicals ........................................................................................................................63
4.3.2 Fish skins preparation .....................................................................................................64
4.3.3 Extraction of gelatin from fish skins ...............................................................................64
4.3.3.1 Removal of non-collagenous proteins ...................................................................64
4.3.3.2 Extraction method for optimization of gelatin extraction ......................................64
4.3.4 Experimental design........................................................................................................65
4.3.4.1 Plackett-Burman design ........................................................................................65
4.3.4.2 Response surface methodology..............................................................................66
4.3.5 Hydroxyproline content ..................................................................................................67
4.3.6 Protein electrophoresis profile analysis ..........................................................................67
4.4 Results and discussion .............................................................................................................68
4.4.1 Screening of significant variables using Plackett–Burman design ................................68
4.4.2 Optimization of significant variables using response surface methodology .................70
4.5 Conclusion ..............................................................................................................................76
XI
CONNECTING STATEMENT 3 ...............................................................................................77
CHAPTER V. Characterization of films prepared using salmon skin gelatin extracted by a
trypsin-aided process ..................................................................................................................78
5.1 Abstract ...................................................................................................................................79
5.2 Introduction .............................................................................................................................80
5.3 Materials and methods ............................................................................................................82
5.3.1 Chemicals .......................................................................................................................82
5.3.2 Extraction of gelatin from salmon skin ..........................................................................82
5.3.3 Preparation of gelatin films ............................................................................................83
5.3.4 Film characterization .....................................................................................................83
5.3.4.1 Mechanical properties ...........................................................................................83
5.3.4.2 Water solubility .....................................................................................................84
5.3.4.3 Light transmission and opacity .............................................................................85
5.3.4.4 Electrophoretic analysis ........................................................................................85
5.3.4.5 Fourier transform infrared (FT-IR) spectra analysis .............................................85
5.3.4.6 Scanning electron microscopy (SEM) ..................................................................86
5.3.4.7 Statistical analysis .................................................................................................86
5.4 Results and discussion .............................................................................................................86
5.4.1 Mechanical properties .....................................................................................................86
5.4.2 Water solubility ..............................................................................................................88
5.4.3 Light barrier properties ...................................................................................................88
5.4.4 Electrophoretic protein patterns ......................................................................................90
5.4.5 FT-IR spectroscopy.........................................................................................................92
5.4.6 Morphology ....................................................................................................................95
5.5 Conclusion ...............................................................................................................................99
CONNECTING STATEMENT 4 .............................................................................................100
CHAPTER VI. Synthesis and characterization of salmon skin gelatin-corn zein composite
films plasticized with canola oil ...............................................................................................101
6.1 Abstract .................................................................................................................................102
6.2 Introduction ...........................................................................................................................103
6.3 Materials and methods ..........................................................................................................104
6.3.1 Materials ......................................................................................................................104
6.3.2 Extraction of salmon skin gelatin .................................................................................104
6.3.3 Preparation of gelatin-zein composite films .................................................................105
6.3.4 Preparation of gelatin-zein composite films synthesized with canola oil and lecithin .105
6.3.5 Film characterization ....................................................................................................106
6.3.5.1 Film thickness .....................................................................................................106
6.3.5.2 Mechanical properties .........................................................................................106
6.3.5.3 Film solubility ......................................................................................................107
6.3.5.4 Water vapor permeability (WVP) .......................................................................107
6.3.5.5 Light transmission................................................................................................108
6.3.5.6 Fourier transform infrared (FT-IR) spectra analysis ............................................108
6.3.5.7 Polarized light microscopy ..................................................................................108
6.3.5.8 Statistical analysis ...............................................................................................108
XII
6.4 Results and discussion ..........................................................................................................108
6.4.1 Effect of zein concentration on the properties of gelatin-zein composite films ...........108
6.4.1.1 Thickness ............................................................................................................108
6.4.1.2 Mechanical properties .........................................................................................109
6.4.1.3 Film solubility ......................................................................................................110
6.4.2 Effect of glycerol/canola oil ratio on the properties of gelatin-zein composite films ..110
6.4.2.1 Thickness .............................................................................................................110
6.4.2.2 Mechanical properties ..........................................................................................111
6.4.2.3 Film solubility ......................................................................................................111
6.4.2.4 Water vapor permeability ....................................................................................112
6.4.2.5 Light transmission................................................................................................112
6.4.2.6 FT-IR spectroscopy..............................................................................................114
6.4.2.7 Polarized light microscopy ..................................................................................116
6.5 Conclusion .............................................................................................................................118
CONNECTING STATEMENT 5 .............................................................................................119
CHAPTER VII. Salmon skin gelatin-corn zein composite films produced via crosslinking
with glutaraldehyde: optimization using response surface methodology .............................120
7.1 Abstract .................................................................................................................................121
7.2 Introduction ...........................................................................................................................122
7.3 Materials and methods ..........................................................................................................124
7.3.1 Materials ......................................................................................................................124
7.3.2 Extraction of salmon skin gelatin .................................................................................124
7.3.3 Experimental design......................................................................................................124
7.3.4 Preparation of the films ................................................................................................126
7.3.5 Measurement of properties ...........................................................................................126
7.3.5.1 Mechanical properties .........................................................................................126
7.3.5.2 Film solubility .....................................................................................................127
7.3.5.3 Statistical analysis ...............................................................................................127
7.4 Results and discussion ...........................................................................................................128
7.4.1 Statistical analysis .........................................................................................................128
7.4.2 Effect on tensile strength ..............................................................................................130
7.4.3 Effect on elongation at break ........................................................................................131
7.4.4 Effect on water solubility ..............................................................................................133
7.4.5 Validation of the predicted model of optimized compositions .....................................135
7.5 Conclusion ............................................................................................................................135
CONNECTING STATEMENT 6 .............................................................................................136
CHAPTER VIII. Characterization of salmon skin gelatin-corn zein composite films
crosslinked with glutaraldehyde ...............................................................................................137
8.1 Abstract .................................................................................................................................138
8.2 Introduction ............................................................................................................................139
8.3 Materials and methods ...........................................................................................................140
8.3.1 Materials .......................................................................................................................140
8.3.2 Extraction of salmon skin gelatin .................................................................................141
XIII
8.3.3 Preparation of the films.................................................................................................141
8.3.4 Film characterization ....................................................................................................142
8.3.4.1 Infrared analysis ...................................................................................................142
8.3.4.2 Thermal analysis ..................................................................................................142
8.3.4.3 Light barrier properties ........................................................................................142
8.3.4.4 Mechanical properties ..........................................................................................143
8.3.4.5 Water barrier properties .......................................................................................143
8.3.4.6 Dynamic mechanical analysis (DMA) .................................................................144
8.3.4.7 Morphological properties .....................................................................................144
8.3.4.8 Statistical analysis ................................................................................................144
8.4 Results and discussion ...........................................................................................................145
8.4.1 Infrared analysis ............................................................................................................145
8.4.2 Thermal analysis ...........................................................................................................146
8.4.3 Light barrier properties .................................................................................................149
8.4.4 Mechanical properties ...................................................................................................151
8.4.5 Water barrier properties ................................................................................................152
8.4.6 Dynamic mechanical analysis .......................................................................................153
8.4.7 Morphological properties ..............................................................................................155
8.5 Conclusion .............................................................................................................................156
CHAPTER IX. General conclusions, contributions to knowledge and recommendations for
future work .................................................................................................................................157
9.1 General Conclusions ..............................................................................................................158
9.2 Contributions to Knowledge ..................................................................................................160
9.3 Recommendations for Future Work.......................................................................................160
REFERENCES ...........................................................................................................................161
XIV
LIST OF FIGURES
Figure 3.1 Hydroxyproline (Hyp) content and yield of gelatin extracted from salmon skin
pretreated with different pre-treatments. ....................................................................52
Figure 3.2 Yield of gelatin from fish skins (A: salmon, B: skate, C: dogfish) pretreated at different
trypsin concentrations (%) and trypsin incubation times (h), and extracted at different
temperatures (°C). .....................................................................................................54
Figure 3.3 SDS-PAGE patterns of gelatins extracted from fish skins (A: salmon, B: skate, C:
dogfish) at 25 U/g trypsin for (lane 1) 8 h at 70 °C; (2) 4 h at 70 °C; (3) 8 h at 50 °C;
(4) 4 h at 50 °C; at 10 U/g trypsin for (5) 8 h at 70 °C; (6) 4 h at 70 °C; (7) 8 h at 50
°C; (8) 4 h at 50 °C. LMW denoted for low molecular weight protein markers. .......56
Figure 3.4 SDS-PAGE patterns of gelatins extracted from fish skins (A: salmon, B: skate, C:
dogfish) incubated with 1 U/g trypsin for 4 h and extracted at 50 °C for 3 h. HMW
denoted for high molecular weight protein markers. ................................................57
Figure 4.1 Three-dimensional response surface plots for optimization of gelatin extracted with
major protein band intensity, as a function of (a) trypsin concentration and extraction
temperature; (b) trypsin concentration and extraction time; (c) extraction temperature
and extraction time. ...................................................................................................74
Figure 4.2 SDS-PAGE patterns of salmon fish skin gelatins extracted in triplicate under optimal
conditions: (lane 1 to 3) gelatins obtained in triplicates; HMW denoted for high
molecular weight protein markers. .............................................................................75
Figure 5.1 Electrophoretic profile of gelatin films prepared with different protein concentrations
(%); HMW denoted for high molecular weight protein markers. ..............................91
Figure 5.2 Electrophoretic profile of gelatin films containing different glycerol concentrations
(%); HMW denoted for high molecular weight protein markers. ..............................92
Figure 5.3 FT-IR spectra of gelatin films prepared with different protein concentrations (%). ..93
Figure 5.4 FT-IR spectra of gelatin films containing different glycerol concentrations (%). ....95
Figure 5.5 SEM micrographs (at 1000x magnification) of surface of salmon gelatin films
prepared with different protein concentrations (%). ..................................................96
Figure 5.6 SEM micrographs (at 1000x magnification) of surface of salmon gelatin films
containing different glycerol concentrations (%). ......................................................98
Figure 6.1 Light transmission of gelatin-zein composite films incorporated with canola oil at
various concentrations. .............................................................................................113
XV
Figure 6.2 FT-IR spectra of gelatin-zein composite films incorporated with canola oil at various
concentrations. ..........................................................................................................114
Figure 6.3 Polarized light microscopy (at 5x magnification) of gelatin-zein composite films
incorporated with canola oil at various concentrations: (a) control film (without canola
oil), (b) 15%, and (c) 30%. .......................................................................................117
Figure 7.1 (a) Predicted versus actual experimental values for TS. (b) Three-dimensional (3D)
response surface contour plot indicating the effect of interaction between zein and
glutaraldehyde concentrations for TS of the resulting films. ...................................131
Figure 7.2 (a) Predicted versus actual experimental values for EAB. (b) Three-dimensional (3D)
response surface contour plot indicating the effect of interaction between zein and
glutaraldehyde concentrations for EAB of the resulting films. ................................132
Figure 7.3 (a) Predicted versus actual experimental values for WS. (b) Three-dimensional (3D)
response surface contour plot indicating the effect of interaction between zein and
glutaraldehyde concentrations for WS of the resulting films. ..................................134
Figure 8.1 FT-IR spectra of gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked with
glutaraldehyde (GZ-gla) films. .................................................................................146
Figure 8.2 DSC thermograms (A) and TGA curves (B) of gelatin (G), gelatin-zein (GZ), and
gelatin-zein crosslinked with glutaraldehyde (GZ-gla) films...................................148
Figure 8.3 Samples of gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked with
glutaraldehyde (GZ-gla) films. .................................................................................150
Figure 8.4 Storage modulus (A) and loss modulus (B) of gelatin-zein (GZ) and gelatin-zein
crosslinked with glutaraldehyde (GZ-gla) films. .....................................................154
Figure 8.5 SEM, at 1500x magnification (A), and polarized light microscopy, at 10x
magnification (B) of gelatin-zein (GZ) and gelatin-zein crosslinked with
glutaraldehyde (GZ-gla) films. .................................................................................155
XVI
LIST OF TABLES
Table 2.1 Literature on the water barrier and mechanical properties of fish gelatin based films
developed via different modification approaches. .......................................................16
Table 2.2 Water barrier properties of fish gelatin films.................................................................22
Table 2.3 Mechanical properties of fish gelatin films. .................................................................27
Table 2.4 Thermal properties of fish gelatin films. ......................................................................34
Table 4.1 Plackett–Burman experiment design with actual experimental values and coded values
(in bracket) and response values for gelatin extraction. ...............................................69
Table 4.2 Effects of the variables on yield of gelatin extracted (based on Hyp content) (Y1) and
statistical analysis of data from the Plackett-Burman design. ......................................69
Table 4.3 Effects of the variables on intensity of α-chains (Y2) in extracted gelatin and statistical
analysis of data from the Plackett-Burman design. ......................................................70
Table 4.4 Box-Behnken experiment design with actual experimental values and coded values (in
bracket) and response values of the yield of gelatin extracted calculated based on αchains band intensity. ...................................................................................................71
Table 5.1 Effect of protein and glycerol concentration on the thickness, mechanical properties and
water solubility of salmon skin gelatin films. ...............................................................87
Table 5.2 Effects of protein concentration on the light transmission and opacity of salmon skin
gelatin films. ..................................................................................................................89
Table 5.3 Effects of glycerol concentration on the light transmission and opacity of salmon skin
gelatin films with 5% protein. .......................................................................................90
Table 6.1 Thickness, tensile strength (TS), elongation at break (EAB) and water solubility of
salmon skin gelatin films blended with zein at different concentrations. ...................109
Table 6.2 Thickness, tensile strength (TS) and elongation at break (EAB) of gelatin-zein
composite films incorporated with canola oil at various concentrations. ...................110
Table 6.3 Water vapor permeability (WVP) and water solubility of gelatin-zein composite films
incorporated with canola oil at various concentrations. ..............................................112
Table 7.1 Independent variables and their actual and coded values (in brackets) used for
optimization of gelatin-zein composite films crosslinked with glutaraldehyde. .........128
Table 7.2 Coefficients and their significance in best fitted regression models of different responses.
.....................................................................................................................................129
Table 7.3 Predicted and experimental response values under the optimum compositions. ........135
XVII
Table 8.1 Thermal degradation temperature (Td, °C) and weight loss (∆w, %) of G, GZ, and GZgla films. ......................................................................................................................149
Table 8.2 Light transmission (%T) of gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked
with glutaraldehyde (GZ-gla) films. ............................................................................150
Table 8.3 Mechanical properties (TS and EAB) and film solubility (water solubility and WVP) of
gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked with glutaraldehyde (GZgla) films......................................................................................................................151
XVIII
LIST OF ABBREVIATIONS
2-ME
AFM
ANOVA
ASTM
BBD
BCA
BSA
BSE
CCD
CO2TR
CV
DDGS
DMA
DMAB
DMA-RH
DSC
EAB
FEG-SEM
FFS
FT-IR
Hyp
LEI
NaCl
NaOH
NMR
OTR
PB
PLM
Pro
RH
RSM
SDS
SDS-PAGE
SEM
T
TEM
TEMED
2-mercaptoethanol
atomic force microscopy
analysis of variance
American Society for Testing and Materials
Box-Behnken design
bicinchoninic acid
bovine serum albumin
bovine spongiform encephalopathy
central composite design
carbon dioxide transmission rate
coefficient of variation
dried distillers grains with soluble
dynamic mechanical analysis
4-dimethylamino-benzaldehyde
humidity-controlled dynamic mechanical analysis
differential scanning calorimetry
elongation at break
field emission gun scanning electron microscope
film forming solution
Fourier-transform-infrared spectroscopy
hydroxyproline
low secondary electron image
sodium chloride
sodium hydroxide
nuclear magnetic resonance spectroscopy
oxygen transmission rate
Plackett-Burman design
polarized light microscopy
proline
relative humidity
response surface methodology
sodium dodecyl sulfate
sodium dodecyl sulfate polyacrylamide gel electrophoresis
scanning electron microscopy
light trasmission
transmission electron microscopy
N,N,N’,N’-tetramethyl ethylene diamine
XIX
TGA
TGase
TS
UV
Vis
WS
WVP
WVTR
XRD
thermogravimetric analysis
transglutaminase
tensile strength
ultraviolet
visible light
water solubility
water vapour permeability
water vapour transmission rate
X-ray diffraction
XX
CHAPTER I. GENERAL INTRODUCTION
1
Gelatin has been extensively studied for its film forming ability as gelatin films can potentially
prolong the shelf life of food products. Protein-based biodegradable films have been synthesized
using mammalian gelatin from pig skin, bovine hide, pork and cattle bones (Gómez-Guillén et al.,
2009). The use of these gelatin sources is limited due to concerns regarding animal diseases (e.g.
bovine spongiform encephalopathy (BSE)), and religious and cultural demands for kosher and
halal foods (Sadowska et al., 2003). Thus, collagen-rich fish wastes such as fish skin and bones,
are of interest as alternative sources of gelatin. Commercial fish wastes account for approximately
7.3 million tonnes per year and are generally discarded or under-utilized as animal feed (Karim &
Bhat, 2009).
Fish gelatins are biopolymers derived through partial hydrolysis of collagen-rich fish wastes. This
water-soluble protein is generally obtained via pre-treating the raw materials with acid, alkaline or
proteases, followed by hot water extraction. Recently, a pepsin-aided process was found to produce
a high yield of fish gelatin (Chomarat et al., 1994; Nalinanon et al., 2008). In comparison to pepsin,
trypsin has a higher degree of hydrolysis and produces a poorer gelatin quality. However, at lower
concentration, a good quality gelatin with higher yield was obtained from trypsin-treated wastes
from the leather industry when compared to pepsin (Cabeza et al., 1997). The higher hydrolytic
behavior of trypsin has become an advantage, in which its narrower specificity and higher
efficiency enable the yield of good quality gelatin, indicating the possibility of a more costeffective gelatin extraction process. However, there are no studies reporting on the trypsin-aided
extraction of fish gelatin. Thus, this has led to the development and optimization of a novel trypsinaided extraction process to obtain gelatin from fish skins. Assessing the properties of films
prepared using the extracted fish gelatins would be of interest to understand the characteristics of
the films formed.
A major drawback with gelatin films is that with high moisture foods or under high humid storage
conditions, the films tend to disintegrate. Hence, gelatin has been studied for its compatibility with
other biopolymers aimed at producing composite packaging films with improved barrier and
mechanical properties (Gómez-Guillén et al., 2011). For this reason, it has led to incorporating
hydrophobic compounds into fish gelatin film matrix. Corn zein is a hydrophobic protein obtained
from corn and under-utilized corn by-products such as dried distillers grains with solubles (DDGS).
It is compatible with other film-forming proteins due to its flexibility and compressibility,
2
producing films with high tensile strength and improved water-resistant properties (Shukla &
Cheryan, 2001). Based on the individual characteristics of fish gelatin and zein, it is anticipated
that their combination would lead to films with better functional properties than those formed by
each protein alone (Hosseini et al., 2013; Mauri & Añón, 2008). It is well known that gelatin is a
water-soluble protein, and that zein is not soluble in water (Chiou et al., 2008; Gu & Wang, 2013).
This implies that the formation of a homogeneous composite film would be a challenge.
Nonetheless, there is a good probability that these proteins would be miscible since zein can
solubilize in 50-90% ethanol (Shukla & Cheryan, 2001) while gelatin dissolves well in aqueous
ethanol up to 50% (Farrugia & Groves, 1999). To date, no studies have explored the incorporation
of zein in fish gelatin films using aqueous ethanol as solvent. Therefore, developing fish gelatinzein composite films will help to evaluate the extent to which zein can improve the water barrier
properties of gelatin-based films. In addition, the incorporation of canola oil to replace glycerol as
plasticizer would be of interest to study the influence of the hydrophilic-hydrophobic nature of the
plasticizer on the properties of the resultant films.
The physical properties of protein-based films have been effectively reinforced by cross-linkers.
The inherent deficiencies in the barrier and mechanical properties of protein films can be resolved
using a cross-linking approach which modifies the film networks to a higher integrity level, and
thereby increases applicability of the networks as food packaging material (Garavand et al., 2017).
Chemical cross-linkers are cheaper and more effective for improving the film properties of fish
gelatin based films, as compared to enzymatic cross-linkers (Cao et al., 2007; Kolodziejska &
Piotrowska, 2007; Kolodziejska et al., 2006). Among cross-linkers, glutaraldehyde is the most
widely used, attributed to its pronounced efficiency to react with collagenous materials (Bigi et al.,
2001; Chen et al., 2014). Glutaraldehyde-induced cross-links markedly improve the water
resistance owing to the tightly formed three-dimensional networks; concomitantly, it also increases
the brittleness and decreases the mechanical strength of the biopolymer films (Garavand et al.,
2017; Schiffman & Schauer, 2007). In addition, the cytotoxicity of glutaraldehyde has limited its
usage in food systems; however, the cytotoxic effects can be reduced by lowering the concentration
(Jayakrishnan & Jameela, 1996) or eliminating glutaraldehyde by washing the film with saline
solution (Cooke et al., 1983). Upon considering the benefits and limitations of glutaraldehyde in
collagenous materials, an investigation would be of interest to discover the extent of its effect on
the performance of fish gelatin films. Currently, limited studies are available pertaining to the
3
improvement of the properties for fish gelatin composite films cross-linked with glutaraldehyde.
In this study, film formulations containing glutaraldehyde and zein have been explored to obtain
fish gelatin films with optimized strength and water resistance.
Various approaches have been employed to assess the importance of structural modifications to
obtain gelatin films with enhanced functional properties. In the last decades, different techniques
have been applied to characterize the modified films, such as differential scanning calorimetry
(DSC) and Fourier-transform-infrared spectroscopy (FT-IR) (Chiou et al., 2008; Denavi et al.,
2009; Ghanbarzadeh & Oromiehi, 2009; Gómez-Guillén et al., 2009; Hoque et al., 2011; Hosseini
et al., 2013; Jongjareonrak et al., 2006; Tongnuanchan et al., 2015). Moreover, scanning electron
microscopy (SEM) is the most effective tool to examine the morphology of films and provide
direct visualization of the structural organization of polymeric matrix corresponding to the
performance of the gelatin films (Ahmad et al., 2012; Arfat et al., 2014; Hoque et al., 2011;
Limpisophon et al., 2010; Nur Hanani et al., 2013; Tongnuanchan et al., 2015). Nevertheless, the
performance of a biopolymer film in real-life application conditions is crucial to enable its
utilization as food packaging material, especially its integrity during storage and handling
conditions at varying humidity. Relative humidity-controlled dynamic mechanical analysis
(DMA-RH) has emerged as a prominent technology to demonstrate the relationship between the
relative humidity and the physical properties of polymeric materials (Adriana et al., 2013;
Bonnaillie & Tomasula, 2015; Cataldo et al., 2017; Gregorová et al., 2015; Milinkovic et al., 2014).
To date, there has been no DMA-RH study conducted on fish gelatin based films.
Given all these points, it was hypothesized that the trypsin-aided extraction process can produce
film-forming fish skin gelatin, and blending gelatin with hydrophobic zein followed by crosslinking with glutaraldehyde can produce films with improved water resistance. This present
research therefore aimed at using trypsin to aid the recovery of gelatin from fish skins, to form
reinforced composite films with zein and glutaraldehyde, and to verify the characteristics of the
resulting films with respect to their barrier, mechanical, thermal, structural and morphological
properties. The specific objectives of this research were as follows:
1. To investigate the use of trypsin for the extraction of gelatin from fish skins.
2. To optimize the trypsin-aided process for extracting fish gelatin with film-forming
attributes.
4
3. To determine the characteristics of fish gelatin films formed in terms of the mechanical,
barrier, structural and morphological properties.
4. To determine the effect of incorporating zein and canola oil (in replacement to glycerol as
plasticizer) on the mechanical, barrier, structural and morphological properties of the fish
gelatin composite films.
5. To determine the optimized formulation for glutaraldehyde-crosslinked fish gelatin-zein
composite films based on the mechanical and water barrier properties.
6. To evaluate the performance of glutaraldehyde-crosslinked fish gelatin-zein composite
films in terms of the mechanical, barrier, thermal, structural and morphological properties.
5
CHAPTER II. LITERATURE REVIEW
6
There is a growing interest in the use of fishery by-products which are rich sources of gelatin as
an alternative to gelatin from mammalian origins due to religious concerns and also animal-derived
diseases (Gómez-Guillén et al., 2009; Muyonga et al., 2004). These fishery by-products (e.g. skins,
bones and fins) are produced abundantly from the fish processing industry, and are usually utilized
as feed or are discarded as waste (Blanco et al., 2007). Fish gelatin is obtained from both warmand cold-water fish by-products (Karim & Bhat, 2009). Cold-water fish accounts for the larger
part of total industrial fish capture and the fish fillet industry. Gelatin sourced from cold-water fish
by-products could therefore become the major source of gelatin (Gómez-Guillén et al., 2009). Fish
gelatin is contributing about 1% of the annual gelatin production (Karim & Bhat, 2009).
In comparison to warm-water fish gelatin, cold-water fish gelatin is often regarded as a lower
quality gelatin that exhibits poorer functional properties (Gómez-Guillén et al., 2011). Much work
has been carried out to study the utilization of cold-water fish gelatins, especially for the
development of biodegradable films for food packaging (Gómez-Guillén et al., 2009). Generally,
gelatin films are known for their poor water resistance ascribed to their hydrophilic nature (GómezGuillén et al., 2009; Guilbert et al., 1996). However, cold-water fish gelatin is more hydrophobic
due to its higher content of hydrophobic amino acids as compared to warm-water fish gelatin
(Avena-Bustillos et al., 2006). This chapter highlights the properties of fish skin gelatin for film
formation, methods used for extracting fish gelatin, processes employed for forming fish gelatin
films, strategies available for improving the properties of fish gelatin films, and assessment of fish
gelatin film properties.
2.1 Fish skin gelatin for film formation
Gelatin is a water-soluble protein with film-forming properties that is derived from partial
hydrolysis of collagen (Cuq et al., 1998). In general, gelatin contains loose monomers in the form
of random coil chains such as α-chains when dissolved in water. When gelatin solution is cooled
and water is evaporated further, these loose chains start to arrange orderly and lose their mobility,
leading to cross-linking and network formation (Harris et al., 2003). The formation of film
networks depends on the amino acid composition and molecular weight distribution of gelatin
(Gómez-Guillén et al., 2009).
7
2.1.1 Amino acid composition
Gelatin contains different amino acids depending on the species and the living habitat of the source
of gelatin (Jongjareonrak et al., 2005; Kittiphattanabawon et al., 2005). The amino acids that are
characteristic to gelatin are the imino acids, proline (Pro) and hydroxyproline (Hyp). These imino
acids are partly responsible for the degree of rigidity of the gelatin structure in films. Higher
amounts of imino acids are associated with improved rheological properties and thermostability of
gelatin films (Gómez-Guillén et al., 2009). It is believed that the hydrogen-bonding ability of Hyp
through its hydroxyl group plays a singular role in facilitating interactions between α-chains, which
leads to stable cross-links and film network (Brinckmann, 2005; Galea et al., 2000). Generally, the
low amount of Pro and Hyp found in cold-water fish skin gelatin results in films with poorer
structural, rheological and thermal properties as compared to films synthesized from warm-water
fish skin gelatin (Gómez-Guillén et al., 2009; Ledward, 1986).
The Hyp content is measured to indicate the amount of collagen and gelatin in a sample. The Hyp
content of some fish gelatins has been measured such as for gelatins from skins of salmon and
herrings (Kołodziejska et al., 2008), bigeye snapper (Nalinanon et al., 2008), and yellowfin tuna
(Cho et al., 2005). The Hyp content is measured by using a hydroxyproline content assay. Hyp is
released from protein and peptides samples by acid hydrolysis, and then oxidized with chloramine
T, followed by reaction with 4-dimethylamino-benzaldehyde (DMAB), resulting in a chromogen
that can be quantified using a UV/Vis spectrophotometer with an absorbance at 560 nm (Reddy &
Enwemeka, 1996).
2.1.2 Molecular weight distribution
The film properties are also influenced by the molecular weight distribution of gelatin. The
predominance of higher molecular weight polymer chains (i.e. α-chains) contributes to a higher
functionality of gelatin (Galea et al., 2000). The molecular weight distribution of gelatin is mainly
affected by the severity of the processing conditions, such as pH, extraction temperature and time
(Gómez-Guillén et al., 2002). Generally, the excessive concentration of acid and/or alkaline and
severe heating conditions increase the gelatin yield, but also degrade the collagen structure,
resulting in a higher proportion of low molecular weight chains in gelatin (Boran & Regenstein,
2010; Gómez-Guillén et al., 2009). These shorter chains hinder the formation of the junction zone
8
and the development of a strong network, owing to the poor interactions among these protein
fractions, leading to lower functional properties of the resulting films (Benjakul et al., 2012).
Gelatin with higher molecular weight proteins need a lower number of cross-links to form a strong
network; in contrast, gelatin with lower molecular weight proteins has limited cross-links to
establish junction zones and therefore form a weaker network (Gilsenan & Ross-Murphy, 2000).
The molecular weight distribution of fish gelatins extracted under various processing conditions
is often examined prior to the preparation of films. This was done for gelatins extracted from skins
of Alaskan pollock and Alaskan pink salmon (Avena-Bustillos et al., 2006), brownbanded bamboo
shark and blacktip shark (Kittiphattanabawon et al., 2010), blue shark skin (Limpisophon et al.,
2009), bigeye snapper (Nalinanon et al., 2008), brownstripe red snapper and bigeye snapper
(Jongjareonrak et al., 2006), and cuttlefish (Hoque et al., 2011). The molecular weight distribution
of gelatin can be determined by means of sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). For this analysis, the sample is first heat-denatured by incubating
with SDS and reducing agents, such as 2-mercapthoethanol or dithiothreitol. The pre-treated
samples are then applied on a polyacrylamide gel and placed under an electric field (Walsh, 2002).
The protein fragments in the sample separate on the basis of their sizes, with the smaller molecules
migrating ahead of the larger ones. The migration of the protein bands in the gel is compared with
those of marker protein bands with known molecular weights (Garfin, 1990).
2.2 Extraction of fish skin gelatin
Fish gelatin is obtained from fish by-products via a series of steps including pre-treatments to
remove non-collagenous materials and to disrupt the collagen structure, followed by warm water
extraction (>40°C) to enable the release of free α-chains to form water-soluble gelatin (GómezGuillén et al., 2002). This is followed by refining/clarification steps including filtration,
concentration, drying and milling to obtain the dried product (Schrieber & Gareis, 2007). The
quality of the gelatin obtained is dependent on the severity of the pre-treatments (Gómez-Guillén
et al., 2002; Gómez-Guillén et al., 2009). To obtain gelatin with desired properties, this requires
optimization of the processing conditions which involve variables such as the pre-treatment agent
concentration and time, extraction time and temperature (Mohtar et al., 2010; Norziah et al., 2014).
Depending on the degree of collagen cross-linking in the raw material, two types of gelatin are
9
produced commercially, which are type A (obtained from acid pre-treatment) and type B (obtained
from alkaline pre-treatment).
2.2.1 Acid pre-treatment
Acid pre-treatment, which is less severe as compared to alkaline pre-treatment, is usually used to
extract gelatin from collagens with a lower cross-linking degree such as fish skin (Gómez-Guillén
et al., 2009; Montero et al., 1990). This pre-treatment yields good quality gelatin by disruption of
acid-labile collagen cross-links without severe protein hydrolysis and amino acid decomposition
(Galea et al., 2000). In addition, this pre-treatment removes other organic substances while
partially inactivating endogenous proteases that catalyze the undesirable formation of short
fragments (Sovik & Rustard, 2006). The type and concentration of acid used can affect the yields
and functional properties of the resulting gelatins. Several mild acids have been used in the
extraction of gelatin from different fish species, such as acetic, phosphoric and citric acids
(Benjakul et al., 2012; Gómez-Guillén et al., 2009). So far, acetic acid treated collagen produces
gelatin with increased gel strength due to a high content in high molecular weight chains (Ahmad
et al., 2010; Giménez et al., 2005; Khiari et al., 2015).
2.2.2 Alkaline pre-treatment
Alkaline pre-treatment is more suitable for collagen having a high degree of crosslinking such as
collagen from mammalian origins (Gómez-Guillén et al., 2002). This process primarily breaks
some cross-links, leading to random hydrolysis of peptide bonds and degradation of protein chains,
further resulting in some amino acids decomposition and lower molecular weight fractions in the
gelatin (Benjakul et al., 2012; Galea et al., 2000; Yoshimura et al., 2000). This pre-treatment
effectively removes considerable amounts of non-collagenous materials (Boran & Regenstein,
2010; Zhou & Regenstein, 2005). To obtain fish gelatin with higher yields and functional
properties, using a combination of weaker and stronger alkali can limit the severe disruption of the
collagen structure (Kaewdang et al., 2016). The most commonly used alkalines for pre-treating
fish skins for gelatin extraction are sodium hydroxide and calcium hydroxide (Karim & Bhat,
2009). Calcium hydroxide is relatively mild and causes less degradation of the gelatin chains
(Benjakul et al., 2012).
10
2.2.3 Other pre-treatments
Other pre-treatments have been used in an attempt to improve the efficiency of extracting fish
gelatin. Fish skins pre-treated with saline solution produced a higher yield of gelatin and higher
gel strength when pre-treated with mild acid and alkaline solutions (Mohtar et al., 2010). This is
attributed to the disruption of the collagen structure by the ions which interacted with the hydrogen
bonds of collagen. This is described as ‘lyotropic hydration’ (Asghar & Henrickson, 1982).
Meanwhile, a pepsin-aided process was found to increase the yield of fish gelatin by approximately
two-fold over the non-pepsin assisted treatment (Nalinanon et al., 2008). Good film-forming
properties, mechanical and water barrier properties were observed for films prepared using gelatins
derived from pepsin-treated giant squid (Giménez et al., 2009). In comparison to pepsin, a lower
concentration of trypsin assisted the extraction of good quality gelatin with improved yield from
wastes of the leather industry (Cabeza, 1997). This is because trypsin has higher specificity upon
collagen as compared to pepsin (Benjakul et al., 2012; Worthington, 2015).
2.3 Fish gelatin film formation
All fish gelatins exhibit good film-forming capacity and formed films that are transparent, water
soluble and which are highly extensible (Avena-Bustillos et al., 2006; Carvalho et al., 2008;
Jongjareonrak et al., 2006; Zhang et al., 2007). The performance of fish gelatin films is not only
dependent on the properties of the gelatin obtained from different species and extraction conditions
but also relies on the film formulation and preparation conditions (Gómez-Guillén et al., 2009).
Generally, the biochemical properties of each protein and the preparation conditions influence the
type of bonds formed such as the hydrophobic, electrostatic, hydrogen and/or covalent bonds
(Gómez-Estaca et al., 2016). Usually, protein films are very brittle (Sothornvit & Krochta, 2001).
A plasticizer is often added to the formulation to reduce the polymer chain-to-chain interactions
and improve the flexibility of the films. The selection of a plasticizer is determined by the degree
of compatibility and the physical properties desired for the films (Cheng et al., 2006).
A protein network is obtained via three main steps, which are the disruption of low-energy
intermolecular bonds, followed by orientation and organization of the polymer chains, and the
establishment of a three-dimensional network via new intermolecular interactions (Cuq et al.,
11
1998). The wet process and the dry process are the main processes used for the synthesis of protein
films (Gómez-Estaca et al., 2016; Hernandez-Izquierdo & Krochta, 2008).
2.3.1 Wet process
The wet process involves the solubilization of proteins and addition of plasticizers and/or other
additives to obtain a film-forming solution. The solution is then casted onto plates. The solvent
must evaporate to obtain the film (Gómez-Estaca et al., 2016). The most commonly used solvents
are water and ethanol (Cuq et al., 1998). Through solvent removal, the film matrix is formed with
the combination of hydrophobic, electrostatic, hydrogen and covalent bonds between the protein
chains (Sothornvit & Krochta, 2001). Plasticizers are often added, such as glycerol and sorbitol,
to reduce the interactions between the protein chains, increase the free-volume and chains mobility,
and decrease the brittleness of the films (Wihodo & Moraru, 2013). This process has been used
frequently for the formation of fish gelatin films (Avena-Bustillos et al., 2006; Carvalho et al.,
2008; Hoque et al., 2011; Jongjareonrak et al., 2006; Limpisophon et al., 2009).
2.3.2 Dry process
The dry process is preferred at the industrial scale since it can be performed in a continuous fashion
(Gómez-Guillén et al., 2009). Under low moisture and high temperature conditions, proteins
exhibit viscoelastic behavior in the presence of plasticizers, enabling them to be shaped into a
desirable film when they are cooled (Cuq et al., 1998). During this process, denaturation of proteins
exposes the embedded functional groups of the proteins, facilitating new interactions and
intermolecular bonding between them, and forming film matrix with modified properties
(Hernandez-Izquierdo & Krochta, 2008). The thermal and mechanical properties of the protein
films are affected by the protein characteristics, such as the functional groups, molecular weight,
molecular organization, types of bonds, as well as the nature and quantity of the plasticizers (Cuq
et al., 1998). The dry process can be further classified into extrusion and thermo-pressing methods,
which are often used together (Gómez-Estaca et al., 2016). The mechanical and water barrier
performance of dry-processed films are generally inferior to that of wet-processed films (GómezEstaca et al., 2016).
12
2.4 Fish gelatin film modification
The main drawbacks of fish gelatin films are their poor water resistance and mechanical
performance that restrict their application as food packaging (Gómez-Guillén et al., 2009).
Improvement of fish gelatin films can be done physically, chemically, or enzymatically. Table 2.1
shows the water barrier and mechanical properties reported on modified fish gelatin based films
using various approaches.
2.4.1 Blending
Polymer blending is an easy approach to modify the properties of protein films without incurring
a high cost (Wang et al., 2009). Blending compatible biopolymers allows each polymer to
complement their advantages while minimizing their individual disadvantages and helps in
achieving the targeted film properties (Galus & Kadzińska, 2015). Particularly, blending fish skin
gelatin with polymers of different molecular weights and hydrophobicity can enhance the
mechanical and barrier properties of the resulting films. For example, films with relatively lower
water vapor permeability and improved mechanical strength were reported when blending soy
protein isolate to cod skin gelatin (Denavi et al., 2009), mungbean protein isolate to cuttlefish skin
gelatin (Hoque et al., 2011), palmitic acid and stearic acid to bigeye snapper and brownstripe red
snapper skin gelatins (Jongjareonrak et al., 2006), sunflower oil to cod skin gelatin (Pérez-Mateos
et al., 2009), and palm oil to tilapia skin gelatin (Tongnuanchan et al., 2015). When blending
hydrophobic compounds with gelatin, an emulsifier is often added to improve the homogeneity of
the blend (Dickinson, 2003).
2.4.2 Lamination
Lamination, also known as bilayer film formation, is a method to form a film in multiple layers in
order to create a composite which will have improved physical properties (Galus & Kadzińska,
2015). The laminate is made either by thermo-pressing or by casting a film solution over a dried
film, and this technique is used to cast the hydrophobic biopolymer onto the protein film (GómezEstaca et al., 2016; Lee & Song, 2017). The resulting laminated films exhibit enhanced properties
as compared to each respective film, with higher water resistance ascribed to the hydrophobic layer
and improved mechanical strength or oxygen barrier attributed to the protein layer (Gómez-Estaca
13
et al., 2016). A recent study reported an increased water resistance for the olive flounder skin
gelatin-polylactic acid bilayer film that was formed using casting technique, making this technique
workable to lower the hydrophilic character of fish gelatin film alone (Lee & Song, 2017).
However, the possibility of poor compatibility between the layers is high due to different polarities.
This method is also time-consuming and therefore is not widely used (Debeaufort & Voilley, 1995;
Gallo et al., 2000).
2.4.3 Cross-linking
Cross-linking agents induce cross-links through the various functional groups of proteins, leading
to a three dimensional networks (De Jong & Koppelman, 2002; Garavand et al., 2017; Yi et al.,
2006). This restricts the mobility of polymer chains and leads to the production of films with
enhanced properties, such as reduced solubility, improved mechanical properties and water
resistance (Kolodziejska & Piotrowska, 2007; Liu et al., 2007).
Chemical or enzymatic cross-linkers are usually added to improve the protein film properties.
Chemical cross-linkers, such as glutaraldehyde, glyceraldehyde, formaldehyde, and glyoxal, have
been added to fish gelatin films (Bigi et al., 2001; Carvalho & Grosso, 2004; Chiou et al., 2008).
Glutaraldehyde is by far the most widely used cross-linker, which is attributed to its low cost and
ability to react with proteins (Bigi et al., 2001; Chen et al., 2014). It forms covalent cross-links
between the aldehyde groups of glutaraldehyde and the free amino groups of lysine or
hydroxylysine (Damink et al., 1995). In a study conducted by Liu et al. (2007), the properties of
pectin-fish skin gelatin composite film were evaluated and compared to glutaraldehyde-treated
pectin-gelatin film. They found that blending pectin and fish gelatin produced films with an
increase in mechanical strength and a decrease in water solubility. These properties were further
enhanced when the films were cross-linked with glutaraldehyde. However, the use of chemical
cross-linkers is limited in food applications due to their toxicity (Chambi & Grosso, 2006).
One enzymatic cross-linker that can be used for food use is transglutaminase (TGase). This enzyme
catalyzes the formation of intra and intermolecular cross-links via acyl transfer between the
carboxylamide groups of glutamine residues (donor) and the amino group of lysine residues
(acceptor) (De Jong & Koppelman, 2002). TGase-modified fish gelatin films have been shown to
exhibit an appreciable increase in tensile strength as the reaction time of gelatin with TGase
14
increased, but a decrease in flexibility owing to the formation of cross-links that hinder the helical
structure formation (Yi et al., 2006). However, chemical cross-linkers are more effective in
improving the water resistance of fish gelatin based films (Kolodziejska et al., 2006; Kolodziejska
& Piotrowska, 2007) and are less expensive than enzymatic cross-linkers (Cao et al., 2007).
2.4.4 Nanoparticle reinforcement
Nanoparticle reinforcement is one of the most recent strategies used for the development of fish
gelatin films (Gómez-Estaca et al., 2016). Nanoparticles act as filler (discontinuous phase) within
the biopolymer matrix (continuous phase) (Wihodo & Moraru, 2013). The nanoparticles disperse
within the biopolymer matrix and limit the polymer chain mobility, which significantly enhance
the mechanical, thermal and the water barrier properties (Castro-Rosas et al., 2016; Kovacevic et
al., 2008). Organic nanoparticles such as starch nanocrystals and cellulose nanoparticles, and
inorganic nanoparticles such as carbon nanotubes and silver nanoparticles can be used as fillers
(Castro-Rosas et al., 2016). The nanoparticles can also be classified according to their dimensions:
iso-dimensional nanoparticles with three nanometric dimensions such as silica spheres, and
whisker nanotubes with two nanometric dimensions, such as elongated and rod-shaped
nanoparticles (Castro-Rosas et al., 2016). Generally, these nanoparticles can be incorporated into
a polymeric matrix through wet or dry processing methods (Gómez-Estaca et al., 2016).
Studies showed that the mechanical properties, water and light barrier properties, and the thermal
properties of fish gelatin nanocomposite films were improved as compared to films composed of
gelatin only. The nanocomposites tested were cellulose whiskers (Santos et al., 2014), sodiummontmorillonite (Bae et al., 2009), and chitosan nanoparticles (Hosseini et al., 2015). A similar
effect was observed when zinc oxide nanoparticles were used (Arfat et al., 2016; Rouhi et al.,
2013). Another study conducted by Bae et al. (2009) showed that the addition of nanoclay
improved the mechanical and barrier properties of fish gelatin films. After cross-linking with
TGase, a decrease in tensile strength and an increase in flexibility were observed, while
maintaining similar barrier properties. However, in spite of several advantages of nanoparticles,
their use in food packaging could be tricky as there could be significant health risks due to possible
migration into the food matrices (Cushen et al., 2012; Honarvar et al., 2016).
15
Table 2.1 Literature on the water barrier and mechanical properties of fish gelatin based films developed via different modification
approaches.
Fish gelatin
Cod skin gelatin
Modification
approach
Blending
Cod skin gelatin
Blending
Catfish skin
gelatin
Blending
Blue shark skin
gelatin
Blending
Cuttlefish skin
gelatin
Blending
Unicorn
leatherjacket skin
gelatin
Blending
Tilapia skin
gelatin
Blending
Tilapia skin
gelatin
Blending
Tilapia skin
gelatin
Blending
Film modification
Cod gelatin (G)/ soybean
protein isolate (S) (100:0,
75:25, 50:50, 25:75, 0:100)
Cod gelatin added with
sunflower oil (0, 0.3, 0.6, 1.0%,
w/v)
Catfish gelatin added with
triacetin (0, 50, 100, 150% of
the gelatin amount)
Blue shark gelatin added with
stearic and oleic acid (0, 25, 50,
100%, w/w)
Cuttlefish gelatin (CG)/
mungbean protein isolate (MPI)
(10:0, 8:2, 6:4, 4:6, 2:8, 0:10)
Unicorn leatherjacket gelatin
added with bergamot (BO) and
lemongrass (LO) oils (0, 5, 10,
15, 20, 25%, w/w)
Tilapia gelatin (4, 6, 8%, w/v)
added with corn oil (55.18%,
w/w)
Tilapia gelatin (FSG)/fish
protein isolate (FPI) (10:0, 5:5,
4:6, 2:8, 0:10)
Tilapia gelatin added with palm
oil (0, 25, 50, 75, 100%, w/w)
16
Main findings on water barrier
and mechanical properties
Lower WVP, lower deformation,
higher breaking force for G:S at
50:50 and 75:25
Higher WVP, lower film solubility,
lower puncture force, lower
puncture deformation
Higher WVP, higher film solubility,
lower TS, higher EAB
Reference
Lower WVP, lower TS, higher EAB
Limpisophon et
al. (2010)
Lower WVP and film solubility,
lower TS, higher EAB for CG:MPI
at 6:4 and 4:6
Hoque et al.
(2011)
For BO: higher WVP, lower film
solubility, lower TS, lower EAB
For LO: lower WVP, lower film
solubility, lower TS, higher EAB
Higher WVP, higher film solubility,
higher TS, lower EAB
Ahmad et al.
(2012)
Higher WVP, lower film solubility,
lower TS, lower EAB
Arfat et al. (2014)
Lower WVP, lower TS, lower EAB
Tongnuanchan et
al. (2015)
Denavi et al.
(2009)
Pérez-Mateos et
al. (2009)
Jiang et al. (2010)
Nur Hanani et al.
(2013)
Olive flounder
skin gelatin
Lamination
Pollock skin
gelatin
Cross-linking
Salmon skin
gelatin
Cross-linking
Fish gelatin
Nanoparticles
reinforcement
Tilapia residue
gelatin
Nanoparticles
reinforcement
Cold-water fish
skin gelatin
Nanoparticles
reinforcement
Warm-water fish
gelatin
Blending and
nanoparticles
reinforcement
Tilapia skin
gelatin
Blending and
nanoparticles
reinforcement
Tilapia skin
gelatin
Blending and
nanoparticles
reinforcement
Olive flounder gelatin (OSG) –
polylactic acid (PLA) bilayer
film
Pollock gelatin cross-linked
with glutaraldehyde (0, 0.25,
0.50, 0.75%, w/w)
Salmon gelatin cross-linked
with glutaraldehyde (0, 0.25,
0.50, 0.75%, w/w)
Fish gelatin incorporated with
zink oxide nanorods (0, 1, 2, 3,
5%, w/w)
Tilapia gelatin incorporated
with cellulose whiskers (0, 5,
10, 15%, w/w)
Fish gelatin incorporated with
chitosan nanoparticles (0, 2, 4,
6, 8%, w/w)
Fish gelatin-egg white added
nanosized sepiolite (C-S), or
clove essential oil (C-CL), or
both (C-CL-S)
Tilapia gelatin (FSG)/fish
protein isolate (FPI)/ basil leaf
essential oil (BEO) (50, 100%,
w/w)/ zink oxide nanoparticles
(ZnONP) (0, 3%, w/w)
Tilapia gelatin (FSG)/fish
protein isolate (FPI)/ zink oxide
nanoparticles (ZnONP) (0, 1, 2,
3, 4%, w/w)
17
Adjustable WVP, film solubility and
TS, higher EAB
Lee & Song
(2017)
Lower WVP, lower TS, lower EAB
Chiou et al.
(2008)
Lower WVP, lower TS, lower EAB
Chiou et al.
(2008)
Higher TS, lower EAB
Rouhi et al.
(2013)
Lower WVP, higher TS, lower EAB
Santos et al.
(2014)
Lower WVP, lower film solubility,
higher TS, lower EAB
Hosseini et al.
(2015)
For C-S film: lower WVP, higher
TS, lower EAB
For C-CL film: higher WVP, lower
TS, higher EAB
For C-CL-S film: higher WVP,
lower TS, lower EAB
For FSG/FPI film added with BEO:
lower WVP, lower TS, higher EAB
For FSG/FPI/BEO film added
ZnONP: lower WVP, higher TS,
lower EAB
Higher WVP, lower TS, lower EAB
Giménez et al.
(2012)
Arfat et al. (2014)
Arfat et al. (2016)
Fish skin gelatin
Blending and
cross-linking
Fish gelatin (FSG) added with
pectin, and cross-linked with
glutaraldehyde (gt)
For FSG-pectin composite film:
Liu et al. (2007)
lower WVP, lower water solubility,
lower TS, lower EAB
For FSG-pectin-gt film: lower WVP,
lower water solubility, higher TS,
lower EAB
Warm-water fish
Nanoparticle
Fish gelatin incorporated with
For nanoclay composite film:
Bae et al. (2009)
gelatin
reinforcement
nanoclay, and cross-linked with lower WVP
and cross2% transglutaminase (for 0, 10, For cross-linked nanocomposite
linking
30, 50 min)
film:
no significant changes on WVP,
lower TS, higher EAB
WVP = water vapour permeability; TS = tensile strength; EAB = elongation at break.
18
2.5 Assessment of fish gelatin film properties
Biopolymeric films designed as food packaging materials are required to protect and maintain the
quality of the food product from the surrounding environment throughout its shelf-life. Therefore,
the following properties must be determined: (i) barrier properties, (ii) mechanical properties, (iii)
thermal properties, (iv) structural properties, and (v) morphological properties (HernandezIzquierdo & Krochta, 2008).
2.5.1 Barrier properties
The barrier properties of a protein film rely on the nature and composition of the film, particularly,
the degree of the organization of the film network and the ratio of non-polar and polar amino acids.
The environmental conditions also affect the barrier properties of protein films, in which an
increase in relative humidity and temperature can increase the water and oxygen permeability
(Fang et al., 2002; Gennadios et al., 1993). It is therefore important to optimize the protein film
formation to extend its functionality as protective barrier layers according to the packaged product
needs. The requirements may differ as products can be sensitive to various gasses, water vapor,
organic vapors, and liquids (Siracusa et al., 2008).
2.5.1.1 Gas barrier properties
The gas barrier properties that are usually tested include oxygen transmission rate (OTR) and
carbon dioxide transmission rate (CO2TR). These tests indicate the transmission of permeates per
unit of area and time through the packaging materials. By correlating the transmission rates with
the thickness of the tested film and the pressure of the permeant across the film, the permeability
efficiency can be determined. Oxygen is one important permeant to be measured as its presence
can induce lipid oxidation in food. Carbon dioxide is another permeant to be examined, particularly
when modified atmospheres are used due to its capability in inhibiting microbial growth inhibition,
thus extending the shelf-life of packaged food products (Siracusa et al., 2008). Under low humidity
conditions, most fish gelatin films exhibit good barrier properties against gases (i.e. oxygen and
carbon dioxide). In contrast, they show poor gas barrier properties when exposed to high humidity
conditions, owing to the increased mobility of the biopolymer chains that permit an increased
permeability of gases (Castro-Rosas et al., 2016; Cuq et al., 1998; Gómez-Guillén et al., 2009).
The addition of other biopolymer or cross-linkers can disrupt the mobility of the biopolymer chains,
19
which is responsible for the alteration of gas barrier properties. Different oxygen permeability
results have been reported for fish skin gelatin films which were plasticized with corn oil (Nur
Hanani et al., 2013), olive flounder skin gelatin films which were laminated with polylactic acid
(Lee & Song, 2017), megrim skin gelatin films charged with sodium-montmorillonite
nanoparticles (Bae et al., 2009), as well as pollock and salmon films cross-linked with
glutaraldehyde (Chiou et al., 2008).
2.5.1.2 Light barrier properties
A packaging film should be a barrier to UV light to decrease the risks of oxidation (Bao et al.,
2009; Elango et al., 2014). The light barrier properties are usually determined through film’s light
transmission and opacity measurement, by exposing a film to UV range (200-400 nm) and visible
range (400-800 nm). The films with lower transmission and higher opacity levels indicate better
light barrier capacities (Jongjareonrak et al., 2006; Limpisophon et al., 2009). The fish gelatin
films have high UV barrier properties due to the absorption of UV light by the peptide bonds of
the gelatin chains, and the hydrophobic residues of tyrosine and tryptophan (Bao et al., 2009;
Denavi et al., 2009; Hosseini et al., 2013). Fish gelatin films are highly transparent but the light
transmitted can vary depending on the interactions between film constituents (Gómez-Guillén et
al., 2009). Different diffractive indexes and light scattering effects resulting from the distribution
of film constituents lead to disrupted film network, affecting the visible light transmission of
gelatin based films (Tongnuanchan et al., 2015; Yang & Paulson, 2000). Additives such as glycerol
(Limpisophon et al., 2009), mungbean protein isolate (Hoque et al., 2011), essential oils (Ahmad
et al., 2012), corn oil (Nur Hanani et al., 2013), palm oil (Tongnuanchan et al., 2015), sunflower
oil (Pérez-Mateos et al., 2009), triacetin (Jiang et al., 2010), and fatty acids (Jongjareonrak et al.,
2006) do affect the transparency of the films.
2.5.1.3 Water barrier properties
The water barrier properties are evaluated by measuring its water vapour transmission rate (WVTR)
or water vapor permeability (WVP) (Avena-Bustillos et al., 2006; Shakila et al., 2012). In this
regard, cold-water fish gelatin films have lower WVP as compared to warm-water fish gelatin
films, ascribed to their higher hydrophobic amino acids content and lower Hyp content (AvenaBustillos et al., 2006). Table 2.2 summarizes the water barrier properties of some fish gelatin films.
Studies showed that the water barrier properties were enhanced for fish gelatin films after inclusion
20
of proteins with higher hydrophobicity, such as soy protein isolate (Denavi et al., 2009) and
mungbean protein isolate (Hoque et al., 2011). Other hydrophobic compounds that had increased
the hydrophobicity of film matrix and led to a reduced water vapour permeability included
sunflower oil (Pérez-Mateos et al., 2009), stearic acid and oleic acid (Limpisophon et al., 2010),
palm oil (Tongnuanchan et al., 2015), as well as lamination with polylactic acid (Lee & Song,
2017). Ahmad et al. (2012) observed that the differences in the hygroscopic nature of oils induced
different ability to attract water to unicorn leatherjacket skin gelatin film networks, resulting in an
increase in water vapour permeability when blended with essential oils of bergamot, but not
lemongrass. Jiang et al. (2010) reported that the addition of triacetin at 50% enhanced water barrier
properties of catfish skin gelatin films, but an increase in water vapour permeability was observed
when added with 100% and 150% triacetin (of the gelatin weight) ascribed to the heterogeneous
distribution of excess triacetin.
Chiou et al. (2008) applied glutaraldehyde as a cross-linker to improve water barrier properties of
pollock and salmon skin gelatin films. Both fish gelatin films showed lower water vapour
permeability values after cross-linking with increasing amount of glutaraldehyde, attributed to the
reduced free volume in film matrix that restricted permeability of water vapour and resulted in
improved water resistibility of films (Table 2.2). Recently, nanoparticles were incorporated to form
strong hydrogen bonds with the gelatin matrix, developing densely linked three-dimensional
networks that limited the diffusivity of water molecules. A decrease in WVP was observed for
warm-water gelatin films when nanoclay was added to the matrix (Bae et al., 2009), and coldwater fish skin gelatin films when charged with chitosan nanoparticles (Hosseini et al., 2015)
(Table 2.2).
21
Table 2.2 Water barrier properties of fish gelatin films.
Source of fish gelatin
I. Blending
Cod skin
Cod skin
Cod skin
Cod skin
Cod skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cod skin
Cod skin
Cod skin
Cod skin
Blue shark skin
Blue shark skin
Blue shark skin
Blue shark skin
Blue shark skin
Blue shark skin
Blue shark skin
Blue shark skin
Film modification
Water vapor permeability
(g / m s Pa)
Film
solubility (%)
Reference
Cod skin gelatin (G)/soy
protein isolate (S)
(100:0)
G/S (75:25)
G/S (50:50)
G/S (25:75)
G/S (0:100)
Cuttlefish skin gelatin
(CG)/mungbean protein
isolate (MPI) (10:0)
CG/MPI (8:2)
CG/MPI (6:4)
CG/MPI (4:6)
CG/MPI (2:8)
CG/MPI (0:10)
Sunflower oil 0%
Sunflower oil 0.3%
Sunflower oil 0.6%
Sunflower oil 1.0%
Stearic acid 0%
Stearic acid 25%
Stearic acid 50%
Stearic acid 100%
Oleic acid 0%
Oleic acid 25%
Oleic acid 50%
Oleic acid 100%
1.03 x 10-11ab
87.66 ± 0.46
Denavi et al. (2009)
0.56 x 10-11ab
0.44 x 10-11ab
0.69 x 10-11ab
0.61 x 10-11ab
1.29 ± 0.03 x 10-10
85.19 ± 3.03
84.63 ± 2.26
81.39 ± 5.64
83.93 ± 2.44
85.87 ± 1.19
Denavi et al. (2009)
Denavi et al. (2009)
Denavi et al. (2009)
Denavi et al. (2009)
Hoque et al. (2011)
1.27 ± 0.03 x 10-10
1.24 ± 0.05 x 10-10
1.20 ± 0.04 x 10-10
1.16 ± 0.02 x 10-10
1.12 ± 0.03 x 10-10
1.20 ± 0.24 x 10-11b
1.03 ± 0.19 x 10-11b
0.86 ± 0.13 x 10-11b
0.90 ± 0.32 x 10-11b
1.30 ± 0.12 x 10-10
0.95 ± 0.07 x 10-10
0.96 ± 0.04 x 10-10
0.70 ± 0.06 x 10-10
1.30 ± 0.12 x 10-10
1.17 ± 0.14 x 10-10
1.01 ± 0.03 x 10-10
0.91 ± 0.06 x 10-10
57.95 ± 3.26
63.54 ± 4.15
67.14 ± 2.83
77.97 ± 3.03
81.54 ± 1.08
87.66 ± 0.46
87.84 ± 1.54
85.73 ± 0.76
80.26 ± 0.07
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
Pérez-Mateos et al. (2009)
Pérez-Mateos et al. (2009)
Pérez-Mateos et al. (2009)
Pérez-Mateos et al. (2009)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
Limpisophon et al. (2010)
22
Catfish skin
Catfish skin
Catfish skin
Catfish skin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Unicorn leatherjacket skin
gelatin
Tilapia skin
Tilapia skin
Tilapia skin
Tilapia skin
Tilapia skin
Olive flounder skin gelatin
Triacetin 0%
Triacetin 50%
Triacetin 100%
Triacetin 150%
Control-essential oils 0%
2.33 ± 0.14 x 10-11b
2.17 ± 0.39 x 10-11b
3.64 ± 0.50 x 10-11b
3.75 ± 0.25 x 10-11b
1.21 ± 0.065 x 10-10
69.5 ± 4.8
75.5 ± 1.8
76.5 ± 7.8
83.3 ± 2.7
97.80 ± 0.78
Jiang et al. (2010)
Jiang et al. (2010)
Jiang et al. (2010)
Jiang et al. (2010)
Ahmad et al. (2012)
Bergamot essential oil
5%
Bergamot essential oil
10%
Bergamot essential oil
15%
Bergamot essential oil
20%
Bergamot essential oil
25%
Lemongrass essential oil
5%
Lemongrass essential oil
10%
Lemongrass essential oil
15%
Lemongrass essential oil
20%
Lemongrass essential oil
25%
Palm oil 0%
Palm oil 25%
Palm oil 50%
Palm oil 75%
Palm oil 100%
Without polylactic acid
1.26 ± 0.028 x 10-10
93.37 ± 0.57
Ahmad et al. (2012)
1.88 ± 0.032 x 10-10
93.14 ± 0.37
Ahmad et al. (2012)
1.94 ± 0.057 x 10-10
93.07 ± 0.45
Ahmad et al. (2012)
1.87 ± 0105 x 10-10
90.04 ± 0.46
Ahmad et al. (2012)
1.84 ± 0.132 x 10-10
89.82 ± 0.96
Ahmad et al. (2012)
1.21 ± 0.040 x 10-10
93.54 ± 0.66
Ahmad et al. (2012)
1.03 ± 0.066 x 10-10
92.30 ± 0.65
Ahmad et al. (2012)
1.00 ± 0.060 x 10-10
92.04 ± 0.57
Ahmad et al. (2012)
1.12 ± 0.082 x 10-10
89.81 ± 0.50
Ahmad et al. (2012)
1.07 ± 0.041 x 10-10
89.16 ± 0.65
Ahmad et al. (2012)
2.54 ± 0.05 x 10-11
1.63 ± 0.12 x 10-11
1.18 ± 0.02 x 10-11
1.11 ± 0.06 x 10-11
0.70 ± 0.02 x 10-11
2.17 ± 0.09 x 10-9
n/a
n/a
n/a
n/a
n/a
16.62 ± 0.72
Tongnuanchan et al. (2015)
Tongnuanchan et al. (2015)
Tongnuanchan et al. (2015)
Tongnuanchan et al. (2015)
Tongnuanchan et al. (2015)
Lee & Song (2017)
23
Olive flounder skin gelatin
II. Nanoparticles
reinforcement
Cold-water fish gelatin
Cold-water fish gelatin
Cold-water fish gelatin
Cold-water fish gelatin
Cold-water fish gelatin
Bilayer with polylactic
acid
0.92 ± 0.09 x 10-9
9.27 ± 0.04
Lee & Song (2017)
Chitosan nanoparticles
0%
Chitosan nanoparticles
2%
Chitosan nanoparticles
4%
Chitosan nanoparticles
6%
Chitosan nanoparticles
8%
Nanoclay 0%
Nanoclay 1%
Nanoclay 3%
Nanoclay 5%
Nanoclay 7%
Nanoclay 9%
3.95 ± 0.24 x 10-7b
71.80 ± 1.51
Hosseini et al. (2015)
2.79 ± 0.47 x 10-7b
68.55 ± 2.67
Hosseini et al. (2015)
2.31 ± 0.11 x 10-7b
63.79 ± 0.15
Hosseini et al. (2015)
1.99 ± 0.10 x 10-7b
62.63 ± 1.14
Hosseini et al. (2015)
2.46 ± 0.35 x 10-7b
65.19 ± 2.32
Hosseini et al. (2015)
3.12 x 10-5ab
2.50 x 10-5ab
1.60 x 10-5ab
1.40 x 10-5ab
1.10 x 10-5ab
0.81 x 10-5ab
n/a
n/a
n/a
n/a
n/a
n/a
Bae et al. (2009)
Bae et al. (2009)
Bae et al. (2009)
Bae et al. (2009)
Bae et al. (2009)
Bae et al. (2009)
2.380 ± 0.197 x 10-7b
2.208 ± 0.158 x 10-7b
2.100 ± 0.183 x 10-7b
2.022 ± 0.241 x 10-7b
3.011 ± 0.247 x 10-7b
2.588 ± 0.166 x 10-7b
2.458 ± 0.144 x 10-7b
2.355 ± 0.130 x 10-7b
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Warm-water fish gelatin
Warm-water fish gelatin
Warm-water fish gelatin
Warm-water fish gelatin
Warm-water fish gelatin
Warm-water fish gelatin
III. Cross-linking
Pollock skin gelatin
Glutaraldehyde 0%
Pollock skin gelatin
Glutaraldehyde 0.25%
Pollock skin gelatin
Glutaraldehyde 0.50%
Pollock skin gelatin
Glutaraldehyde 0.75%
Salmon skin gelatin
Glutaraldehyde 0%
Salmon skin gelatin
Glutaraldehyde 0.25%
Salmon skin gelatin
Glutaraldehyde 0.50%
Salmon skin gelatin
Glutaraldehyde 0.75%
n/a: not available
a
Numerical values are estimated from graph.
b
Data are converted to the same unit.
24
The resistance to water uptake is another important parameter. This is because some films may be
destabilized under high moisture conditions, such as exposure to high humidity storage conditions.
In this context, films would encounter the risk of solubilization, or water absorption, which could
lead to swelling and rupture of the films. Protein films that comprise of tightly linked threedimensional network exhibit low water solubility, due to the interactions of covalent bonds and
cross-links that form a dense network structure which obstructs the migration of water molecules
(Garavand et al., 2017). Thus, film moisture resistance property, i.e. solubility, is examined by
exposing films to water or exposed under high humidity conditions, allowing absorption of water
as a function of time, followed by measurements of the film’s weight changes. Several examples
of gelatin matrices sourced from fish which have been modified by the addition of other additives
have demonstrated good water resistance properties (Table 2.2). Such examples include films
prepared with cod skin gelatin/soy protein isolate blend (Denavi et al., 2009), cuttlefish skin
gelatin/mungbean protein isolate blend (Hoque et al., 2011), cod skin gelatin and sunflower oil
(Pérez-Mateos et al., 2009), catfish skin gelatin and triacetin (Jiang et al., 2010), unicorn
leatherjacket skin gelatin and essential oils (Ahmad et al., 2012), olive flounder skin gelatin and
polylactic acid (Lee & Song, 2017), and cold-water fish gelatin and chitosan nanoparticles
(Hosseini et al., 2015).
2.5.2 Mechanical properties
The mechanical properties of protein films are influenced by the amino acid composition,
molecular weight of the protein chains, the type and the density of intra- and intermolecular
interactions that stabilize the film network (Cuq et al., 1998; Hoque et al., 2011). For fish gelatin,
the low imino acid content (Pro + Hyp) impart a low ability in forming intra- and intermolecular
hydrogen bonds, as well as hydrophobic and ionic interactions between protein chains, leading to
the production of films with a high deformability (Brinckmann, 2005; Galea et al., 2000; GómezEstaca et al., 2009). However, the predominance of high molecular weight protein chains in fish
gelatin facilitates intermolecular interactions and cross-links formation, resulting in films with
increased tensile strength. Since fish gelatin also contains low molecular weight protein chains, it
can disrupt the establishment of a film network, yielding fish gelatin films with high elongation
and low tensile strength (Gómez-Guillén et al., 2009; Habitante et al., 2005; Ledward, 1986). Table
2.3 lists the tensile strength and elongation at break values of some fish gelatin films.
25
Jongjareonrak et al. (2006) observed a lower tensile strength and elongation at break for films
produced from bigeye snapper skin gelatin as compared to films prepared from brownstripe red
snapper skin gelatin. They found that the lower content of high molecular weight fractions of
bigeye snapper skin gelatin contributed to the lower mechanical properties of the resulting films.
Carvalho et el. (2008) obtained gelatin from Atlantic halibut skins with a predominance in low
molecular weight fractions due to heat degradation of the protein during the evaporation step (at
60 °C). They reported a lower tensile strength and higher elongation at break of the resulting films
when compared to films produced from gelatin without the evaporation step. Hoque et al. (2011)
prepared films from cuttlefish skin gelatin that was obtained from different degree of hydrolysis.
They found that an increased degree of hydrolysis induced higher degradation of gelatin and
formation of shorter chains, which led to films with lower tensile strength and elongation at break.
An increase in the gelatin concentration also affects the mechanical properties of the films. The
tensile strength and elongation at break of films increase with increasing protein concentration,
attributed to an increase in the intermolecular interactions induced by the increase in protein chains
per surface unit at higher concentrations (Cuq et al., 1996). Jongjareonrak et al. (2006) prepared
films from bigeye snapper skin and brownstripe red snapper skin gelatins at increasing protein
concentration. They observed that the tensile strength and elongation at break increased with
increasing protein concentration from 2 to 3%. Similar results were also reported for blue shark
skin gelatin films prepared from 1 to 3% protein concentration (Limpisophon et al., 2009).
26
Table 2.3 Mechanical properties of fish gelatin films.
Source of fish gelatin
I. Gelatin preparation
Atlantic halibut skin
Atlantic halibut skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
II. Gelatin concentration
Bigeye snapper skin
Bigeye snapper skin
Brownstripe red snapper
skin
Brownstripe red snapper
skin
Blue shark skin
Blue shark skin
Blue shark skin
III. Plasticizer concentration
Bigeye snapper skin
Bigeye snapper skin
Bigeye snapper skin
Bigeye snapper skin
Brownstripe red snapper
skin
Film modification
Tensile strength Elongation at
(MPa)
break (%)
Reference
Gelatin prepared with
evaporation at 60 °C
Gelatin prepared without
evaporation at 60 °C
Gelatin with 1.20% of
hydrolysis
Gelatin with 0.80% of
hydrolysis
Gelatin with 0.40% of
hydrolysis
Gelatin without hydrolysis
3.8 ± 0.8
294.5 ± 47.8
Carvalho et el. (2008)
11.1 ± 2.6
170.3 ± 36.4
Carvalho et el. (2008)
12.99 ± 1.18
2.45 ± 0.29
Hoque et al. (2011)
11.64 ± 1.70
2.89 ± 0.45
Hoque et al. (2011)
18.96 ± 1.84
3.65 ± 0.33
Hoque et al. (2011)
36.57 ± 2.56
5.39 ± 0.16
Hoque et al. (2011)
2% protein concentration
3% protein concentration
2% protein concentration
28.28 ± 6.76
44.28 ± 10.06
41.09 ± 9.81
2.68 ± 0.64
7.00 ± 1.85
7.02 ± 1.46
Jongjareonrak et al. (2006)
Jongjareonrak et al. (2006)
Jongjareonrak et al. (2006)
3% protein concentration
58.10 ± 8.45
8.20 ± 1.16
Jongjareonrak et al. (2006)
1% protein concentration
2% protein concentration
3% protein concentration
12.58 ± 1.25
27.29 ± 1.90
20.53 ± 1.09
61.13 ± 11.72
72.43 ± 12.69
74.17 ± 4.00
Limpisophon et al. (2009)
Limpisophon et al. (2009)
Limpisophon et al. (2009)
0% glycerol concentration
25% glycerol concentration
50% glycerol concentration
75% glycerol concentration
0% glycerol concentration
57.34 ± 15.08
44.28 ± 10.06
15.41 ± 2.93
7.97 ± 1.19
67.78 ± 14.67
3.40 ± 1.14
7.00 ± 1.85
24.42 ± 6.68
50.30 ± 5.72
5.24 ± 1.56
Jongjareonrak et al. (2006)
Jongjareonrak et al. (2006)
Jongjareonrak et al. (2006)
Jongjareonrak et al. (2006)
Jongjareonrak et al. (2006)
27
Brownstripe red snapper
skin
Brownstripe red snapper
skin
Brownstripe red snapper
skin
Blue shark skin
Blue shark skin
Blue shark skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
IV. Blending
Cod skin
Cod skin
Cod skin
Cod skin
Cod skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
Cuttlefish skin
V. Nanoparticles
reinforcement
Fish gelatin
Fish gelatin
Fish gelatin
25% glycerol concentration
58.10 ± 8.45
8.20 ± 1.16
Jongjareonrak et al. (2006)
50% glycerol concentration
33.58 ± 4.43
39.75 ± 6.09
Jongjareonrak et al. (2006)
75% glycerol concentration
18.28 ± 3.10
95.04 ± 10.27
Jongjareonrak et al. (2006)
0% glycerol concentration
25% glycerol concentration
50% glycerol concentration
10% glycerol concentration
15% glycerol concentration
20% glycerol concentration
45.90 ± 1.86
38.93 ± 2.96
23.30 ± 2.03
45.63 ± 1.10
40.39 ± 3.45
36.57 ± 2.56
1.57 ± 0.03
6.24 ± 2.10
80.40 ± 4.41
3.91 ± 0.64
4.29 ± 0.59
5.39 ± 0.16
Limpisophon et al. (2009)
Limpisophon et al. (2009)
Limpisophon et al. (2009)
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
Cod skin gelatin (G)/soy
protein isolate (S) (100:0)
G/S (75:25)
G/S (50:50)
G/S (25:75)
G/S (0:100)
Cuttlefish skin gelatin
(CG)/mungbean protein
isolate (MPI) (10:0)
CG/MPI (8:2)
CG/MPI (6:4)
CG/MPI (4:6)
CG/MPI (2:8)
CG/MPI (0:10)
4.2a
100a
Denavi et al. (2009)
7.2a
5.0a
3.0a
2.8a
5.94 ± 0.59
82a
34a
16a
10a
140.43 ± 7.11
Denavi et al. (2009)
Denavi et al. (2009)
Denavi et al. (2009)
Denavi et al. (2009)
Hoque et al. (2011)
4.50 ± 1.10
3.64 ± 1.03
2.75 ± 1.16
1.69 ± 0.41
1.09 ± 0.42
177.90 ± 7.62
193.67 ± 6.39
201.91 ± 8.38
165.93 ± 4.52
156.87 ± 5.54
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
Hoque et al. (2011)
0% zinc oxide nanorods
1% zinc oxide nanorods
2% zinc oxide nanorods
15.0a
17.0a
18.5a
42a
30a
23a
Rouhi et al. (2013)
Rouhi et al. (2013)
Rouhi et al. (2013)
28
Fish gelatin
Fish gelatin
Cold-water fish skin gelatin
Cold-water fish skin gelatin
Cold-water fish skin gelatin
Cold-water fish skin gelatin
Cold-water fish skin gelatin
Tilapia residue gelatin
Tilapia residue gelatin
Tilapia residue gelatin
Tilapia residue gelatin
VI. Blending and
nanoparticles reinforcement
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
Tilapia skin gelatin
VII. Cross-linking
Pollock skin gelatin
Pollock skin gelatin
Pollock skin gelatin
Pollock skin gelatin
Salmon skin gelatin
3% zinc oxide nanorods
5% zinc oxide nanorods
0% chitosan nanoparticles
2% chitosan nanoparticles
4% chitosan nanoparticles
6% chitosan nanoparticles
8% chitosan nanoparticles
0% cellulose whiskers
5% cellulose whiskers
10% cellulose whiskers
15% cellulose whiskers
19.2a
20.5a
7.44 ± 0.17
7.99 ± 1.46
8.77 ± 1.11
10.57 ± 0.19
11.28 ± 1.02
16.30a
17.00a
16.97a
16.80a
25a
17a
102.04 ± 28.38
70.09 ± 11.93
64.72 ± 24.59
44.71 ± 11.80
32.73 ± 7.38
14.0a
13.2a
13.5a
13.5a
Rouhi et al. (2013)
Rouhi et al. (2013)
Hosseini et al. (2015)
Hosseini et al. (2015)
Hosseini et al. (2015)
Hosseini et al. (2015)
Hosseini et al. (2015)
Santos et al. (2014)
Santos et al. (2014)
Santos et al. (2014)
Santos et al. (2014)
Tilapia gelatin (FSG)/fish
protein isolate (FPI)-zink
oxide nanoparticles
(ZnONP) 0% pH 3
FSG/FPI-ZnONP 1% pH 3
FSG/FPI-ZnONP 2% pH 3
FSG/FPI-ZnONP 3% pH 3
FSG/FPI-ZnONP 4% pH 3
FSG/FPI-ZnONP 0% pH 11
FSG/FPI-ZnONP 1% pH 11
FSG/FPI-ZnONP 2% pH 11
FSG/FPI-ZnONP 3% pH 11
FSG/FPI-ZnONP 4% pH 11
11.66 ± 0.77
70.33 ± 5.13
Arfat et al. (2016)
12.21 ± 0.86
13.09 ± 0.76
14.18 ± 0.69
8.97 ± 0.57
13.07 ± 0.60
14.29 ± 0.63
15.83 ± 0.86
17.76 ± 0.93
9.43 ± 0.53
66.19 ± 4.37
61.57 ± 4.57
53.33 ± 5.36
43.47 ± 3.59
64.31 ± 5.64
61.43 ± 4.55
56.82 ± 4.48
49.20 ± 4.14
39.43 ± 4.63
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
Arfat et al. (2016)
0% glutaraldehyde
0.25% glutaraldehyde
0.50% glutaraldehyde
0.75% glutaraldehyde
0% glutaraldehyde
50.1 ± 4.9
49.0 ± 6.7
47.3 ± 7.4
45.9 ± 6.3
51.2 ± 4.7
3.44 ± 0.25
3.43 ± 0.43
3.24 ± 0.42
3.23 ± 0.33
3.57 ± 0.32
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
29
Salmon skin gelatin
Salmon skin gelatin
Salmon skin gelatin
VII. Blending and crosslinking
Fish skin gelatin
0.25% glutaraldehyde
0.50% glutaraldehyde
0.75% glutaraldehyde
Fish skin gelatin/pectin-0%
glutaraldehyde
Fish skin gelatin
Fish skin gelatin /pectin0.1% glutaraldehyde
Fish gelatin
Fish gelatin/nanoclay-2%
transglutaminase 0 min
Fish gelatin
Fish gelatin/nanoclay-2%
transglutaminase 10 min
Fish gelatin
Fish gelatin/nanoclay-2%
transglutaminase 30 min
Fish gelatin
Fish gelatin/nanoclay-2%
transglutaminase 50 min
a
Numerical values are estimated from graph.
49.6 ± 9.1
60.0 ± 10.9
49.7 ± 8.2
3.37 ± 0.45
3.80 ± 0.55
3.36 ± 0.48
Chiou et al. (2008)
Chiou et al. (2008)
Chiou et al. (2008)
43.5 ± 7.6
3.0 ± 1.5
Liu et al. (2007)
54.2 ± 6.9
2.1 ± 0.4
Liu et al. (2007)
61a
16a
Bae et al. (2009)
58a
16a
Bae et al. (2009)
56a
15a
Bae et al. (2009)
57a
13a
Bae et al. (2009)
30
Film matrix formed with a high degree of molecular organization requires the addition of
plasticizer to increase the extensibility and flexibility. Low molecular weight plasticizers reduce
the intermolecular forces within the matrix, resulting in an increase in polymer chains’ mobility
and free volume (Vieira et al., 2011). Thus, the addition of plasticizer usually increases the
elongation at break and decreases the tensile strength of a film. The most commonly used
plasticizers for the formation of fish gelatin films are glycerol and sorbitol (Elango et al., 2014;
Gómez-Guillén et al., 2009; Hoque et al., 2011). Jongjareonrak et al. (2006) investigated the
plasticization effect of glycerol on bigeye snapper skin and brownstripe red snapper skin gelatin
films. The addition of glycerol ranging from 0 to 75% (of the gelatin weight) increased the
elongation at break by 47 to 89% and decreased the tensile strength by 73 to 86% of the resulting
films. Similar results were obtained by Limpisophon et al. (2009) who utilized from 0 to 50% of
glycerol (of the gelatin weight) on blue shark skin gelatin films, as well as 10 to 20% glycerol (of
the gelatin weight) on cuttlefish skin gelatin films by Hoque et al. (2011).
The blending of vegetable-based protein biopolymer with fish gelatin for film formation was
studied. The biopolymers change the degree of organization and molecular packing of fish gelatin,
altering the mechanical properties of the resulting films, which vary depending on the type and
ratio of proteins, as well as the interaction among them (Hoque et al., 2011; Siew et al., 1999).
Denavi et al. (2009) developed films from a blend of cod skin gelatin and soy protein isolate. They
observed that increasing the ratio of soy protein isolate caused a lower breaking force and greater
deformation of gelatin films than for gelatin only films. Hoque et al. (2011) added mungbean
protein isolate in films of cuttlefish skin gelatin. As the mungbean protein isolate concentration
increased, a decrease in tensile strength and an increase in elongation at break was observed.
A uniform dispersion of nanoparticles within a film matrix acts as filler and reinforces the
mechanical properties (Castro-Rosas et al., 2016; Kovacevic et al., 2008). Rouhi et al. (2013)
developed bio-nanocomposite fish gelatin films by incorporating zinc oxide nanorods from 0 to 5%
(w/v). An increase in tensile strength and a decrease in elongation at break were observed as the
nanorod concentration increased. Hosseini et al. (2015) also reinforced cold-water fish skin gelatin
films with chitosan nanoparticles at concentration ranging from 0 to 8% (w/v). As the
concentration of nanoparticles increased, the tensile strength improved and the elongation at break
values decreased. However, Santos et al. (2014) observed that the properties of charged matrices
31
can reach a plateau. For their experiments, no further increase in tensile strength and decrease in
elongation at break were observed for tilapia gelatin films when the concentration of cellulose
whiskers reached above 5%. In a study conducted by Arfat et al. (2016), the mechanical properties
of fish protein isolate and fish gelatin blends reinforced with zinc oxide nanoparticles improved
when the concentration of nanoparticles ranged from 1 to 3% (w/w), but a sharp decrease was
observed for both tensile strength and elongation at break when the films were charged with 4%
nanoparticles. They suggested that the decrease in mechanical strength was due to the uneven
dispersion of nanoparticles within the protein matrix.
The addition of a cross-linker generally increases the rigidity of the film and its mechanical
strength and decreases the elongation at break. Liu et al. (2007) developed composite films from
pectin, fish skin gelatin, and glutaraldehyde as a cross-linker. The composite films exhibited
increased tensile strength and decreased elongation at break, attributed to the cross-linking effect
that reduced the interstitial spaces among protein molecules. Chiou et al. (2008) prepared crosslinked pollock and salmon skin gelatin films by adding glutaraldehyde at concentration ranging
from 0 to 0.75% (w/w). They reported a minor cross-linking effect of glutaraldehyde on films from
both species which exhibited comparable tensile strength and elongation at break. Bae et al. (2009)
studied the cross-linking effect of transglutaminase on warm-water fish gelatin films for which the
mechanical properties had been reinforced with nanoclay. It was observed that the tensile strength
and elongation at break values of the films treated films with 2% transglutaminase for a period
varying from 0 to 50 min decreased with the increase in treatment time. They proposed that the
decreased rigidity and extensibility resulted from the cross-links prior to the interaction between
gelatin molecules and nanoclay.
2.5.3 Thermal properties
The thermal properties of films vary depending on the structure, molecular weight distribution and
organization of the films. The phase transitions of protein films can be examined at varying
atmospheric conditions, cooling and heating rates, and at different temperatures (Garavand et al.,
2017). One important phase transition to be measured is the glass transition temperature (Tg), in
which polymeric materials undergo a phase transition from the glassy state to the rubbery state.
This change in state is attributed to the increase in free volume, and mobility of the polymeric
32
chains (Cuq et al., 1997). Common techniques used for thermal analysis include differential
scanning calorimetry (DSC) and thermogravimetric analysis (TGA).
The thermal transitions of protein films are affected by the protein concentration, the molecular
weight distribution, the addition of plasticizers and cross-linkers, and the addition of other
biopolymers within the protein matrix (Hernandez-Izquierdo & Krochta, 2008; Garavand et al.,
2017). The thermal behavior of some fish gelatin films has been characterized using DSC (Table
2.4). In a study, the addition of glycerol induced a decrease in the endothermic melting transition
temperature (Tmax) for the bigeye snapper and the brownstripe red snapper skin gelatin films, due
to the plasticizing effect of glycerol (Jongjareonrak et al., 2006). The effect of palm oil on the
thermal properties of tilapia skin gelatin films was studied by Tongnuanchan et al. (2015). They
found that the presence of palm oil hindered the protein-protein interactions and increased the
mobility of gelatin chains, leading to a decrease in the order of the structure formed in the film,
which caused a decrease in the Tg and the melting transition enthalpy (∆H) values. However, an
increase in the Tmax value was observed due to the lower water content of the films.
Denavi et al. (2009) investigated the thermal properties of a blend of cod skin gelatin and soy
protein isolate. They observed an increase in denaturation temperatures as the gelatin concentration
increased. This was due to the increase interactions and higher level of organization between the
gelatin and the soy protein isolate. Consistent with these results, Hoque et al. (2011) reported a
broad Tg peak at approximately 30 °C rather than two Tg peaks for cuttlefish skin gelatin and
mungbean protein isolate blend films, confirming the miscibility and molecular interaction of both
protein molecules. Chiou et al. (2008) reported that the addition of 0.25 to 0.75% (w/w)
glutaraldehyde to pollock and salmon skin gelatin had no effect on the melting temperature of the
films, which was consistent with the little effect observed on the tensile properties of the films.
However, Yi et al. (2006) found that the melting temperatures increased when fish gelatin films
were covalently cross-linked with transglutaminase.
33
Table 2.4 Thermal properties of fish gelatin films.
Source of fish gelatin
Bigeye snapper skin
Brownstripe red
snapper skin
Tilapia skin
Unicorn leatherjacket
skin
Film modification DSC
Added glycerol (0, Decreased transition temperature
25, 50, 75%, w/w) from 96.42 °C to 53.14 °C, and
decreased transition enthalpy from
265.98 J/g to 29.23 J/g
Added glycerol (0, Decreased transition temperature
25, 50, 75%, w/w) from 100.28 °C to 59.89 °C, and
decreased transition enthalpy from
217.88 J/g to 18.80 J/g
Added palm oil (0, Decreased glass transition
25, 50, 75, 100%,
temperature from 41.02 °C to 35.85
w/w)
°C, decreased transition enthalpy
from 11.76 J/g to 2.45 J/g, and
increased transition temperature
from 117.43 °C to 123.52 °C
Added bergamot
n/a
essential oil (0, 15,
25%, w/w)
Unicorn leatherjacket
skin
Added lemongrass
essential oil (0, 15,
25%, w/w)
n/a
Cod skin
Blended with soy
protein isolate
(100:0, 75:25,
50:50, 25:75,
0:100, w/w)
Increased denaturation
temperatures of 3-7 °C with
increasing gelatin concentrations
34
TGA
n/a
Reference
Jongjareonrak et al.
(2006)
n/a
Jongjareonrak et al.
(2006)
Decreased decomposition
temperatures 200.74 °C to
186.43 °C, and 287.49 °C
to 275.92 °C
Tongnuanchan et
al. (2015)
Higher decomposition
Ahmad et al. (2012)
temperature from 157.41
°C to 257.19 °C, and lower
weigh loss from 61.23% to
56.97%
Higher decomposition
Ahmad et al. (2012)
temperature from 157.41
°C to 302.14 °C, and lower
weigh loss from 61.23% to
56.44%
n/a
Denavi et al. (2009)
Cuttlefish skin
Blended with
mungbean protein
isolate (6:4, w/w)
Broad Tg at approximately 30.08
°C
Tilapia skin
Blended with fish
protein isolate
(5:5, w/w) pH 11
n/a
Tilapia skin
Blended with fish
protein isolate
(5:5, w/w) pH 3
n/a
Pollock skin
Cross-linked with
glutaraldehyde
(0.25, 0.50, 0.75%,
w/w)
Cross-linked with
glutaraldehyde
(0.25, 0.50, 0.75%,
w/w)
Cross-linked with
2%
transglutaminase
(w/w) from 0 to 50
min
Salmon skin
Fish gelatin
n/a: not available
Hoque et al. (2011)
No effect on melting temperatures
Higher decomposition
temperatures from 161.85
°C to 188.79 °C, and
296.38 °C to 310.28 °C
Higher decomposition
temperatures from 199.11
°C to 204.47 °C, and
300.88 °C to 305.50 °C
Higher decomposition
temperatures from 195.07
°C to 198.10 °C, and
294.01 °C to 301.01 °C
n/a
No effect on melting temperatures
n/a
Chiou et al. (2008)
Increased from 124.78
± 7.34 ◦C to 158.49
± 2.68 ◦C
n/a
Yi et al. (2006)
35
Arfat et al. (2014)
Arfat et al. (2014)
Chiou et al. (2008)
The thermal stability of fish gelatin films can also be assessed using TGA (Table 2.4). Films with
higher thermal resistance exhibit a lower weight loss and a higher decomposition temperature
(Stevens, 1999; Uragami et al., 1994). Ahmad et al. (2012) investigated the effect of the addition
of essential oils on the thermal stability of unicorn leatherjacket skin gelatin films. They observed
a higher decomposition temperature and a lower weight loss, which due to the interaction between
the gelatin and essential oils that resulted in films with stronger network and higher heat resistance.
In contrast, Tongnuanchan et al. (2015) found that the addition of palm oil to tilapia skin gelatin
films decreased the decomposition temperature of the films. They concluded that that the presence
of palm oil had lowered the gelatin’s intermolecular interaction, resulting in a poorer film network
with a lower heat resistance and mechanical properties. Hoque et al. (2011) and Arfat et al. (2014)
blended fish skin gelatins with mungbean protein isolate and fish protein isolate, respectively. Both
studies concluded that the interaction between fish gelatins and protein isolates yielded stronger
film network, resulting in higher heat resistance of the films.
2.5.4 Structural properties
Several techniques are used to determine the structural properties of films such as Fourier
transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), nuclear magnetic resonance
(NMR) spectroscopy, and SDS-PAGE (Garavand et al., 2017).
FT-IR is one the most commonly used technique to effectively examine the structural properties
of films, by demonstrating the possible functional chemical groups and their molecular interactions
(Garavand et al., 2017). In fish gelatin film studies, this technique reveals the extent of interaction
of the fish gelatins with other polymers of different molecular weights, including additives,
hydrophobic and hydrophilic compound and nanoparticles. The interactions are determined by
comparing the presence of new bands, observing changes in the intensity of the bands, as well as
the shifting of wavenumber of the bands. FT-IR spectra of fish gelatin based films reported major
bands in amide region, including amide-A ranging from 3270 to 3280 cm-1 (arising from the
stretching vibration of N-H group), amide-B from 2926 to 3086 cm-1 (representing CH stretching
and -NH2), amide-I from 1630 to 1633 cm-1 (representing C=O stretching/hydrogen bonding
coupled with COO), amide-II from 1536 to 1538 cm-1 (attributed to the bending vibration of N-H
groups and stretching vibrations of C-N groups), and amide-III from 1235 to 1238 cm-1 (attributed
36
to the vibrations in plane of C-N and N-H groups of bound amide or vibrations of CH2 groups)
(Arfat et al., 2014; Hoque et al., 2011; Tongnuanchan et al., 2015).
Changes in wavenumber and amplitude of these bands indicate that different conformations and
orientations occur between the polymeric chains due to the incorporation of other compounds,
further revealing the extent of intermolecular interactions (Ahmad et al., 2012). Hoque et al. (2011)
found that the higher amplitudes of amide-A and amide-B bands resulted from the addition of
glycerol, which lowered the interaction between gelatin molecules, and therefore resulted in a
higher concentration of -NH2 groups. Studies that investigated blends of protein isolates into fish
gelatin films showed a shift of the amide-A band to a lower wavenumber and lower amplitude as
the protein isolates content increased, suggesting increased interactions and hydrogen bonding
between fish gelatins and protein isolates (Arfat et al., 2014; Hoque et al., 2011). A study by
Denavi et al. (2009) showed that a higher intensity of the new peaks at 1687 cm-1 and 1656 cm-1
indicated the possible interactions between soy protein isolates and fish gelatin, when the mixture
of these two proteins at ratio of 25:75 was used. They concluded that the higher interaction between
proteins at this ratio produced films which were more compact, of reduced thickness, less
deformable, and exhibited decreased water vapour permeability.
Pérez-Mateos et al. (2009) developed films made from cod skin gelatin and sunflower oil blends
and observed a peak at around 1700 cm-1 that could correspond to the carbonyl groups in the ester
bonds formed between the fatty acids of sunflower oil and cod skin gelatin. Ahmad et al. (2012)
studied the addition of essential oils into gelatin films from the skin of unicorn leatherjacket. They
found a shift to a lower wavenumber for amide-I and amide-II peaks, suggesting a decrease in
molecular order and an increase in the interaction between gelatin molecules and essential oil at
increasing concentrations. This resulted in a decrease in films’ tensile strength performance. A
study by Tongnuanchan et al. (2015) reported that the incorporation of palm oil decreased the
interaction between gelatin molecules from tilapia skin, as evidenced by a shift of amide-A and
amide-B peaks to higher wavenumbers. In a study on fish gelatin films charged with nanoparticles,
Hosseini et al. (2015) found that a shift to higher wavenumbers for the amide-A and amide-II
bands was attributed to the possible formation of hydrogen bonds between the gelatin and the
chitosan nanoparticles.
37
XRD is another technique that can be used to reveal the effect of the addition of additives and
other biopolymers into gelatin films. The use of additives, such as cross-linker and nanoparticles,
can disrupt or inhibit the assembly of gelatin molecules into triple-helices (da Silva et al., 2015;
Huang et al., 2017). The magnitude of these changes is reflected by the degree and intensity of the
diffraction peaks at a specific scattering angle (2θ, degree), which is displayed on the diffractogram.
A sharp-defined diffraction peak represents the crystalline structure, while a broad diffraction peak
represents the amorphous structure. The appearance of the amorphous structure may be associated
to the stable interaction between the additives and the gelatin molecules, or to an increase in
plasticizers and moisture in the film matrix which hinders the formation of semi-crystalline regions
and limits the re-crystallization (Bergo & Sobral, 2007). Liu et al. (2012) studied the effect of
adding chitosan in a gelatin film from walleye pollock skin. The intensity of the diffraction peak
found at 2θ values of 7-8° decreased and the shape of the peak became broad with the addition of
chitosan. This confirmed a decrease in the crystallinity of the matrix due to the presence of
significant hydrogen bonding interactions. In another study, the diffraction peaks at 2θ values of
7.0° and 20.5°, representing the partial crystallization of gelatin, decreased in intensity and became
broader in shape when titanium dioxide nanoparticles were added in a shark skin gelatin film
matrix (He et al., 2016).
SDS-PAGE can be used to indicate the compatibility among film constituents, by examining the
changes in the molecular weight by examining the intensity of the protein bands. When cod gelatin
films were blended with soy protein isolate, Denavi et al. (2009) found that the protein bands
associated to gelatin showed a decrease in intensity at increasing concentration of soy protein
isolate, suggesting the presence of interactions between proteins, which further lead to the
formation of films with lower water solubility. Hoque et al. (2011) studied composite films
composed of cuttlefish skin gelatin films and mungbean protein isolate, and the compatibility of
both proteins in the film network was proposed based on the decreased intensity of protein band
associated to mungbean. Ahmad et al. (2012) developed unicorn leatherjacket skin gelatin films
and observed the disappearance of the gelatin band while a new band formed upon addition of
essentials oils. The resulting film had poor elongation at break values.
38
2.5.5 Morphological properties
A scanning electron microscope (SEM) is often used to study the morphology of gelatin films.
The morphological properties of films are found to be related to the functional properties of the
biopolymer films, such as the mechanical and barrier properties (Hernandez-Izquierdo & Krochta,
2008). Arfat et al. (2014) observed no distinct separation from the SEM micrographs of the
composite films of tilapia skin gelatin and yellow stripe trevally protein isolate, suggesting that
the compatibility of both proteins had contributed to an increase in the mechanical properties of
the resulting films. The smooth and homogenous surface was also observed by SEM on blue shark
skin gelatin films plasticized with fatty acids, which indicated the compatibility between the film
constituents. These films showed interesting WVP properties (Limpisophon et al., 2010). A study
on tilapia skin gelatin film plasticized with palm oil showed that oil droplets on top layer of film
that could be observed by SEM. It was proposed that these droplets could act as water barrier and
contributed to the reduction of WVP (Tongnuanchan et al., 2015). Relatively dense structures were
visualized by SEM on films when chitosan nanoparticles were added to cold-water fish skin gelatin
films. The addition of chitosan improved the mechanical and barrier properties of the films
(Hosseini et al., 2015). In another study, SEM images were also used to explain the improved
dispersion of cellulose whiskers in tilapia gelatin films when the film forming solutions were
subjected to sonication prior to the film formation step (Santos et al., 2014).
The poor compatibility of polymers and the reduced level of molecular organization can form
rough surface films with pores, pin holes or cracks. These structural defects can be visualized
through SEM and are used to relate to the lower mechanical or barrier properties of the resultant
films. For example, Hoque et al. (2011) proposed that the rough surface of films observed through
SEM were formed from the poor interaction between cuttlefish skin gelatin and mungbean protein
isolate, resulting in films with poor tensile strength (TS). Ahmad et al. (2012) observed holes or
pores on unicorn leatherjacket skin gelatin films plasticized with essential oil of bergamot,
suggesting the possibility of the volatility of oil droplets in the film structure that led to the high
WVP values. The surface micrographs of tilapia gelatin-corn oil films showed an irregular surface,
which led to poor water barrier properties (Nur Hanani et al., 2013).
Other microscopic techniques can also be used to assess morphological information of films.
Transmission electron microscopy (TEM) is used for film samples with thickness of no more than
39
0.5 µm (Jiang et al., 2010; Jonge & Ross, 2011). For example, Bae et al. (2009) developed warmwater fish gelatin films charged with clay nanoparticles. The exfoliation of the nanoclay was
observed by TEM. Polarized light microscopy (PLM) is used in providing information on the
structure and composition of film constituents due to their difference in optical properties (Oster,
1955). For example, Mousia et al. (2001) used PLM to examine the homogeneity of highly
birefringent maize starch granules dispersed inside the gelatin film matrix. Atomic force
microscopy (AFM) can be used to produce three dimensional images of a film’s surface under
high resolution and real time, and provide direct information on the structure in relation to the
properties of the film. For example, the interaction between fish gelatin matrix and zinc oxide
nanorods was shown by AFM. The addition of zinc oxide improved the physical properties of the
resultant fish gelatin bio-nanocomposites (Rouhi et al., 2013). Moreover, the size and distribution
of chitosan nanoparticles were revealed through AFM imaging for cold-water fish skin gelatin bionanocomposite films (Hosseini et al., 2015).
2.6 Conclusion
In the development of biodegradable films, fish gelatins are potential biopolymers to be used as
filmogenic materials in replacement to mammalian gelatins for food packaging applications. The
properties of fish gelatin films not only rely on the gelatin’s amino acid composition due to fish
species, but also the peptide size due to varying molecular weight distribution resulting from the
extraction conditions. While acid and alkaline pretreatments have been extensively studied in the
past two decades, enzymatic pretreatments show promising results. Particularly, few studies
showed that pretreatment using protease facilitate the extraction of fish gelatins. Due to the
different specificities of proteases, more research in the field is required.
Gelatin films generally exhibit poor water resistance and mechanical properties. In comparison,
fish gelatin films are known to have inferior physical properties than those synthesized from
mammalian gelatins. Various strategies have been studied extensively to improve the properties
of fish gelatin films. The addition of hydrophobic biopolymers, such as proteins and
polysaccharides, has been used for mechanical and water resistance enhancement. Moreover, the
inclusion of nanoparticles is mainly contributing to improvement in mechanical properties. The
40
addition of lipids as plasticizer can increase the water barrier property of the films. Another
strategy is by introducing a crosslinker to the film network to overcome the deficiencies in both
mechanical and water barrier properties of fish gelatin films.
Overall, the success in developing fish gelatin films with improved mechanical and water
resistance is highly dependent on the interactions within the film matrix. Therefore, detailed
characterization need to be carried out to assess their potential use.
41
CONNECTING STATEMENT 1
Chapter III presents the investigation comparing the effect of different extraction methods on the
yield of Atlantic salmon (Salmo salar) fish skin gelatin. The study compared extraction methods
with saline, saline in combination with alkaline, and trypsin-aided pre-treatments. The study
further investigated the effect of different trypsin-aided process conditions on the yield and
molecular weight distribution of gelatins from three fish skins, salmon, skate and dogfish skins.
The results of this study have been prepared to be considered for publication as:
Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Trypsin supplementation process for the
extraction of gelatin from different fish skins.
42
CHAPTER III. TRYPSIN SUPPLEMENTATION PROCESS FOR THE
EXTRACTION OF GELATIN FROM DIFFERENT FISH SKINS
43
3.1 Abstract
Gelatin was extracted from Atlantic salmon (Salmo salar) skin, skate (Leucoraja erinacea) and
Atlantic dogfish (Squalus acanthias) skins, using different trypsin-aided process conditions.
Different fish skins pre-treated with trypsin at 250 U/g of fish skin for 8 h followed by a heat
extraction step at 70 °C for 3 h produced high gelatin yields (27-78%), but their protein chains
were degraded as evidenced by the concomitant disappearance of high molecular weight protein
chains. When a lower concentration of trypsin (1 U/g) was used in pre-treating all fish skins for 4
h and followed by lower extraction temperature at 50 °C for 3 h, the degradation of gelatin was
reduced with co-production of high molecular weight α-chains, but the corresponding yields of
gelatins were low. The results demonstrated the viability of the trypsin supplementation process
to obtain good quality fish gelatin.
Keywords: Fish skins; gelatin; trypsin; yield; molecular weight distribution
44
3.2 Introduction
Gelatin is a denatured protein extracted through thermal hydrolysis of collagen (Djabourov et al.,
1993; Stainsby, 1987). Gelatin exhibits good film forming ability and the properties of gelatin
films are affected by the molecular weight distribution and the amino acid composition of gelatin
(Bigi et al., 2000; Carvalho et al., 2008; Gómez-Guillén et al., 2009). The predominance of highmolecular weight chains in gelatin results in the formation of films with improved physical and
structural properties (Gómez-Guillén et al., 2002). During film formation, gelatin chains tend to
interact between themselves and further form a tree-dimensional network where zones of
intermolecular microcrystalline junctions are formed (Arvanitoyannis, 2002; Slade & Levine,
1987). The formation of the gelatin network relies on the presence of amino acids, specifically
hydroxyproline (Hyp), due to its ability to form intra- and intermolecular hydrogen bonds
(Brinckmann, 2005). Moreover, hydrophobic and ionic interactions between high-molecular
weight fractions (α-chains) improve the network stability (Galea et al., 2000).
Fish gelatin has gained popularity as an alternative source in replacement of gelatins from
mammals (Karim & Bhat, 2009). Gelatin extracted from industry processing wastes has been
characterized for its influence on the mechanical and barrier properties of the resulting films, which
are largely dependent on the amino acid composition and molecular weight distribution of the
gelatin (Gómez-Guillén et al., 2009). Few studies investigated the yield aspect, the molecular
weight distribution, and the Hyp content (Kolodziejska et al., 2008; Muyonga et al., 2004;
Nalinanon et al., 2008; Zhang et al., 2007). Studies showed that the extraction process affects the
yield and the molecular weight distribution of the extracted gelatin, while Hyp content was found
to vary depending on the species and the living habitat of the fish (Jongjareonrak et al., 2005;
Karim & Bhat, 2009; Kittiphattanabawon et al., 2005). Hyp content is lower in fish gelatin as
compared to mammalian gelatin, and is particularly low in gelatin from cold-water fish species
(Gudmundsson & Hafsteinsson, 1997; Haug et al., 2004; Zhou et al., 2006).
The extraction of gelatin generally involves three successive steps as follows: pre-treatment of the
raw material, followed by the extraction of the gelatin, and finally a concentration or a drying step
prior storage (Benjakul et al., 2012). Generally, the raw material is pre-treated with acid, alkaline
or a saline solution, to disrupt the collagen structure via the breakdown of non-covalent bonds,
resulting in the solubilization of the collagen (Giménez et al., 2005; Gómez-Guillén et al., 2009;
45
Stainsby, 1987). Subsequently, heat treatment is applied to induce cleavage of the hydrogen and
covalent bonds, converting the helical structure of collagen into gelatin, which comprises of
loosely coiled protein chains (Djabourov et al., 1993). Hence, the severity of the conditions used
(i.e., concentration of pre-treatment agents, pH, temperature and time) for both the pre-treatment
and the extraction steps are found to influence the gelatin yields and the proteins’ molecular weight
distribution (Karim & Bhat, 2009).
Various studies have been conducted to evaluate the influence of different pre-treatment methods
on the general properties of extracted gelatin. By using an effective pre-treatment method, high
quality gelatin can be produced using lower extraction temperatures (Johnston-Bank, 1990).
Generally, the selection of a pre-treatment agent depends on the source of the materials, and the
collagen type (Benjakul et al., 2012; Gómez-Guillén et al., 2009). Milder pre-treatment using acids
(e.g., 0.05 M acetic acid or 0.2 M-1.0 M diluted sulfuric or hydrochloric acid) are found to be more
suitable for solubilizing less crosslinked collagen from fish or pig skins. In contrast, an alkaline
pre-treatment (e.g., 0.025N sodium hydroxide solution with supersaturated solution of calcium
hydroxide) is usually used for highly crosslinked collagen of bovine origins (Benjakul et al., 2012;
Schrieber & Gareis, 2007). In some fish gelatin extraction studies, acetic acid showed a superior
effect in extracting gelatin regarding yields, viscoelastic properties and gel strength as compared
to citric, lactic, propionic, malic, and tartaric acids (Giménez et al., 2005; Gómez-Guillén et al.,
2001; Khiari et al., 2015). Meanwhile, in other studies where an alkali pre-treatment was used,
higher alkali concentration facilitated the extraction of fish gelatin having high purity but the yield
was lower (Yang et al., 2007). Moreover, the fish gelatin was more viscous as compared to alkaliacid mixture (Yoshimura et al., 2000), and the gel strength and the yield were higher using strong
and weak alkali mixture (Kaewdang et al., 2016). Saline solution pre-treatments are capable of
solubilizing collagen structure effectively by interacting with its structurally bound water
molecules. This leads to an improved yield of fish skin gelatin extracted while preserving the high
molecular weight of the protein chains (Giménez et al., 2005).
In comparison to chemical pre-treatments, proteases were found to specifically cleave the interchain cross-links of collagen but not its domain structure, resulting in the improved collagen
solubilization (Galea et al., 2000). Studies reported that pepsin-aided pre-treatment yielded
approximately two-fold higher amounts of gelatin, as compared to those without pepsin (Chomarat
46
et al., 1994; Nalinanon et al., 2008). Trypsin is found to be more effective than pepsin in assisting
the extraction of gelatin from wastes from leather industry, due to the higher substrate specificity
of trypsin (Cabeza et al., 1997). To date, trypsin has not been reported as a pre-treatment for fish
skins with the intent of extracting the gelatin. Thus, this study was conducted to compare the effect
of different extraction methods on yield of Atlantic salmon skin gelatin, which used saline, saline
in combination with alkaline, and trypsin-aided pre-treatments. Subsequently, the influence of
different trypsin-aided process conditions on the yield and molecular weight distribution of gelatin
were investigated by using three fish skins, namely salmon, skate and Atlantic dogfish skins.
3.3 Materials and methods
3.3.1 Materials
Atlantic salmon, skate and Atlantic dogfish were obtained from a local fish market, Montreal,
Canada. Trypsin from porcine pancreas (EC 3.4.21.4; powdered; 90.97 U/mg) was obtained from
ICN Biomedicals Inc. (Ohio, USA); isopropanol, methanol, sodium dodecyl sulfate (SDS) and
Tris base were purchased from Fisher Scientific (Fair Lawn, NJ, USA); glacial acetic acid and
hydrochloric acid were purchased from Fisher Scientific (Nepean, Ontario, Canada); sodium
hydroxide (NaOH) was purchased from Merck (KGaA, Darmstadt, Germany); sodium chloride
(NaCl) was purchased from BDH Inc. (Toronto, Ontario, Canada); 2-mercaptoethanol (2-ME),
activated charcoal, bromophenol blue, chloramine-T hydrate, Coomassie brilliant blue R-250,
Ehrlich’s reagent solution, N,N,N’,N’-tetramethyl ethylene diamine (TEMED) and trans-4hydroxy-L-proline (Hyp) were purchased from Sigma Chemical Co. (St. Louis, MO, USA);
Laemmli sample buffer was purchased from Bio-Rad Laboratories (Hercules, CA, USA); lowmolecular-weight protein markers (14kDa – 97kDa) and high-molecular weight markers (53kDa
– 220kDa) were purchased from GE Healthcare (Buckinghamshire, UK). All chemicals and
reagents used were of analytical grade.
3.3.2 Fish skins handling
Fish skins were manually removed at the fish market, packed in polyethylene bags and kept in ice
with a skin/ice ratio of 1:2 (w/w) in a polystyrene box. Fish skins were transported to the laboratory
within 1 h, and any residual meat was removed manually from the skin. The skins were then cut
into small pieces (ca 1.5 x 1.5 cm2) with scissors and washed with tap water. The skins were placed
47
in polyethylene bags and stored at -20 °C. The skins were thawed overnight in a refrigerator before
use.
3.3.3 Studies of gelatin extraction methods
Among previous studies on gelatin extraction, three gelatin extraction methods that involved the
use of saline solution with or without additional alkaline solution and trypsin-supplementation
were chosen. These methods were performed to compare the Hyp content and the yield of gelatin.
The yield was calculated based on the Hyp content of the lyophilized gelatin as compared to the
Hyp content of the wet fish skins, by using the following equation:
𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 =
𝑚𝑚𝑚𝑚
𝐻𝐻𝐻𝐻𝐻𝐻 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑒𝑒𝑒𝑒𝑒𝑒 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 � 𝑔𝑔 � 𝑥𝑥 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 (𝑔𝑔)
𝐻𝐻𝐻𝐻𝐻𝐻 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑜𝑜𝑜𝑜 𝑓𝑓𝑓𝑓𝑓𝑓ℎ 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 �
𝑚𝑚𝑚𝑚
� 𝑥𝑥 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑜𝑜𝑜𝑜 𝑓𝑓𝑓𝑓𝑓𝑓ℎ 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 (𝑔𝑔)
𝑔𝑔
𝑥𝑥 100 %
(1)
3.3.4 Extraction method with saline solution pre-treatment
The method described by Kołodziejska et al. (2008) was used with slight modifications. Fish skins
were pre-treated by gently stirring in 0.45 M NaCl (1:6, w/v) at 4 °C for 3 min, then rinsed five
times with distilled water (1:6, w/v), and the gelatin extraction was conducted using distilled water
(1:6, w/v) at 45 °C for 60 min. The protein solution was then centrifuged using a laboratory
centrifuge (Model B-22M, IEC, MA, USA) at 10 000 g for 30 min at 15 °C and the supernatant
obtained was lyophilized using a freeze dryer (Modulyod-115, ThermoSavant, Holbrook, NY,
USA) at 120 mBar for 48 h at -50 °C. The lyophilized proteins were kept at -20 °C until further
analysis.
3.3.5 Extraction with saline and alkaline solutions pre-treatment
The method followed by Rahman et al. (2008) was used with slight modifications. Fish skins were
washed with distilled water and pre-treated with 0.45 M NaCl 1:6 (w/v) at 4 °C for 3 min. The
samples were then soaked in 0.1 M NaOH 1:6 (w/v) at room temperature (22-25 °C) for 40 min
and washed five times with distilled water (1:6, w/v). The extraction was performed using distilled
water (1:6, w/v) at 50 °C for 18 h. The protein solution was centrifuged (10 000 g, at 15 °C, for 30
min) and the supernatant was then lyophilized (120 mBar, at -50 °C, for 48 h) and kept at -20 °C
until further analysis.
48
3.3.6 Extraction method with trypsin solution pre-treatment
The method followed by Cabeza et al. (1997) was used with slight modifications. Fish skins were
pre-treated with trypsin at 250 U/g (of fish skin) in Tris-HCl buffer (pH 8.0; 1:6, w/v) for 8 h at
room temperature (22-25 °C). The samples were then filtered and rinsed five times with distilled
water (1:6, w/v), and the extraction was conducted with distilled water (1:6, w/v) at 50 °C for 3 h.
The protein solution was centrifuged and the supernatant was lyophilized as described above.
3.3.7 Gelatin extraction from fish skins using trypsin-aided process
Studies showed that salmon skins pre-treated with trypsin solution produced the highest gelatin
yield and Hyp content. Thus, this extraction method was used to investigate the effects of different
trypsin concentrations, trypsin incubation times and extraction temperatures on the gelatin
extracted from salmon, skate and dogfish.
Fish skins were soaked in Tris-HCl buffer (pH 8.0) with a skin/solution ratio of 1:6 (w/v) in the
presence of trypsin at 250 U/g and 10 U/g of fish skins. The mixture was incubated with continuous
stirring at room temperature (22-25 °C) for 8 and 4 h. The fish skins were then filtered with a
Whatman No. 4 filter paper and rinsed five times with distilled water (1:6, w/v). The gelatin
extraction was conducted with distilled water (1:6, w/v) at 70 and 50 °C for 3 h using a shaking
water bath (model 25, Precision Scientific, USA). The skin/solution ratio was 1:6 (w/v). The
gelatin extract was centrifuged using a laboratory centrifuge at 10 000 g for 30 min at 15 °C. The
supernatant was lyophilized using a freeze dryer at -50 °C and 120 mBar for 48 h. All the
lyophilized gelatin was evaluated for Hyp content, extraction yield and protein electrophoretic
patterns via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The
extraction yield was calculated using equation as described earlier.
3.3.8 Hydroxyproline content
The Hyp content of gelatin was determined according to the method of Nalinanon et al. (2008)
with slight modifications. In a typical experiment, a sample (1.0 g) was hydrolyzed with 6 M HCl
(8.0 ml) in an oven at 105 °C for 24 h. The hydrolysate was then clarified with activated charcoal
(200 mg) and filtered using a Whatman No. 4 filter paper. The filtrate was neutralized to pH 6.0–
6.5 with 10.0 M, 1.0 M and 0.1 M NaOH. The neutralized sample (0.1 ml) was transferred into an
49
amber tube and isopropanol (0.2 ml) was added and mixed well. To the mixture, 0.1 ml of an
oxidant solution (a mixture of 7% (w/v) chloroamine T (w/v) and acetate/citrate buffer, pH 6, at a
ratio of 1:4 (v/v)) was added and mixed thoroughly. Subsequently, 1.3 ml of Ehrlich's reagent
solution (a mixture of 2 g 4-dimethylamino-benzaldehyde in 98 ml of 8% (v/v) hydrochloric acid)
and isopropanol at a ratio of 3:13 (v/v)) were added. The mixture was mixed and heated in a
shaking water bath at 60 °C for 25 min and cooled in running tap water for 2–3 min. The solution
was diluted to 5 ml with isopropanol (99.9%). The absorbance was measured within 30 min at
A558 nm using an UV/Vis spectrophotometer (model DU 800, Beckman Coulter, USA). A Hyp
standard curve was prepared using absorbance readings from standard solutions with
concentrations ranging from 10 to 60 ppm. Distilled water was used as the blank. Hyp content was
calculated and expressed as mg/g sample.
3.3.9 Protein electrophoretic profile analysis
SDS–PAGE was performed to determine the gelatin electrophoretic profile according to the
method of Laemmli (1970) with minor modifications. The gelatin samples (0.01 g) were dissolved
completely in distilled water (1.0 ml). Solubilized samples were mixed at a 1:1 (v/v) ratio with
Laemmli sample buffer (containing 62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS and 0.01%
Bromophenol Blue) in the presence of 10% 2-ME, and heated at 100 °C for 10 min. Fifteen
micrograms of protein of each sample were loaded onto each well (15 µg/well) of pre-cast gradient
polyacrylamide gels of thickness of 1.0 mm x 10 wells (Bio-Rad Mini-PROTEAN® TGXTM, a 415% Polyacrylamide gel, USA). Electrophoresis was conducted using a Mini Protein II unit (BioRad, USA) at constant voltage of 100 V for approximately 95 min of total running time. The gel
was stained with 1 g Coomassie brilliant blue R-250 in a 4.5:4.5:1 solution of methanol-wateracetic acid and de-stained several times by gentle shaking with a 8:1:1 solution of water-methanolacetic acid. Low-molecular-weight markers ranging from 14kDa – 97kDa and high-molecular
weight markers ranging from 53kDa – 220kDa (GE Healthcare UK) were used to estimate the
molecular weight of the protein fractions.
50
3.4 Results and discussion
3.4.1 Effect of the different extraction methods on the hydroxyproline content and yield of
gelatin
The Hyp content and the yield of gelatin extracted using three different methods are shown in Fig.
3.1. The method using trypsin pre-treatment produced the highest Hyp content (7.41 ± 0.49 mg
Hyp/g treated skin) and the highest yield (53.05 ± 4.38%) of gelatin from salmon skins, as
compared to the other two pre-treatments used. The difference in Hyp and yield of gelatin extracted
may possibly be due to the effectiveness of the protease in collagen hydrolysis, in contrast to the
random hydrolysis of collagen by chemical pre-treatments. In general, saline solution is used to
solubilize myofibrillar proteins and the remaining muscle adhered to the skins. The saline solution
also randomly disrupts the hydrogen bonds that stabilize the collagen structure to facilitate the
extraction of gelatin (Giménez et al., 2005). The alkaline pre-treatment randomly hydrolyzes
peptide bonds and cleaves some inter-chain cross-links of the collagen protein which further
disrupts the collagen structure (Galea et al., 2000; Yoshimura et al., 2000). Collagen cross-links
are readily cleaved by proteases, and protease-aided processes have been successfully used to
enhance collagen solubilization with improved gelatin yields (Chomarat et al., 1994; Nalinanon et
al., 2008). Therefore, the higher Hyp content and the higher gelatin yield obtained suggest an
increased cleavage of collagen cross-links by trypsin, resulting in a higher degree of collagen
solubilization and enhanced gelatin extraction efficiency. This is consistent with the higher yields
of gelatin reported from bigeye snapper skins treated with pepsin as compared to skins without
pepsin treatment (Nalinanon et al., 2008).
51
70
10.00
Hyp
9.00
Yield
60
50
7.00
6.00
40
5.00
30
4.00
3.00
20
2.00
10
1.00
0.00
Yielda (%)
Hyp (mg/g treated skin)
8.00
Pre-treatment with saline solution
Pre-treatment with saline and
alkaline solutions
Pre-treatment with trypsin
solution
0
a
Yield was calculated based on the Hyp content of the lyophilized gelatin compared to the Hyp
content of the wet fish skin.
Fig. 3.1 Hydroxyproline (Hyp) content and yield of gelatin extracted from salmon skin pretreated
with different pre-treatments.
3.4.2 Effects of trypsin concentrations, incubation times and extraction temperatures on the
yield of gelatin
The yields of gelatin obtained from the three fish skins using different extraction conditions are
presented in Fig. 3.2 (A: salmon, B: skate, C: dogfish). The lowest yields of gelatin were observed
with lower trypsin concentrations (10 U/g) and shorter incubation times (4 h), and the highest
gelatin yields were obtained with higher trypsin concentration (250 U/g) and a longer incubation
time (8 h) for all fish skins. The types of pre-treatment used affected the degree of collagen
solubilization and gelatin extractability (Benjakul et al., 2012; Boran & Regenstein, 2010).
Protease-aided pre-treatments have been found to effectively cleave collagen inter-chain crosslinks, resulting in higher disruption of the collagen structure and an increased conversion of
collagen into gelatin (Galea et al., 2000). Similarly, Nalinanon et al. (2008) and Cabeza et al.
(1997) showed that increasing the protease concentration during pre-treatments produced higher
gelatin yields from bigeye snapper fish skin and chrome shavings, respectively.
52
A higher gelatin yield was obtained when using an extraction temperature of 70 °C. A higher heat
applied during extraction imposes higher transition of the helix-to-coil of collagen to gelatin,
leading to an increased gelatin solubilization and a higher yield (Kittiphattanabawon et al., 2010).
Lower extraction temperatures, on the other hand, lead to low yields (Fig. 3.2) (Boran &
Regenstein, 2010). The results in this study are consistent with the higher extraction yields of
gelatin reported from Schrieber and Gareis (2007) who obtained a higher gelatin yield by
25.0
20.0
15.0
10.0
5.0
0.0
250 U/g
10 U/g
Trypsin concentration
53
4 h, 50 °C
4 h, 50 °C
Yield (%)
30.0
8 h, 50 °C
35.0
4 h, 70 °C
40.0
8 h, 70 °C
45.0
8 h, 50 °C
50.0
4 h, 70 °C
A
8 h, 70 °C
increasing the extraction temperature from 50 °C to 70 °C.
B
10.0
4 h, 50 °C
8 h, 50 °C
15.0
4 h, 70 °C
20.0
8 h, 70 °C
4 h, 50 °C
Yield (%)
25.0
8 h, 50 °C
4 h, 70 °C
30.0
8 h, 70 °C
35.0
5.0
0.0
250 U/g
10 U/g
40.0
30.0
4 h, 50 °C
50.0
8 h, 50 °C
60.0
8 h, 50 °C
Yield (%)
70.0
20.0
4 h, 50 °C
80.0
4 h, 70 °C
90.0
8 h, 70 °C
100.0
4 h, 70 °C
C
8 h, 70 °C
Trypsin concentration
10.0
0.0
250 U/g
10 U/g
Trypsin concentration
Fig. 3.2 Yield of gelatin from fish skins (A: salmon, B: skate, C: dogfish) pretreated at different
trypsin concentrations (U/g) and trypsin incubation times (h), and extracted at different
temperatures (°C).
54
3.4.3 Effects of trypsin concentrations, incubation times and extraction temperatures on the
protein electrophoretic patterns of gelatin
The protein patterns of the gelatin extracted from the fish skins are shown in Fig. 3.3 (A: salmon,
B: skate, C: dogfish). From comparing the pre-treatment conditions, the results showed that the
trypsin concentrations used had a marked effect on the molecular weight distribution of the gelatin
polypeptide chains. For instance, the high molecular weight protein components associated with
the α-chains of gelatin (100-120 kDa) were absent for all gelatin extracted from all fish skins.
These major chains were completely degraded when using 250 U/g trypsin as pre-treatment (lane
1 to 4). Furthermore, there was concomitant disappearance of major gelatin components with the
formation of low molecular weight protein fragments for all gelatins extracted from fish skins
pretreated with trypsin at 10 U/g (lane 5 to 8). From the results, higher trypsin concentration caused
excessive enzymatic hydrolysis of gelatin and cleaved the major polypeptide chains, resulting in
an increased degradation of the gelatin. This is in agreement with results reported by Nalinanon et
al. (2008) where the degradation of major protein chains in gelatin obtained from bigeye snapper
fish skins increased with the increasing concentration of pepsin. A similar trend was also observed
by Cabeza et al. (1997) for a gelatin extraction study using pepsin and trypsin pre-treatments.
Both results of yields and protein patterns of gelatin revealed distinct effects of the trypsin
concentrations used in pre-treatment, where higher trypsin concentration produced gelatin with
higher yield but also had a greater effect on the degradation of the major protein chains of gelatin.
The degradation of the gelatin major protein chains into low molecular weight chains is
undesirable in the production of high quality gelatin (Galea et al., 2000), as the functional
properties of gelatin are influenced by their molecular weight distribution (Muyonga et al., 2004).
Thus, an increased trypsin concentration used in this study had the effect of decreasing the quality
of the gelatin extracted.
55
A
kDa
97
66
45
31
22
14
LMW
B
1
2
3
4
5
6
7
8
kDa
97
66
45
31
22
14
LMW
C
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
kDa
97
66
45
31
22
14
LMW
1
Fig. 3.3 SDS-PAGE patterns of gelatins extracted from fish skins (A: salmon, B: skate, C: dogfish)
at 250 U/g trypsin for (lane 1) 8 h at 70 °C; (2) 4 h at 70 °C; (3) 8 h at 50 °C; (4) 4 h at 50 °C; at
10 U/g trypsin for (5) 8 h at 70 °C; (6) 4 h at 70 °C; (7) 8 h at 50 °C; (8) 4 h at 50 °C. LMW
denoted for low molecular weight protein markers.
56
A further investigation was conducted to examine the yield and molecular weight distribution of
gelatin extracted from all three fish skins incubated with a lower trypsin concentration of 1 U/g for
4 h, and extracted at 50 °C for 3 h. Gelatin yields were markedly decreased using these processing
conditions for all fish skins (8.05 ± 0.16% from salmon, 4.49 ± 0.14% from skate, 5.30 ± 0.09%
from dogfish). Lower yields were reported when using lower pepsin concentration (i.e. 5 units/g
treated skin) was used in pre-treating fish skin for gelatin extraction studies (Nalinanon et al.,
2008), extracting times and temperatures (Kolodziejska et al., 2008). Nonetheless, all gelatin
obtained in this investigation showed the presence of major polypeptide chains (α-chains) (Fig.
3.4). Hence, a lower trypsin concentration was found to successfully minimize the degradation of
gelatin extracted from all fish skins. This is in agreement with the findings from a study of gelatin
extraction conducted by Cabeza et al. (1997), which reported that a very small amount of trypsin
was sufficient to produce good quality gelatin as a result of the high efficiency of trypsin in
solubilizing collagen.
kDa
220
170
116
α-chains
76
53
HMW
A
B
C
Fig. 3.4 SDS-PAGE patterns of gelatins extracted from fish skins (A: salmon, B: skate, C: dogfish)
incubated with 1 U/g trypsin for 4 h and extracted at 50 °C for 3 h. HMW denoted for high
molecular weight protein markers.
57
3.5 Conclusion
Extraction of salmon skin gelatin with trypsin supplementation induced a higher collagen
solubilization and higher yield of gelatin as compared to extraction methods using chlorides and
alkaline solutions pre-treatments. However, all trypsin concentrations, trypsin incubation times
and extraction temperatures used in this study produced noticeable degradation of the gelatin’s
major protein chains from all fish skins investigated. Consequently, a very low level of trypsin (1
U/g) with milder processing conditions produced gelatin having a higher molecular weight,
however, lower yields were observed.
58
CONNECTING STATEMENT 2
The previous chapter demonstrated the viability of the very low-level trypsin supplementation
process to aid the recovery of good quality gelatins, however, the yield was low. The results also
showed that the yield and the intensity of high molecular weight molecules of salmon skin gelatin
were higher than gelatins obtained from skate and dogfish skins (Chapter III). Therefore, the
optimization of the trypsin-aided extraction process to obtain higher yield gelatins with high
molecular weight protein chains from salmon skins using response surface methodology (RSM)
was performed. In Chapter IV, the trypsin concentration, trypsin pre-treatment time, gelatin
extraction temperature and time were tested through a 12-run Plackett-Burman (PB) design
experiments. The yield of gelatin (based on Hyp content) and the intensity of gelatin’s α-chains
were used as responses. The significant variables affecting the yield and intensity of α-chains of
gelatin were identified and further optimized using a 3-factors 3-levels Box-Behnken design
(BBD). The optimum conditions in extracting the highest yield of gelatin (based on intensity of αchains) were determined.
The results of this study were published in Journal of Food Science and Technology as:
Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2017). Extraction of gelatin from salmon (Salmo salar)
fish skin using trypsin-aided process: optimization by Plackett-Burman and response surface
methodological approaches. Journal of Food Science and Technology, 54(12), 4000-4008.
59
CHAPTER IV. EXTRACTION OF GELATIN FROM SALMON (SALMO SALAR)
FISH SKIN USING TRYPSIN-AIDED PROCESS: OPTIMIZATION
BY PLACKETT-BURMAN AND RESPONSE SURFACE
METHODOLOGICAL APPROACHES
60
4.1 Abstract
Gelatin from salmon (Salmo salar) skin with high molecular weight protein chains (α-chains) was
extracted using trypsin-aided process. Response surface methodology (RSM) was used to optimise
the extraction parameters. Yield, hydroxyproline content and protein electrophoretic profile via
sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (SDS-PAGE) of gelatin were
used as responses in the optimization study. The optimum conditions were determined as follow:
trypsin concentration (X1) at 1.49 U/g; extraction temperature (X2) at 45 °C; and extraction time
(X3) at 6 h 16 min. This response surface optimized model was significant (p <0.05) and produced
an experimental value (202.04 ± 8.64%) in good agreement with the predicted value (204.19%).
Two-fold higher yields of gelatin with high molecular weight protein chains were achieved in the
optimized process with trypsin treatment when compared to the process without trypsin.
Keywords: Fish gelatin; trypsin; Plackett-Burman design; response surface methodology; yield
61
4.2 Introduction
Gelatin is a denatured polypeptide extracted by thermal hydrolysis from pretreated collagen
sources, mainly animal skins and bones (Kim & Mendis, 2006). Collagen has a molecular weight
of approximately 300 kDa, which comprises of three discrete polypeptide α-chains (molecular
weights ranging from 80 to 125 kDa) twisted around one another to form a triple-helical structure
(Boran & Regenstein, 2010). During the gelatin extraction, pre-treatments enhance the collagen
extractability, followed by thermal hydrolysis during extraction which disrupts the collagen triplehelical structure and induces helix-to-coil transition, producing gelatin comprised of loosely coiled
protein chains (Gómez-Guillén et al., 2002).
Fish skin has been extensively studied as a source of gelatin (Boran & Regenstein, 2010). The
quality of fish gelatin is mostly affected by its amino acid composition and molecular weight
distribution. Its unique amino acid composition, particularly the content of imino acids (proline
and hydroxyproline), varies depending on the species and living habitat of the fish. High levels of
imino acids are correlated with better physicochemical properties of gelatin (Boran & Regenstein,
2010; Jongjareonrak et al., 2005). Meanwhile, the quality of gelatin is determined by the length of
its protein chains. Longer protein chains of higher molecular weight produce gelatin with better
functional properties (Benjakul et al., 2012). The molecular weight distribution of gelatin is greatly
influenced by the extraction conditions (Gómez-Guillén et al., 2009). Milder extraction conditions
(i.e. lower temperature and shorter reaction time) reduce the degradation level of the protein, thus
producing gelatin with high molecular weight protein chains. However, milder extraction
conditions result in lower gelatin yields (Boran & Regenstein, 2010; Galea et al., 2000; Harris et
al., 2003). In contrast, severe extraction conditions (i.e. higher temperatures and longer reaction
times) produce gelatin with improved yields. However, molecular weight distribution is broader
due to the greater cleavage of the protein chains into shorter chain fragments (Boran & Regenstein,
2010; Kittiphattanabawon et al., 2010).
Lately, pre-treatment conditions with better efficiency have been studied to improve gelatin yield
while allowing milder extraction conditions (Benjakul et al., 2012). In this regard, relatively new
protease-aided pre-treatment using pepsin has successfully enhanced the conversion of collagen to
gelatin and yielded approximately two-fold higher amounts of fish skin gelatin as compared to
those without pepsin-aided treatment (Nalinanon et al., 2008). The study also reports the use of
62
another protease, namely trypsin, which was found to be more effective than pepsin for extracting
gelatin from wastes from the leather industry (Cabeza et al., 1997). To the best of the authors’
knowledge, there is no study reporting on the use of trypsin for the extraction of gelatin from fish
skin.
To maximize the yield and the quality of extracted gelatin, several optimization studies have been
conducted involving processing variables such as the concentration of pre-treating agent, and the
temperature and time of pre-treatment and extraction. These optimization studies were performed
on New Zealand hoki skins (Mohtar et al., 2010; Mohtar et al., 2013), surimi processing waste
(Norziah et al., 2014), and African catfish skin (Alfaro et al., 2014). Recently, statistical
experimental approaches including Plackett-Burman (PB) design and RSM have been used to
optimize multiple parameters that affect the extraction efficiency (Alfaro et al., 2014; Reddy et al.,
2008; Sai-Ut & Benjakul, 2014; Zhou et al., 2011). PB design enables an effective determination
of significant factors from a large number of process variables for subsequent optimization studies
(Plackett & Burman, 1944). RSM helps in evaluating the process factors to build models of
variables with optimized conditions, and further exposes responses with the highest desirability
(Sai-Ut & Benjakul, 2014).
Atlantic salmon (Salmo salar) that is mostly farm-raised, is a salmon of high commercial
importance. Because of the increased demand of skinless salmon food products, large amount of
skins (5 wt.% of the whole fish) are produced as by-products that can become a potential source
of gelatin (Gómez-Guillén et al., 2002; Gómez-Guillén et al., 2009). However, there is no efficient
protocol reporting the extraction of gelatin from salmon fish skin using trypsin. Therefore, the
objective of this study was to optimize the extraction conditions of gelatin from salmon skin using
trypsin-aided process in order to maximize the yield of gelatin with high molecular weight protein
chains.
4.3 Materials and methods
4.3.1 Chemicals
Atlantic salmon (Salmo salar) skins were obtained from the Jean-Talon fish market, Montreal,
Canada. Trypsin from porcine pancreas (EC 3.4.21.4; powdered; 90.97 U/mg) was obtained from
ICN Biomedicals Inc. (Ohio, USA); 2-propanol, citric acid anhydrous, glycine, methanol, sodium
63
dodecyl sulfate (SDS) and Tris base were purchased from Fisher Scientific (Fair Lawn, NJ, USA);
acetic acid and hydrochloric acid were purchased from Fisher Scientific (Nepean, Ontario,
Canada); sodium hydroxide was purchased from Merck (KGaA, Darmstadt, Germany); 2mercaptoethanol (2-ME), activated charcoal, chloramine-T hydrate, Coomassie Brilliant Blue R250, Ehrlich’s solution, N,N,N’,N’-tetramethyl ethylene diamine (TEMED) and trans-4-hydroxyL-proline (Hyp) were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Laemmli
sample buffer was purchased from Bio-Rad Laboratories (Hercules, CA, USA); high-molecularweight protein markers of 53kDa – 220 kDa were purchased from GE Healthcare
(Buckinghamshire, UK). All chemicals and reagents used were of analytical grade.
4.3.2 Fish skins preparation
Fish skins were manually removed at the fish market and immediately packed in polyethylene
bags, kept in ice with a skin/ice ratio of 1:2 (w/w) in a polystyrene box, and transported to the
Department of Food Science, McGill University, within 1 h. Upon arrival, residual meat was
removed manually from the skin and washed with tap water. The skins were cut into small pieces
(1.5 x 1.5 cm2) with scissors and placed in polyethylene bags. The skins were stored at -20 °C for
further use.
4.3.3
Extraction of gelatin from fish skins
4.3.3.1 Removal of non-collagenous proteins
The frozen fish skins were thawed overnight in the refrigerator before use. The samples were
degreased by tumbling in warm (35 °C) water (Muyonga et al., 2004). Non-collagenous proteins
from the skin were removed by stirring in 0.45 M NaCl at 4 °C for 3 min, and washed with distilled
water (Rahman et al., 2008). The skin/solution mass ratio was 1:6 (w/v).
4.3.3.2 Extraction method for optimization of gelatin extraction
As pre-treatment, collagenous-rich fish skins were soaked in 50 mM tris-HCl buffer (pH 8.0) in
the presence of trypsin at different concentrations, and stirred continuously at room temperature
(22-25 °C) for different periods of time, then filtered with a Whatman No. 4 filter paper and washed
with distilled water. The extraction step was conducted by gently stirring the mixture of pretreated
skins and distilled water using a shaking water bath (model 25, Precision Scientific, USA) at
64
different temperatures for different periods of time. The protein solutions were then centrifuged
(7000 g, 15 °C, 30 min) and the supernatant was lyophilized using a freeze dryer (Modulyod-115,
ThermoSavant, Holbrook, NY, USA) at -50 °C and 120 mBar for 48 h. The lyophilized proteins
were stored at -20 °C. The skin/solution ratio was 1:6 (w/v) throughout the process.
4.3.4
Experimental design
4.3.4.1 Plackett-Burman design
In order to select significant variables for gelatin extraction, the concentration of trypsin used in
pre-treatment, the pre-treatment time, extraction temperature and extraction time were tested
through PB design experiments. A 12-run PB design was applied to evaluate eleven factors
inclusive of four selected variables (A to D) and seven dummy variables (E to K). Each variable
was represented at two levels, coded as -1 for the low level and +1 for the high level.
Each level’s experimental value was established based on preliminary experimental results. Briefly,
gelatin with high molecular weight protein chains were successfully obtained from fish skins pretreated at a trypsin concentration of 1.0 U/g for 4 h and extracted at 50 °C for 3 h; however, low
yields were obtained (data not shown). In this study, each variable and their corresponding levels
to be studied were generated. Trypsin concentration ranging from 0.5 U/g (-1) to 1.5 U/g (+1) was
generated by referring 1.0 U/g as center level. Trypsin pre-treatment time ranging from 1.5 h (-1)
to 5 h (+1) was used to minimize the effect of longer pre-treatment time that can lead to a decreased
yield of fish skin gelatin extracted (Yang et al., 2007). Extraction temperature ranging from 40 °C
(-1) to 70 °C (+1) was generated to induce the transition of collagen to gelatin which occurs at 40
°C and above (Eastoe & Leach, 1977), while excessive cleavage of the protein chains happens at
80 °C and beyond (Djagny et al., 2001). Extraction times ranging from 5 h (-1) to 15 h (+1) was
performed to increase gelatin yield by increasing extraction times (Muyonga et al., 2004).
Two responses were recorded, namely the yield of the extracted gelatin (calculated based on Hyp
content) (Response 1, Y1) expressed in equation (2), and the intensity of the protein chains (αchains)
quantified
densitometrically
via
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis (SDS-PAGE) analysis (Response 2, Y2). The response values (Y1 and Y2) were the
average of the experiments performed in triplicates. Analysis of the regression coefficients and
65
significant variables with confidence levels above 95% were conducted to the yield (Y1) and
intensity of α-chains (Y2) in extracted gelatin.
𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 (𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑜𝑜𝑜𝑜 𝐻𝐻𝐻𝐻𝐻𝐻 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) =
𝑚𝑚𝑚𝑚
� 𝑥𝑥 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 (𝑔𝑔)
𝑔𝑔
𝑚𝑚𝑚𝑚
𝐻𝐻𝐻𝐻𝐻𝐻 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑜𝑜𝑜𝑜 𝑓𝑓𝑓𝑓𝑓𝑓ℎ 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 � � 𝑥𝑥 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑜𝑜𝑜𝑜 𝑓𝑓𝑓𝑓𝑓𝑓ℎ 𝑠𝑠𝑘𝑘𝑘𝑘𝑘𝑘 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 (𝑔𝑔)
𝑔𝑔
𝐻𝐻𝐻𝐻𝐻𝐻 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑡𝑡 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑖𝑖𝑛𝑛 �
4.3.4.2 Response surface methodology
(2)
𝑥𝑥 100 %
A Box-Behnken Design (BBD) of RSM was adopted to determine the effects of the independent
variables and to generate the optimum gelatin extraction conditions for maximum response. A total
of 18 experiments were conducted with six replicates of the center point. Three significant
variables identified from the Plackett–Burman (PB) design were used: trypsin concentration (X1),
extraction temperature (X2), and time (X3). Each variable was assessed at three coded levels (-1, 0,
+1), by employing the optimal levels estimated from the PB design as level zero. In this regard,
the pre-treatment time was fixed at 4 h 58 min, which was the estimated optimal level. The yield
of gelatin extracted based on the intensity of the α-chains (Y) was taken as the response calculated
by using equation (3). Gelatin extracted according to the method described by Kołodziejska et al.,
(2008) without enzyme-aided process was used as control for the calculation of the response.
Briefly, fish skins were pretreated by stirring in 0.45 M NaCl at 4 °C for 3 min, and subjected to
gelatin extraction in distilled water at 45 °C for 60 min. The response value (Y) was the average of
the experiments performed in triplicates.
𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌𝑌 𝑜𝑜𝑜𝑜 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔 (𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑜𝑜𝑜𝑜 𝛼𝛼 − 𝑐𝑐ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎) =
𝐼𝐼1 𝑥𝑥 𝑊𝑊1
𝐼𝐼2 𝑥𝑥 𝑊𝑊2
𝑥𝑥 100 %
(3)
where I1 is the intensity of α-chains in gelatin with trypsin-assisted process (%); W1, the weight of
gelatin with trypsin-assisted process (g); I2, the intensity of α-chains in gelatin without trypsinassisted process (%); W2, the weight of gelatin without trypsin-assisted process (g).
Values obtained from experimental runs of RSM on gelatin extraction were subjected to analysis
of variance (ANOVA), using the Design Expert (Version 7.0.0) software. A quadratic model was
used to fit the response (dependent variable) to the independent variables expressed as coded
factors (X1, X2 and X3). The coefficient of determination (R2) was used to evaluate the fit of the
response surface quadratic model. The three-dimensional response surface plots were built as a
66
function of the independent variables (X1, X2 and X3) to assess the interactive relationship among
significant variables and to generate optimal conditions for the gelatin extraction.
4.3.5 Hydroxyproline content
The hydroxyproline (Hyp) content of gelatin was determined according to the method of Bergman
and Loxley (1963) with minor modifications. The sample (1.0 g) was hydrolyzed with 6 M HCl
(8.0 ml) at 105 °C in an oven for 24 h. The hydrolysate was clarified with 200 mg of activated
charcoal and filtered. The filtrate was neutralized with 10.0 M, 1.0 M and 0.1 M NaOH to pH 6.0–
6.5. An aliquot of 0.1ml neutralized sample was transferred into an amber tube and isopropanol
(0.2 ml) was added and mixed well. To the mixture, 0.1 ml of an oxidant solution (a mixture of
7% (w/v) chloroamine T and acetate/citrate buffer, pH 6, at a ratio of 1:4 (v/v)) was added and
mixed thoroughly. Subsequently, 1.3 ml of Ehrlich's reagent solution (a mixture of 2 g 4dimethylamino-benzaldehyde in 98 ml of 8% (v/v) hydrochloric acid) and isopropanol at a ratio
of 3:13 (v/v) was added. The mixture was mixed and heated at 60 °C for 25 min in a shaking water
bath and cooled in running water for 2–3 min. The solution was diluted to 5 ml with isopropanol.
Absorbance was measured within 30 min at A558 nm using an UV/Vis spectrophotometer (model
DU 800, Beckman Coulter, USA). Hydroxyproline standard curve was prepared using absorbance
readings obtained from standard solutions with concentrations ranging from 10 to 60 ppm.
Distilled water was used as the blank. The Hyp content was calculated and expressed as mg/g
sample.
4.3.6 Protein electrophoretic profile analysis
SDS–PAGE was used for gelatin electrophoretic profile analysis according to the method
of Laemmli (1970) with minor modifications. The gelatin samples (0.01 g) were dissolved
completely in 1.0 ml distilled water. Solubilized samples were mixed at a 1:1 (v/v) ratio with
Laemmli sample buffer (62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS and 0.01%
Bromophenol Blue) in the presence of 5% 2-ME, and heated at 100 °C for 10 min. Fifteen
micrograms of protein of each sample were loaded onto each well (15 µg/well) of polyacrylamide
gels (1.5 mm thickness) comprising of 5% stacking gel and 10% resolving gel, and subjected to
electrophoresis using a Mini-PROTEAN II unit (Bio-Rad, USA). Electrophoresis was conducted
at constant voltage of 80 V for stacking gel and 120 V for resolving gel for approximately 90 min
of total running time. The gel was stained with 1 g Coomassie Brilliant blue R-250 in a 4.5:4.5:1
67
solution of methanol-water-acetic acid and de-stained several times by gentle shaking with a 8:1:1
solution of water-methanol-acetic acid. High-molecular weight markers of 53kDa – 220kDa (GE
Healthcare UK) were used to estimate the molecular weight of the protein fractions. The intensity
of the protein fractions was quantified densitometrically using the Quantity One version 4.6.2
software from Bio Rad (USA).
4.4
Results and discussion
4.4.1 Screening of significant variables using Plackett–Burman design
The PB experimental design matrix and the corresponding responses are shown in Table 4.1. The
effects of the variables on the yield of gelatin extracted based on Hyp content (Y1) and statistical
analysis of the PB design are shown in Table 4.2. The concentration of trypsin (A) and extraction
temperature (C) were found to significantly increase the yield of extractability of the gelatin, while
the effects of trypsin incubation time (B) and extraction time (D) were insignificant. Cabeza et al.,
(1997) reported that increased concentrations of trypsin may affect the cleavage of the cross-links
of collagen and further enhance the gelatin extractability. Meanwhile, increased extraction
temperatures produced higher gelatin yield from fresh salmon skin as a result of an increase in
collagen solubility (Kolodziejska et al., 2008). In comparison, the effect of the extraction
temperature was found to be slightly more significant with a higher contribution level of 32.42%
and a lower probability value (p-value) of 0.0106 when compared to trypsin concentration with
contribution of 32.34% and a p-value of 0.0182 (data of p-value not shown).
The effects of the variables on the intensity of the α-chains are shown in Table 4.3. The intensity
of the α-chains (Y2) from extracted gelatin showed that the extraction temperature (C) and time (D)
imposed significant influence with negative effects (lower intensity of α-chains measured), while
trypsin concentration (A) and trypsin incubation time (B) were insignificant. This is in agreement
with the production of shorter chain fragments from greater gelatin degradation attributed to the
exposure of higher extraction temperature and longer extraction time (Galea et al., 2000;
Kittiphattanabawon et al., 2010). In comparison, the effect of the extraction temperature was found
to be more significant with higher contribution of 37.59% and a lower p-value of 0.0091, as
compared to the effect of the extraction time with 17.65% of contribution and a p-value of 0.0258.
68
Table 4.1 Plackett–Burman experiment design with actual experimental values and coded values
(in bracket) and response values for gelatin extraction.
Exp
Run
Trypsin
(U/g),
A
Incubation
time (h),
B
Extrac.
Temp
(°C), C
Extrac.
Time (h),
D
E
F
G
H
I
J
K
Yield (by
Hyp)
(%),
Y1
Intensity
of αchains
(%), Y2
1
1.5
(1)
1.5
(-1)
40
(-1)
5
(-1)
1
-1
1
1
-1
1
1
31.7
55.18
2
1.5
(1)
5
(1)
40
(-1)
15
(1)
1
1
-1
-1
-1
1
-1
43.2
49.80
3
0.5
(-1)
5
(1)
70
(1)
5
(-1)
1
1
1
-1
-1
-1
1
36.7
32.76
4
1.5
(1)
1.5
(-1)
70
(1)
15
(1)
1
-1
-1
-1
1
-1
1
42.0
22.56
5
1.5
(1)
1.5
(-1)
70
(1)
15
(1)
-1
1
1
1
-1
-1
-1
45.2
19.47
6
0.5
(-1)
5
(1)
70
(1)
15
(1)
-1
-1
-1
1
-1
1
1
36.8
20.65
7
0.5
(-1)
1.5
(-1)
40
(-1)
15
(1)
-1
1
1
-1
1
1
1
27.3
45.13
8
0.5
(-1)
1.5
9
1.5
(1)
10
0.5
11
0.5
12
1.5
(1)
(-1)
70
(1)
5
(-1)
1
1
-1
1
1
1
-1
32.9
41.57
5
(1)
70
(1)
5
(-1)
-1
-1
1
-1
1
1
-1
56.8
53.25
(-1)
5
(1)
40
(-1)
15
(1)
1
-1
1
1
1
-1
-1
30.1
45.42
(-1)
1.5
(-1)
40
(-1)
5
(-1)
-1
-1
-1
-1
-1
-1
-1
11.1
54.08
(1)
40
(-1)
5
(-1)
-1
1
-1
1
1
-1
1
26.2
54.56
5
Table 4.2 Effects of the variables on yield of gelatin extracted (based on Hyp content) (Y1) and
statistical analysis of data from the Plackett-Burman design.
Intercept
Trypsin concentration (U/g), A
Pre-treatment time (h), B
Extraction temp (°C), C
Extraction time (h), D
E (dummy)
F (dummy)
G (dummy)
H (dummy)
J (dummy)
K (dummy)
AB
R2 = 0.8811; Adj-R2 = 0.7384
a
5% significance level
Effect
11.70
6.60
16.57
7.97
5.30
-2.60
9.03
0.73
-1.33
3.13
9.30
Coefficient
35.00
5.85
3.30
8.28
3.98
2.65
-1.30
4.52
0.37
-0.67
1.57
4.65
69
Sum of Squares
% Contribution
410.67
130.68
411.68
95.20
42.13
10.14
122.40
0.81
2.67
14.73
28.83
32.34a
10.29
32.42a
7.50
3.32
0.80
9.64
0.064
0.21
1.16
2.27
Table 4.3 Effects of the variables on intensity of α-chains (Y2) in extracted gelatin and statistical
analysis of data from the Plackett-Burman design.
Intercept
Trypsin concentration (U/g), A
Pre-treatment time (h), B
Extraction temp (°C), C
Extraction time (h), D
E-F
F-G
G-H
H-J
J-K
K-L
AB
R2 = 0.9491; Adj-R2 = 0.8601
a
5% significance level
Effect
2.54
3.07
-13.53
-9.27
5.48
-6.77
6.79
2.00
-0.37
0.66
16.37
Coefficient
41.20
1.27
1.54
-6.76
-4.64
2.74
-3.38
3.39
1.00
-0.18
0.33
8.19
Sum of Squares
% Contribution
19.28
28.37
274.46
128.90
45.10
68.68
69.16
6.02
0.20
0.66
89.38
2.64
3.88
37.59a
17.65a
6.18
9.41
9.47
0.82
0.028
0.09
12.24
Based on both response analyses from PB experimental designs, three significant variables were
identified, namely trypsin concentration during pre-treatment (A), extraction temperature (C) and
extraction time (D). Those variables were further optimized in the subsequent studies. The optimal
levels of these variables estimated from PB with the highest desirability were used as zero levels
in the subsequent optimization studies. To estimate the optimal levels, variables were set as ‘in
range’ for goal, while both responses were set as ‘maximize’ for goal, with the intensity of the αchains set as the highest importance (+++++), and the yield of the extracted gelatin (based on Hyp
content) set as moderate importance (+++). The optimal levels estimated were trypsin
concentration at 1.5 U/g, extraction temperature at 40 °C, and extraction time of 5 h.
4.4.2 Optimization of significant variables using response surface methodology
The BBD of RSM design matrix and the corresponding results of RSM experiments, together with
the experimental values and coded levels of independent variables are shown in Table 4.4. The
intensity of the α-chains of all extracted gelatins were determined using SDS-PAGE analysis and
are used as part of the result calculation. It was found that high molecular weight protein chains
(α-chains) with molecular weight ranging from 95-117 kDa were observed at different intensities
(figure not shown). However, no α-chain or other protein fractions were obtained for all gelatin
70
extracted at 30 °C and 40 °C for 1 h, which showed insufficient extraction heat and time applied
for the conversion of water-insoluble collagen into water-soluble gelatin (Benjakul et al., 2012;
Gómez-Guillén et al., 2002).
Table 4.4 Box-Behnken experiment design with actual experimental values and coded values (in
bracket) and response values of the yield of gelatin extracted calculated based on α-chains band
intensity.
Exp
Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Trypsin concentration
(U/g),
X1
1.5 (0)
2.5 (1)
1.5 (0)
1.5 (0)
2.5 (1)
1.5 (0)
1.5 (0)
1.5 (0)
2.5 (1)
2.5 (1)
0.5 (-1)
0.5 (-1)
1.5 (0)
0.5 (-1)
1.5 (0)
1.5 (0)
1.5 (0)
0.5 (-1)
Extraction
Temperature (°C),
X2
40 (0)
30 (-1)
40 (0)
30 (-1)
40 (0)
50 (1)
40 (0)
40 (0)
50 (1)
40 (0)
30 (-1)
40 (0)
40 (0)
40 (0)
30 (-1)
40 (0)
50 (1)
50 (1)
71
Extraction Time
(h),
X3
5 (0)
5 (0)
5 (0)
9 (1)
9 (1)
1 (-1)
5 (0)
5 (0)
5 (0)
1 (-1)
5 (0)
9 (1)
5 (0)
1 (-1)
1 (-1)
5 (0)
9 (1)
5 (0)
Yield (by α-chains
intensity) (%),
Y
162.26
0.79
215.69
3.74
114.82
62.60
185.52
210.00
110.92
6.82
0.67
90.72
142.71
4.40
0.44
192.18
143.61
135.13
The ANOVA analysis for the response surface quadratic model for the gelatin yield based on αchains intensity indicated that the model was significant (p <0.05) at the 95% probability level,
with p-value of 0.0004. The coefficient of determination (R2) for the model was 0.9449 (a value >
0.75 indicates fitness of the model), indicating that the model is capable of explaining 94.49% of
the variation in response. The lack of fit for the model was insignificant (p >0.05) with p-value of
0.5131, suggesting that the experimental data obtained was a good fit with the model.
The ANOVA analysis of the optimization study indicated that the model terms, the linear
coefficients of X2, X3, and the quadratic coefficients of X12, X22 and X32 were significant (p < 0.01).
The linear coefficients of X1 (trypsin concentrations) were not significant, indicating that these
trypsin concentrations could not influence the yields of gelatin with α-chains. This suggested that
trypsin, similar to some proteases, enabled collagen structure disruption by cleaving the crosslinks at the terminal telopeptide regions, but did not cause cleavage of the collagen triple-helical
domain allowing production of gelatin with high molecular weight protein chains (Cabeza, 1997;
Galea et al., 2000). This was observed with the relatively high yields of gelatin with α-chains
obtained when high trypsin concentration (2.5 U/g) was used (refers to Table 4.4 exp run 5 and 9).
Meanwhile, the extraction temperature and time affected the response significantly, where more
severe extraction conditions (higher temperature and time) decreased the yields of gelatin with αchains. This indicated the random cleavage of gelatin structures, leading to the production of
shorter chain fragments and the lower yields (Benjakul et al., 2012; Kittiphattanabawon et al.,
2010). From this study, the gelatin yield (Y) can be expressed in terms of the following regression
equations as follow:
Y =184.73 + 0.30 X 1 + 55.83 X 2 + 34.83 X 3 − 6.08 X 1 X 2 + 5.42 X 1 X 3 + 19.43 X 2 X 3 − 60.63 X 12 − 62.22 X 22 − 69.91X 32
(4)
The optimal levels of each variable for a maximum yield of gelatin extracted with high α-chains
band intensity were determined by creating three-dimensional response surface plots (Fig. 4.1a, b
and c). These plots were made with the response (Y) on the Z-axis against any two independent
variables, while keeping another variable at the centre point value (coded value = 0). The yield of
gelatin with α-chains increased with the increase in trypsin concentration (X1) and extraction
temperature (X2) (Fig. 4.1a), trypsin concentration (X1) and extraction time (X3) (Fig. 4.1b), and
72
extraction temperature (X2) and extraction time (X3) (Fig. 4.1c). However, an increment of these
variables beyond the optimum value imposed depletion of the response, indicating excessive
destabilization and cleavage of the extracted gelatin, resulting in the decrease in gelatin’s α-chains
intensity. Excessive extraction conditions, particularly high extraction temperatures and prolonged
extraction times, imposed extensive degradation by cleaving gelatin’s high molecular weight
protein chains (α-chains), which resulted in the concomitant formation of shorter chain fragments.
An increase in shorter chain fragments and a decrease in intensity of the gelatin high molecular
weight protein chains were observed in gelatin extracted from skin of brown-banded bamboo shark,
blacktip shark (Kittiphattanabawon et al., 2010) and African catfish (Alfaro et al., 2014) when
using higher extraction temperatures.
73
a
210
Yield (alpha-chain)
155
100
45
-10
50.00
2.50
45.00
2.00
40.00
B: Extract. temp
1.50
35.00
1.00
30.00
0.50
A: Trypsin conc.
b
9
Yield (alpha-chain)
210
160
110
60
10
9.00
2.50
7.00
2.00
5.00
C: Extract time
1.50
3.00
1.00
1.00
0.50
A: Trypsin conc.
c
Yield (alpha-chain)
220
160
100
40
-20
9.00
50.00
7.00
45.00
5.00
C: Extract time
40.00
3.00
35.00
1.00
30.00
B: Extract. temp
Fig. 4.1 Three-dimensional response surface plots for optimization of gelatin extracted with major
protein band intensity, as a function of (a) trypsin concentration and extraction temperature; (b)
trypsin concentration and extraction time; (c) extraction temperature and extraction time.
74
The optimum levels of each variable obtained from RSM analysis were: trypsin concentration (X1)
= 1.49 U/g, extraction temperature (X2) = 45 °C, and extraction time (X3) = 6h 16 min, with
predicted gelatin yield value of 204.19%. This value was further verified by conducting
experiments in triplicates under the optimal conditions. A gelatin yield of 202.04 ± 8.64% was
obtained and was not significantly different (p >0.05) when compared to the predicted value, which
demonstrated the validity of the model. The electrophoretic profile analysis (Fig. 4.2) shows that
under these optimal conditions, gelatin contained distinct bands of α-chains. This indicated that
gelatin with high molecular weight protein chains can be obtained under these optimized
conditions.
kDa
220
170
α-chains
(95 - 117 kDa)
116
76
53
HMW
1
2
3
Fig. 4.2 SDS-PAGE patterns of salmon fish skin gelatins extracted in triplicate under optimal
conditions: (lane 1 to 3) gelatins obtained in triplicates; HMW denoted for high molecular weight
protein markers.
75
4.5 Conclusion
The optimized conditions for trypsin-aided extraction of gelatin from salmon fish skin has been
achieved using the PB and BBD of RSM. The optimum levels of three significant variables (trypsin
concentration, extraction temperature, and extraction time) successfully maximized the yield of
gelatin with high molecular weight protein chains. Approximately two fold higher yields of gelatin
with high molecular weight protein chains were attained with the optimized process using trypsin
treatment when compared to the process without trypsin.
76
CONNECTING STATEMENT 3
An optimized trypsin-aided extraction process to yield salmon skin gelatins with high molecular
weight protein chains was achieved (Chapter IV). In order to understand the performance of gelatin
films formed, the effect of different protein and glycerol concentrations on the resultant gelatin
films’ properties were investigated in Chapter V. The characteristics of the films with respect to
their mechanical properties, water solubility, light barrier properties, molecular weight distribution,
structural and morphological properties were determined and are reported in this present chapter.
The results of this study have been prepared for publication as:
Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Characterization of films prepared using
salmon skin gelatin extracted by a trypsin-aided process.
77
CHAPTER V. CHARACTERIZATION OF FILMS PREPARED USING SALMON SKIN
GELATIN EXTRACTED BY A TRYPSIN-AIDED PROCESS
78
5.1 Abstract
Properties of films prepared using salmon (Salmo salar) skin gelatin extracted by a trypsin-aided
process were investigated. Films with increasing protein concentration (from 1 to 5%, w/v)
exhibited higher thickness, tensile strength (TS), and elongation at break (EAB), but a marked
decrease in EAB was observed for films with 6 and 7% protein concentrations. Films with 5%
proteins showed higher thickness, lower TS and higher EAB as the concentration of glycerol
increased (from 10 to 50% of proteins). All films exhibited high water solubility, and as the protein
and glycerol contents increased in films, a decrease in light transmission accompanied by an
increase in opacity were observed. An electrophoretic study showed that the increase in the
mechanical properties of the films was correlated with the increase in protein concentration, owing
to the increased content of high molecular weight chains. Furthermore, Fourier transform infrared
(FT-IR) spectroscopy and scanning electron microscopy (SEM) revealed the interaction between
the proteins and glycerol for all films. Thus, this study can be a harbinger to other studies to make
salmon gelatin suitable for food packaging applications.
Keywords: fish protein, trypsin, film, mechanical properties
79
5.2 Introduction
Gelatin is a denatured polypeptide extracted from collagen from animal skins and bones via
thermal hydrolysis (Kim & Mendis, 2006). To date, the main sources of commercial gelatin are
bovine and porcine skins and bones. However, the potential of transmission of pathogenic vectors
from mammalian gelatin has created concerns among users of mammalian collagen and gelatin
products (Sadowska et al., 2003). Gelatin from aquatic animals is therefore gaining prominence in
recent years, not only as an alternative to mammalian gelatin, but also to add value to the
underutilized by-products from the fish processing industry. It is estimated that approximately 30%
of fish by-products in the form of bones and skins are produced from filleting processes (Blanco
et al., 2007). Atlantic salmon (Salmo salar) is a cold-water fish that is in high demand for fillet
production, thus contributing a large quantity of by-products that can serve as a rich source of
gelatin.
Gelatin has attracted much attention for the development of edible films for food packaging due
to its film forming ability (Bigi et al., 2000). Films made from fish gelatin have been extensively
studied over the years, including gelatin from the skins of bigeye red snapper and brownstripe red
snapper (Jongjareonrak et al., 2006), tuna (Gómez-Guillén et al., 2007), Atlantic halibut (Carvalho
et al., 2008), blue-shark (Limpisophon et al., 2009), as well as red snapper and grouper (Elango et
al., 2014). A comparison study was conducted among mammalian, warm- and cold-water fish
gelatins, and significant differences in physical and chemical properties of resulting films were
reported (Avena-Bustillos et al., 2006). This comparative study showed that cold-water fish gelatin
films exhibited lower water vapor permeability, suggesting its applicability as potential
biopolymer for encapsulating drugs or for packaging frozen food systems.
The physical and structural properties of gelatin films are affected by the gelatin’s amino acid
composition, which is species-specific, and its molecular weight distribution, which depends on
the extraction conditions (Carvalho et al., 2008; Gómez-Guillén et al., 2009). Generally, milder
processing conditions induce minimal degradation, and favor the production of gelatin with a high
content of high molecular weight polypeptide fractions. This could contribute towards the
formation of films with improved mechanical and light barrier properties (Gómez-Guillén et al.,
2002; Jongjareonrak et al., 2006; Limpisophon et al., 2009). Recently, fish skins were pre-treated
with pepsin to facilitated the extraction of gelatin with a high yield and minimal degradation
80
(Nalinanon et al., 2008). In another study, gelatin with high molecular weight protein chains was
produced from chrome shavings when treated with a lower concentration of trypsin as compared
to pepsin (Cabeza et al., 1997). Therefore, a trypsin-aided extraction process could be studied to
produce gelatin from fish skins for film-formation.
Gelatin chains tend to interact via crosslinks to form a three-dimensional network with zones of
intermolecular microcrystalline junctions in a polymeric system (Arvanitoyannis, 2002; Slade &
Levine, 1987). However, extensive intermolecular interactions together with dehydration of this
system may produce brittle films (Vanin et al., 2005). To overcome the brittleness of films,
relatively small molecular weight plasticizers are often added to the formulation. Plasticizers
compete for hydrogen bonding and electrostatic interactions with protein polymeric chains and
increase the free-volume or intermolecular spacing, resulting in an increased molecular mobility
and improved flexibility and extensibility (Limpisophon et al., 2009; Sothornvit et al., 2002). The
plasticizing effect on films is associated with the plasticizer’s ability to attract water, which also
acts as a plasticizer. This is influenced by the composition, size and shape of the plasticizer as well
as its compatibility with the polymer (Sothornvit & Krochta, 2001). Among different plasticizers
that can be added, glycerol and sorbitol are mainly used in gelatin-based films (Arvanitoyannis &
Biliaderis, 1998; Carvalho & Grosso, 2004; Menegalli et al., 1999; Sakanaka et al., 2001; Sobral
et al., 2001). However, sorbitol can crystallize in the films when stored at low and intermediate
relative humidity conditions, affecting its plasticizing effect (Sakanaka et al., 2001).
Few studies have investigated the effects of protein and plasticizer concentrations on the properties
of fish gelatin films, especially gelatin from cold water fish skin such as Atlantic salmon. In
addition, only few studies have reported on the physical properties of films using spectroscopic
methods and morphological analyses. Thus, the objectives of this study were to evaluate the
physical properties of films formed with salmon skin gelatin extracted by a trypsin-aided process,
prepared at different protein and glycerol concentrations, and to further correlate their mechanical
properties with their protein patterns via electrophoretic analysis, molecular interactions using FTIR spectroscopy and morphological analyses using scanning electron microscopy (SEM).
81
5.3 Materials and methods
5.3.1 Chemicals
Atlantic salmon (Salmo salar) skins were obtained from a local fish market (Jean-Talon, Montreal,
Canada). Sodium chloride was purchased from BDH Inc. (Toronto, Ontario, Canada); porcine
pancreas trypsin (EC 3.4.21.4; powdered; 90.97 U/mg) was obtained from ICN Biomedicals Inc.
(Ohio, USA); glycerol, methanol, potassium carbonate and Tris base were purchased from Fisher
Scientific (Fair Lawn, NJ, USA); bicinchoninic acid (BCA) protein assay reagents and bovine
serum albumin (BSA) standard were purchased from Pierce (Rockford, Illinois, USA); Laemmli
sample buffer was purchased from Bio-Rad Laboratories (Hercules, CA, USA); 2mercaptoethanol (2-ME) and Coomassie Brilliant Blue R-250, were purchased from Sigma
Chemical Co. (St. Louis, MO, USA); glacial acetic acid and hydrochloric acid were purchased
from Fisher Scientific (Nepean, Ontario, Canada); high-molecular-weight protein markers of
53kDa – 220 kDa were purchased from GE Healthcare (Buckinghamshire, UK). All other
chemicals and reagents used were of analytical grade.
5.3.2 Extraction of gelatin from salmon skin
Fish skins were manually removed at the fish market and immediately packed in polyethylene bags
and kept in ice in a polystyrene box. Fish skins were transported to the laboratory within 1 h,
residual meat was removed manually and skins were washed with tap water. The skins were cut
into 1.5 x 1.5 cm2 pieces with scissors and stored at -20 °C. Prior to gelatin extraction, the frozen
fish skins were thawed overnight in the refrigerator. The samples were degreased by tumbling in
warm (35 °C) water (Muyonga et al., 2004). Non-collagenous proteins from the skin were removed
by stirring in 0.45 M NaCl at 4 °C for 3 min, and washed with distilled water (Rahman et al., 2008).
The skins were then pretreated by soaking in 50mM tris-HCl buffer (pH 8.0) in the presence of
trypsin at 1.49 U/g, and stirred continuously at room temperature (22-25 °C) for 5 h, then filtered
with a Whatman No. 4 filter paper and washed with distilled water. Gelatin was extracted by gently
stirring the mixture of pretreated skins and distilled water using a shaking water bath (model 25,
Precision Scientific, USA) at 45 °C for 6 h 15 min. The skin/solution ratio was 1:6 (w/v)
throughout the process. The protein solutions were then centrifuged (7000 g at 15 °C, for 30 min)
and the supernatant was lyophilized using a freeze dryer (Modulyod-115, ThermoSavant,
82
Holbrook, NY, USA) at -50 °C and 120 mBar for 48 h. The lyophilized proteins were referred as
‘gelatin powder’ and were stored at -20 °C.
Protein concentration of the gelatin powder was determined using a standard BCA protein assay.
Gelatin solution (1000 µg/ml) was prepared in distilled water. To 0.1 ml of the gelatin solution,
2.0 ml of the BCA working reagents were added and mixed thoroughly. The mixture was incubated
at 37 °C for 30 min using a shaking water bath and then cooled to room temperature (22-25 °C).
The absorbance was measured within 10 min at A562 nm using an UV/Vis spectrophotometer
(model DU 800, Beckman Coulter, USA) and distilled water was used as the blank. The protein
concentration was determined by referring to a standard curve, which was prepared using
absorbance readings obtained from bovine serum albumin (BSA) standard solutions with
concentrations ranging from 25 to 2000 µg/ml, and were treated as described as for the gelatin
samples.
5.3.3 Preparation of gelatin films
In the first set of experiment, gelatin film forming solutions (FFS) (20 ml) were prepared by mixing
gelatin powder in distilled water to obtain protein concentrations of 1, 2, 3, 4, 5, 6, 7% (w/v). The
glycerol plasticizer was added into FFS at concentration of 30% (w/w) of protein. The FFS was
stirred gently for 30 min at room temperature (22-25 °C), filtered with a Whatman No. 1 filter
paper and cast onto a rimmed silicone plates (55 x 120 mm). The plates were placed on a leveled
surface in a fume hood to evaporate the solvent for a period of 48 h at room temperature (22-25
°C). The dried films were manually peeled off for characterization.
In the second set of experiment, gelatin films of 5% protein concentration were used to evaluate
the effect of glycerol concentration on gelatin films. Gelatin FFS with glycerol concentrations of
10, 20, 30, 40 and 50% (w/w) of protein were prepared. The FFS were then cast and dried as
previously described.
5.3.4 Film characterization
5.3.4.1 Mechanical properties
Prior to the determination of the mechanical properties, the thickness of the films was measured
with a hand-held digital micrometer (Marathon Part No. 030025, Marathon Watch Company Ltd.,
83
Ontario, Canada) with an accuracy of 0.002 mm. Six measurements were taken at random positions
for each film specimen, and the average thickness was used to estimate the cross-sectional area of
the specimen. The tensile strength (TS) and elongation at break (EAB) values were determined
according to ASTM method D 882-10 (ASTM 2010) using an Instron Universal Testing Machine
(model 4500, Instron Corporation, Canton, MA, USA). The films were conditioned at 23 ± 2 °C
in a desiccator containing saturated solutions of potassium carbonate (50 ± 2% relative humidity)
for at least 40 h before testing. The films were fixed on the grips of the device with an initial grip
separation of 30 mm, and pulled apart at a mechanical crosshead speed of 10 mm/min and preload
of 2 N. At least five replicates were tested for each film and the average was taken as the results.
TS (MPa) and EAB (%) were calculated by the following equations:
(5)
TS (MPa) = F max / A
where F max = maximum load (N) needed at the moment of rupture, A = cross-sectional area (m2)
of the samples.
𝐸𝐸
(6)
𝐸𝐸𝐸𝐸𝐸𝐸 (%) = �30� 𝑥𝑥 100
where E = film elongation (mm) at the moment of rupture, 30 = initial grip length (mm) of samples.
5.3.4.2 Water solubility
The water solubility of the films was determined according to the method of Shakila et al. (2012).
Films of surface area of 4 cm2 were cut and weighed (± 0.0001 g) to determine the initial weight
(Wi). Films were immersed separately in 15 ml of distilled water, gently shaken at room
temperature (22-25 °C) for 15 h and then filtered through a Whatman No. 1 filter paper. The
unsolubilized film fraction collected on the filter paper was dried in a hot air oven at 105 °C for
24 h and weighted (Wf). Three replicates were tested for each film and the average values were
taken as the result. The solubility of the film was calculated by the following equation:
Solubility (%) = �
𝑊𝑊𝑖𝑖 − 𝑊𝑊𝑓𝑓
𝑊𝑊𝑖𝑖
(7)
� 𝑥𝑥 100
where Wi = initial weight of the film specimen, Wf = weight of unsolubilized film fraction.
84
5.3.4.3 Light transmission and opacity
The barrier properties of gelatin films against ultraviolet (UV) and visible light were measured at
selected wavelengths (200 – 800 nm) using an UV/Vis spectrophotometer, according to the method
of Fang and others (2002). The films were cut in rectangular pieces (12 x 43 mm), directly placed
into a quartz cuvette and measured. An empty cuvette was used as the blank. The test was
performed in triplicate for each film and the averages were taken as the results. Light transmission
(T) was recorded using transmittance (%) measured at each wavelength for each film, and the
opacity (%) was calculated by the following equation:
(8)
Opacity (%) = 100% − 𝑇𝑇
where T = transmittance (%) at each wavelength.
5.3.4.4 Electrophoretic analysis
The protein patterns of gelatin films were determined using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS–PAGE) according to the method of Laemmli (1970)
with minor modifications. The gelatin films (0.01 g) were dissolved completely in distilled water
(1.0 ml). Solubilized samples were mixed at a 1:1 (v/v) ratio with Laemmli sample buffer
(containing 62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS and 0.01% Bromophenol Blue)
in the presence of 5% 2-ME, and heated at 100 °C for 10 min. Ten microliters of each sample were
loaded into each well of polyacrylamide gels (1.5 mm thickness) comprising of 5% stacking gel
and 10% resolving gel. Electrophoresis was conducted using a Mini Protein II unit (Bio-Rad, USA)
at constant voltage of 80 V for stacking gel and 120 V for resolving gel for approximately 90 min
of total running time. The gels were stained with 1 g Coomassie brilliant blue R-250 in a 4.5:4.5:1
solution of methanol-water-acetic acid, and de-stained several times by gentle shaking with an
8:1:1 solution of water-methanol-acetic acid. High-molecular weight markers ranging from 53kDa
– 220kDa (GE Healthcare UK) were used to estimate the molecular weight of the protein fractions.
5.3.4.5 Fourier transform infrared (FT-IR) spectra analysis
The differences in frequencies of functional groups in gelatin films prepared with different protein
and glycerol concentrations were determined using a Nicolet iS5 FT-IR spectrometer (Thermo,
Madison, WI, USA). Films were placed onto the crystal cell and the cell was clamped into the
mount of a FT-IR spectrometer. The spectra were collected in 32 scans with a resolution of 4 cm85
1
over the range of 4000-400 cm-1, and the data were rationed against a background spectrum
recorded from the clean empty cell at 25 °C. The spectra were analyzed using the OMNIC 8.2
software package (Thermo Fisher Scientific Inc., USA).
5.3.4.6 Scanning electron microscopy (SEM)
The morphology of the upper surface of the film samples was studied using a field emission gun
scanning electron microscope (FEG-SEM) (JSM-7600TFE, JEOL, Tokyo, Japan). The samples
were mounted on specimen stubs using double sided adhesive tape, and made conductive by
sputter-coating with gold-palladium. This step was repeated twice for 15 s using a sputter-coater
under vacuum for 30 s under a current of 15 mA. After coating, the samples were observed at an
accelerating voltage of 2 kV using a LEI (low secondary electron image) detector at low current.
5.3.4.7 Statistical analysis
Data were statistically analyzed using the General Linear Models procedure of SAS (Release 9.4,
SAS Institute Inc., Cary, NC, USA) software. Mean comparisons were carried out by Duncan’s
multiple range test (p < 0.05) (Steel & Torrie, 1980).
5.4 Results and discussion
5.4.1 Mechanical properties
The TS and EAB of films prepared with different protein concentrations are shown in Table 5.1.
The TS of the films increased (from 6.31 to 44.00 MPa) when the protein concentration increased
from 1 to 7%. As shown in Table 5.1, there was a marked increase in the TS values of the films (p
< 0.05) when the protein concentration increased from 5 (16.42 MPa) to 6% (48.87 MPa). The TS
values of films with 6 and 7% protein concentrations (48.87 and 44.00 MPa, respectively) were
significantly higher (p < 0.05) than the other films. The increase in the TS values was due to the
increase in the number of protein chains per unit surface, resulting in an increase in the potential
intermolecular interactions that contribute towards higher TS values (Cuq et al., 1996). Similar
effect was also observed for films prepared from bigeye red snapper and brownstripe red snapper
skin gelatins (Jongjareonrak et al., 2006), and blue shark skin gelatin (Limpisophon et al., 2009).
Meanwhile, the EAB of the films increased (from 2.67 to 58.43%) with increasing protein
concentration of the FFS from 1 to 5%, where the EAB values of the films at 4 and 5% protein
86
concentration were significantly higher (p < 0.05) than those for the other films. These higher EAB
values indicated that an increase in protein concentration increased the protein chain-to-chain
interactions, resulting in an enhanced flexibility of the films (Hoque et al., 2011; Jongjareonrak et
al., 2006; Limpisophon et al., 2009). However, decreased EAB values were obtained for films
beyond 5% protein concentration. For films with 6 and 7% protein (14.12% and 6.26%,
respectively), a remarkable decrease in the EAB values accompanied with a significant increase
in TS values were observed. This could be due to possible extensive protein intermolecular
interactions and cross-links formation as a result from the excessive protein concentrations in FFS.
This led to reduced mobility of the protein chains, forming films with high strength but low
elasticity. Films with 6 and 7% protein concentration were thick, hard and brittle (Table 5.1).
Table 5.1 Effect of protein and glycerol concentration on the thickness, mechanical properties and
water solubility of salmon skin gelatin films.1,2
Protein
concentration
(% in FFS3)
1%
2%
3%
4%
5%
6%
7%
Glycerol
concentration
(% of protein)
30%
Thickness
(mm)
TS
(MPa)
EAB
(%)
Water solubility
(%)
6.31 ± 1.80c
11.31 ± 0.63bc
11.77 ± 1.94bc
11.90 ± 2.78bc
16.42 ± 3.34b
48.87 ± 8.11a
44.00 ± 4.01a
2.67 ± 0.39b
12.31 ± 3.43b
27.28 ± 5.52b
57.66 ± 7.26a
58.43 ± 4.16a
14.12 ± 3.43b
6.26 ± 1.10b
94.91 ± 5.01a
92.10 ± 3.94a
91.58 ± 6.43a
90.14 ± 6.71a
89.07 ± 1.69a
90.07 ± 0.49a
90.64 ± 1.28a
10%
0.049 ± 0.007c
68.84 ± 12.63a
22.00 ± 1.17b
c
a
20%
0.056 ± 0.008
56.37 ± 4.29
31.97 ± 2.40b
b
b
30%
0.079 ± 0.004
16.42 ± 3.34
58.43 ± 4.16a
b
b
40%
0.079 ± 0.018
17.28 ± 3.98
66.03 ± 4.12a
a
c
50%
0.101 ± 0.016
1.49 ± 0.47
78.01 ± 5.52a
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
3
FFS means film-forming solution.
84.80 ± 1.59a
85.72 ± 5.30a
89.07 ± 1.69a
81.29 ± 7.89a
84.26 ± 6.32a
0.039 ± 0.006e
0.042 ± 0.005e
0.059 ± 0.007d
0.070 ± 0.006cd
0.079 ± 0.004bc
0.090 ± 0.013b
0.112 ± 0.012a
5%
As shown in Table 5.1, the TS values decreased (from 68.84 to 1.49 MPa) and the EAB values
increased (from 22.00 to 78.01%) when the glycerol concentration increased from 10 to 50% (w/w,
of protein) for the same protein concentration (5%). Glycerol is a relatively small molecule that
flows through the protein chains and form hydrogen bonds with the amide groups and the amino
acid side chains of the proteins. As a result, increasing the glycerol concentration in FFS causes a
reduced intermolecular interaction in the protein chains, leading to an increased mobility of the
87
protein chains and elasticity of the films (Gontard et al., 1993). In this study, significant differences
(p < 0.05) were observed for both TS and EAB values for films at 20 and 30% glycerol
concentrations.
5.4.2 Water solubility
The water resistance and integrity of a film can be measured by film solubility (Rhim et al., 2000).
Gelatin films are known for their low water resistance because of their hydrophilic nature
(McHugh & Krochta, 1994). Water solubility of gelatin films is shown in Table 5.1. No significant
differences (p > 0.05) were found for the solubility of the films (from 89 to 95%) prepared with
protein concentrations varying from 1 to 7%. The results were consistent with the findings obtained
previously in other fish gelatin films (Carvalho et al., 2008; Hoque et al., 2011; Jiang et al., 2010).
Meanwhile, the water solubility ranged from 81 to 89% for films having a glycerol concentration
ranging from 10 to 50% (of protein); however, the differences observed were also not significant
(p > 0.05). Glycerol, is a hydrophilic plasticizer capable of attracting water to the plasticized
protein system due to the presence of three hydroxyl groups (Sothornvit & Krochta, 2001).
Consequently, the addition of glycerol can increase the hydrophilicity and water solubility of
protein-based films (Cuq, 2002; Nemet et al., 2010). An increase in film solubility was reported
for gelatin-based composite films having a glycerol concentration ranging from 0.2 to 0.8%, but
differences were insignificant (Nur Hanani et al., 2013).
5.4.3 Light barrier properties
The transmission of UV, visible light and opacity of the films at varying protein concentrations
are presented in Table 5.2. As the protein concentration increased from 1 to 7%, the light
transmission decreased and the opacity increased (wavelength from 200 to 800 nm). The lowest
transmission and the highest opacity were recorded for films with the highest protein concentration
(7%). Films with higher protein concentration absorbed light more effectively than those with
lower protein concentration, owing to their greater thickness (Jongjareonrak et al., 2006) and the
presence of more peptide bonds in the gelatin chains (Bao et al., 2009). Meanwhile, noticeable low
values of light transmission (0.1 to 0.3%) accompanied by high opacity (92.8 to 99.9%) were
recorded for all films in the UV light range of 200 to 280 nm (Table 5.2). Higher UV light barrier
capacity was also reported for gelatin films by Jongjareonrak et al. (2006) and Hoque et al. (2011).
These results suggested a possible reduction in UV-induced lipid oxidation when applied to food
88
systems (Gómez-Guillén et al., 2007). Similar to increasing protein concentration, the light
transmission decreased and the opacity increased as the glycerol concentration increased from 10
to 50% (of protein) (Table 5.3). The lowest light transmission with the highest opacity was
recorded for films having a glycerol concentration of 50%. An increase in glycerol concentration
was found to the improve light barrier properties of gelatin films. This is possibly due to the
different diffractive index between gelatin and glycerol (Limpisophon et al., 2009).
Table 5.2 Effects of protein concentration on the light transmission and opacity of salmon skin
gelatin films. 1,2
Protein
concentration 200 nm
280 nm
350 nm
of FFS3 (%)
Light transmission (%T) at different wavelength
0.3 ± 0.0a
7.2 ± 0.1a
75.6 ± 0.1a
1%
a
b
0.3 ± 0.0
4.1 ± 0.1
76.0 ± 0.1a
2%
0.2 ± 0.1a
1.1 ± 0.1c
59.4 ± 0.8b
3%
b
d
0.1 ± 0.0
0.1 ± 0.0
45.8 ± 0.1e
4%
b
d
0.1 ± 0.0
0.2 ± 0.0
50.4 ± 0.0c
5%
b
d
0.1 ± 0.0
0.1 ± 0.0
47.7 ± 0.0d
6%
b
d
0.1 ± 0.1
0.1 ± 0.0
26.2 ± 0.1f
7%
400 nm
500 nm
600 nm
700 nm
800 nm
82.2 ± 0.1a
80.7 ± 0.1b
69.6 ± 1.4c
55.6 ± 0.1f
58.0 ± 0.0d
56.5 ± 0.1e
38.2 ± 0.1g
87.1 ± 0.1a
84.5 ± 0.0b
78.1 ± 2.6c
68.5 ± 0.1d
67.0 ± 0.0d
65.2 ± 0.1e
51.9 ± 0.0f
88.1 ± 0.1a
85.7 ± 0.0b
81.2 ± 2.7c
72.4 ± 0.1d
69.9 ± 0.0e
68.6 ± 0.0e
57.6 ± 0.1f
89.0 ± 0.0a
86.7 ± 0.0b
83.3 ± 2.8c
75.7 ± 0.0d
72.4 ± 0.0e
70.8 ± 0.0e
61.4 ± 0.1f
89.8 ± 0.0a
87.6 ± 0.0b
84.7 ± 3.1c
78.4 ± 0.0d
74.5 ± 0.0e
72.9 ± 0.1e
64.4 ± 0.1f
11.9 ± 0.1f
14.3 ± 0.0e
18.8 ± 2.7d
27.6 ± 0.1c
30.1 ± 0.0b
31.4 ± 0.0b
42.4 ± 0.1a
11.0 ± 0.0f
13.3 ± 0.0e
16.7 ± 2.8d
24.3 ± 0.0c
27.6 ± 0.0b
29.2 ± 0.0b
38.6 ± 0.1a
10.2 ± 0.0f
12.4 ± 0.0e
15.3 ± 3.1d
21.6 ± 0.0c
25.5 ± 0.0b
27.1 ± 0.1b
35.6 ± 0.1a
Opacity4 (%) at different wavelength
99.7 ± 0.0b 92.8 ± 0.1d
24.4 ± 0.1f 17.8 ± 0.1g 12.9 ± 0.1f
1%
b
c
99.7 ± 0.0
95.9 ± 0.1
24.0 ± 0.1f 19.3 ± 0.1f 15.5 ± 0.0e
2%
b
b
99.8 ± 0.1
98.9 ± 0.1
40.6 ± 0.8e 30.4 ± 1.4e 21.9 ± 2.6d
3%
a
a
99.9 ± 0.0
99.9 ± 0.0
54.2 ± 0.1b 44.4 ± 0.1b 31.5 ± 0.1c
4%
99.9 ± 0.0a
99.8 ± 0.0a 49.6 ± 0.0d 42.0 ± 0.0d 33.0 ± 0.0c
5%
a
99.9
±
0.0
99.9 ± 0.0a
52.3 ± 0.0c 43.5 ± 0.1c 34.8 ± 0.1b
6%
a
a
99.9 ± 0.1
99.9 ± 0.0
73.8 ± 0.1a 61.8 ± 0.1a 48.1 ± 0.0a
7%
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
3
FFS means film-forming solution.
4
Opacity (%) = 100% - T (T, transmittance (%) at each wavelength).
89
Table 5.3 Effects of glycerol concentration on the light transmission and opacity of salmon skin
gelatin films with 5% protein. 1,2
Glycerol
concentration 200 nm
280 nm
350 nm
in FFS3 (%)
Light transmission (%T) at different wavelength
10%
0.2 ± 0.0a
5.5 ± 0.1a
80.1 ± 0.0a
a
b
20%
0.2 ± 0.1
2.4 ± 1.8
69.3 ± 8.0b
a
c
30%
0.1 ± 0.0
0.2 ± 0.0
50.4 ± 0.0c
40%
0.1 ± 0.0a
0.1 ± 0.0c
44.5 ± 0.1cd
50%
0.1 ± 0.0a
0.1 ± 0.0c
40.8 ± 0.0d
400 nm
500 nm
600 nm
700 nm
800 nm
84.8 ± 0.0a
77.0 ± 6.1b
58.0 ± 0.0c
57.0 ± 0.1c
55.7 ± 0.0c
88.0 ± 0.0a
83.8 ± 4.6a
67.0 ± 0.0b
67.5 ± 0.1b
66.9 ± 0.0b
89.0 ± 0.0a
85.7 ± 2.9a
69.9 ± 0.0b
68.9 ± 0.0b
68.0 ± 0.0b
89.7 ± 0.0a
82.4 ± 1.8b
72.4 ± 0.0c
72.9 ± 0.0c
71.6 ± 0.0c
90.3 ± 0.0a
88.4 ± 2.2a
74.5 ± 0.0b
74.5 ± 0.0b
73.0 ± 0.0b
11.0 ± 0.0b
14.0 ± 3.5b
30.1 ± 0.0a
31.1 ± 0.0a
32.0 ± 0.0a
10.3 ± 0.0b
17.6 ± 1.8a
27.6 ± 0.0a
27.1 ± 0.0a
28.4 ± 0.0a
9.7 ± 0.0b
11.6 ± 2.1b
25.5 ± 0.0a
25.5 ± 0.0a
27.0 ± 0.0a
Opacity4 (%) at different wavelength
10%
99.8 ± 0.0a 94.5 ± 0.1c 19.9 ± 0.0d 15.2 ± 0.0c
12.0 ± 0.0b
a
b
c
b
20%
99.8 ± 0.1
97.6 ± 1.8 30.7 ± 8.0
22.8 ± 7.5
16.2 ± 4.6b
a
a
b
a
30%
99.9 ± 0.0
99.8 ± 0.0 49.6 ± 0.0
42.0 ± 0.0
33.0 ± 0.0a
a
a
ab
a
40%
99.9 ± 0.0
99.9 ± 0.0 55.5 ± 0.1
43.0 ± 0.1
32.5 ± 0.1a
50%
99.9 ± 0.0a 99.9 ± 0.0a 59.2 ± 0.0a 44.3 ± 0.0a
33.1 ± 0.0a
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
3
FFS means film-forming solution.
4
Opacity (%) = 100% - T (T, transmittance (%) at each wavelength).
5.4.4 Electrophoretic protein patterns
The electrophoretic profiles for all films displayed the presence of α-chains in gelatins but at
different intensities (Fig. 5.1), confirming no excessive hydrolysis by trypsin on gelatin molecules.
It was observed that increasing the protein concentration (from 1 to 7%) produced films with
increased band intensity for the high molecular weight α-chains (α1 and α2-chains). The α-chains
of gelatin can form inter- and intra-molecular crosslinks mainly via hydrogen bonds, producing
gelatin networks which are directly involved in film formation (Galea et al., 2000). Hence, the
increased content of α-chains in films prepared with high protein concentrations probably caused
an increase in the crosslinking density, leading to improved strength and elasticity of the films.
This was evidenced by an increase in the TS and EAB values of the films (Table 5.1). Protein
chains with different molecular weights affect the formation of the film network and resulting
properties (Hoque et al., 2011). A high content in α-chains improves the functional properties (e.g.
viscoelastic properties and gelling strength) of gelatin (Gómez-Guillén et al., 2002). In contrast, a
decrease in high molecular weight protein chains and/or an increase in low molecular weight
protein chains yield weaker film network (e.g. low TS and EAB) (Hoque et al., 2011;
Jongjareonrak et al., 2006).
90
kDa
220
170
α1
α2
116
76
53
HMW
1%
2%
3%
4%
5%
6%
7%
Fig. 5.1 Electrophoretic profile of gelatin films prepared with different protein concentrations (%);
HMW denoted for high molecular weight protein markers.
As shown in Fig. 5.2, there was no difference in protein pattern observed for all films with
increasing glycerol concentration (from 10 to 50% of protein). High molecular weight proteins (αchains) with no difference in their band intensities were observed in all gelatin films at varying
glycerol concentrations. Similar observation was reported for films prepared from blue shark skin
gelatin (Limpisophon et al., 2009) and cuttlefish skin gelatin (Hoque et al., 2011). However, a
decrease in TS values and an increase in EAB values were observed for films prepared with
increasing glycerol concentration (Table 5.1). These results are due to a decrease in intermolecular
interactions between protein chains (Jongjareonrak et al., 2006).
91
kDa
220
170
α1
α2
116
76
53
HMW
10%
20%
30%
40%
50%
Fig. 5.2 Electrophoretic profile of gelatin films containing different glycerol concentrations (%);
HMW denoted for high molecular weight protein markers.
5.4.5 FT-IR spectroscopy
FT-IR spectra for gelatin films prepared with different protein concentrations (1 to 7%) are shown
in Fig. 5.3. Similar spectra were recorded for all films ranging from wavenumbers 1800-600 cm-1,
covering the amide-I, II and III bands. All films displayed major absorption bands at around 1634
cm-1 (amide-I, representing C=O stretching/hydrogen bonding coupled with COO), 1539 cm-1
(amide-II, attributed to the bending vibration of N-H groups and stretching vibrations of C-N
groups), and 1239 cm-1 (amide-III, attributed to the vibrations in plane of C-N and N-H groups of
bound amide or vibrations of CH2 groups of glycine) (Aewsiri et al., 2009; Muyonga et al., 2004).
Arfat et al. (2014) reported similar results for fish gelatin films, where the amide-I, amide-II and
amide-III absorption bands were found at wavenumbers 1633, 1536 and 1238 cm-1, respectively.
In addition, the shift to a higher wavenumber (from 1634 to 1635 cm-1) of amide-I band (Fig. 5.3)
was coherent with the FT-IR spectra displayed for films prepared with increasing gelatin
concentrations (Nur Hanani et al., 2013). The band corresponding to the glycerol was found at
around 1038 cm-1 (Fig. 5.3) (Arfat et al., 2014; Bergo & Sobral, 2007; Hoque et al., 2011).
92
7%
6%
5%
4%
3%
2%
1%
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig. 5.3 FT-IR spectra of gelatin films prepared with different protein concentrations (%).
The FT-IR results showed that the amide-A band at wavenumbers around 3286-3289 cm-1, and the
amide-B band at 2916-2930 cm-1 were present for all films (Fig. 5.3). Arfat et al. (2014) reported
that amide-A and amide-B bands at wavenumbers of 3270-3280 cm-1 and 2926-2928 cm-1
respectively, were observed in all yellow stripe trevally skin gelatin films. Moreover, from Fig.
5.3, as the protein concentration increased from 1 to 7%, an increase in the amplitude of the amideA band and a decrease in the amplitude of the amide-B band were observed, with noticeable
changes for film made from 4% protein concentration. The amide-A band represents the stretching
vibrations of N-H groups, whilst the amide-B band represents the stretching vibrations of CH and
NH3+ groups (Ahmad & Benjakul, 2011; Muyonga et al., 2004). The higher amplitude of amide
bands indicates the higher availability of amino groups, reflecting the lower interaction between
gelatin molecules, and vice versa (Hoque et al., 2011). Meanwhile, the shift of wavenumbers of
amide bands to lower frequencies demonstrates the higher involvement of N-H group in a
hydrogen bond, indicating a higher interaction between the functional groups of peptide chains
(Ahmad et al., 2012; Doyle et al., 1975). Particularly at amide-B region, the shift to lower
93
wavenumber (from 2930 to 2916 cm-1) and lower amplitude of the amide-B band (Fig. 5.3) were
shown for films prepared at increasing protein concentrations from 1 to 7%, suggesting the
increased interaction of -NH3 group between gelatin molecules (Ahmad et al., 2011; Ahmad et al.,
2012).
Thus, the FT-IR results in this study confirmed the influence of protein concentrations in the film
network on the mechanical properties of the resulting films. At increasing protein concentrations,
the noticeable changes of amide bands’ amplitudes and wavenumbers could support the increase
in elasticity (EAB) of the films, particularly films prepared with 4 and 5% protein concentration
(Table 5.1). Furthermore, the FT-IR spectra of films at higher protein concentrations (6 and 7%)
demonstrated higher changes in amplitudes and wavenumbers of amide bands, suggesting that the
excess of a certain threshold amount of protein could lead to the possible extensive protein
intermolecular interactions, which was reflected by the significant increase in TS values and a
decrease in EAB of the films (Table 5.1).
The FT-IR spectra of gelatin films containing glycerol concentrations ranging from 10 to 50% (of
proteins) are shown in Fig. 5.4. Similar to films with increasing protein concentration, major
absorption bands of amide-I, II and III were located at wavenumbers 1634 cm-1, 1539 cm-1, and
1239 cm-1, respectively. The amplitude of the band located at around 1038 cm-1 increased with
increasing glycerol concentration (Bergo & Sobral, 2007; Hoque et al., 2011). This is consistent
with the findings observed on the effect of increasing glycerol content on pigskin gelatin films
(Bergo & Sobral, 2007) and beef skin gelatin films (Nur Hanani et al., 2013). In addition, the
amplitudes of the amide-A band (located at wavenumbers around 3286–3289 cm-1) and the amideB band (located at 2916-2918 cm-1) increased as the glycerol concentration increased in films. An
increase in amplitudes for both amide peaks formed are attributed to the higher availability of the
amino groups, reflecting a decrease in interactions between gelatin chains in the presence of
increased concentrations in glycerol (Hoque et al., 2011). On the other hand, an increase in the
EAB values accompanied by a decrease in the TS values was observed for films with increasing
glycerol concentration (Table 5.1).
94
50%
40%
30%
20%
10%
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1)
Fig. 5.4 FT-IR spectra of gelatin films containing different glycerol concentrations (%).
5.4.6 Morphology
SEM micrographs of the surface of gelatin films prepared with different protein concentrations are
shown in Fig. 5.5. Gelatin films prepared with 1 to 3% protein concentration showed smooth
surfaces, indicating a homogenous structure of films. Rough surface was noticed for films prepared
with 4 to 7% protein concentrations, particularly for films with 6 and 7% protein concentrations.
The roughness and compact structure of the films could be attributed to the increased number of
interactions between the biopolymer chains via covalent and non-covalent bonding (Hoque et al.,
2011; Prodpran et al., 2007). Moreover, the rough surface for films with 6 and 7% protein
concentrations could be indicative of extensive protein intermolecular interactions and cross-links
formation, resulting in films with high mechanical strength and brittleness, as evidenced by their
high TS and low EAB values (Table 5.1).
95
1%
4%
2%
5%
3%
6%
7%
Fig. 5.5 SEM micrographs (at 1000x magnification) of surface of salmon gelatin films prepared
with different protein concentrations (%).
96
SEM micrographs of the surface of gelatin films having different glycerol concentrations are
shown in Fig. 5.6. Smooth surface was observed for films having 10 and 20% glycerol
concentration. Meanwhile, protein chains organization was more pronounced on the surface of
films when the glycerol content increased from 30 to 50%, with a more ordered arrangement for
glycerol concentrations of 40 and 50%. Interactions between small molecular weight compounds
and gelatin produced uncoiled and elongated protein chains (Shakila et al., 2012). Consequently,
higher glycerol concentrations increased gelatin molecules’ elongation and mobility, contributing
to an increased elasticity of films as evidenced by lower TS and higher EAB values (Table 5.1).
97
10%
30%
20%
40%
50%
Fig. 5.6 SEM micrographs (at 1000x magnification) of surface of salmon gelatin films containing
different glycerol concentrations (%).
98
5.5 Conclusion
Properties of films prepared using salmon gelatin extracted by a trypsin-aided process were studied
at different protein and glycerol concentrations. The TS and EAB values of films increased with
an increase in protein concentration from 1 to 5%. However, the EAB value reduced markedly for
films with 6 and 7% protein concentrations, indicating the possible extensive protein
intermolecular interactions and cross-links formation, plausibly attributed to the excess of a certain
threshold amount of protein. Meanwhile, the decrease in the TS coupled with the increase in the
EAB values was affected by the increased plasticizing effect as the concentration of glycerol
increased. The increasing protein and glycerol concentrations had no effect on the water solubility
for all films, but a decrease light transmission accompanied by an increase in opacity were
observed. The electrophoretic study displayed the presence of α-chains that confirmed no
hydrolysis by trypsin on gelatin molecules, and the increased in mechanical properties was
attributed to the increased content of high molecular weight chains in gelatin as the concentration
of protein increased. Meanwhile, the FT-IR spectra and morphological analysis revealed the
interaction behavior between protein chains and protein chain with glycerol as the protein and
glycerol content increased in films.
99
CONNECTING STATEMENT 4
The characterization of salmon skin gelatin films (Chapter V) revealed their properties at varying
protein and glycerol concentrations. It was demonstrated that the gelatin films prepared with 5%
protein and plasticized with 30% glycerol exhibited good tensile strength and elasticity, as
compared to gelatin films prepared with higher protein concentrations which were hard and brittle.
However, these films were highly water soluble (Chapter V). Chapter VI explored the effect of the
addition of corn zein in order to improve the water barrier properties of salmon gelatin films.
Subsequently, the gelatin-zein composite film formulation was further investigated for the effect
of canola oil in replacement of glycerol as plasticizer. The mechanical, water barrier and light
barrier properties, as well as structural and morphological properties of the resultant films, were
determined and discussed in this Chapter.
The results of this study have been prepared for publication as:
Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Synthesis and characterization of salmon skin
gelatin-corn zein composite films plasticized with canola oil.
100
CHAPTER VI. SYNTHESIS AND CHARACTERIZATION OF SALMON
SKIN GELATIN-CORN ZEIN COMPOSITE FILMS
PLASTICIZED WITH CANOLA OIL
101
6.1 Abstract
Composite films were prepared by blending gelatin (5%, w/v) and zein (1, 3, 5%, w/v) at a ratio
of 1:1 using an ethanol-water mixture (1:1, v/v). The films were plasticized with glycerol (30%,
w/w of protein) or canola oil (10 – 30%, w/w of protein). As the zein concentration increased, the
resultant films showed an increase in the film thickness, a decrease in tensile strength (TS) and
elongation at break (EAB), and a decrease in water solubility. Gelatin blended with 5% zein had
the highest thickness value and the lowest TS, EAB and water solubility values (p < 0.05). Gelatinzein composite films plasticized with 30% of canola oil were the thickest and exhibited the lowest
TS, EAB, water solubility, water vapor permeability (WVP) and light transmission values. Fourier
transform infrared (FT-IR) spectra and polarized light microscopy (PLM) images revealed the
protein-lipid interactions of the composite films. The data obtained indicate that the concentration
of zein and canola oil used directly affected the physical and mechanical properties of the gelatin
films.
Keywords: fish gelatin, zein, glycerol, canola oil, composite films
102
6.2 Introduction
Gelatin is a natural biopolymer obtained from pretreated collagen via thermal hydrolysis (Kim &
Mendis, 2006). As an alternative to mammalian gelatin, fish gelatin extracted from fish bones and
skins has gained importance in recent years. For example, fish gelatin has attracted increased
attention for the development of edible films for food packaging and preservation due to its good
film forming ability, high transparency and gas barrier properties (Chiou et al., 2008). However,
gelatin films have poor water vapor barrier properties, attributed to their high content in
hydrophilic amino acids, limiting their potential for food packaging applications (Chiou et al.,
2008; Denavi et al., 2009; Hoque et al., 2010; Jongjareonrak et al., 2006).
The mechanical and barrier properties of gelatin films are highly influenced by the microstructure
of gelatin, which varies depending on their structure and inter / intra molecular interactions
(Denavi et al., 2009). Specifically, the amino acid composition and molecular weight of the gelatin
protein molecules play significant roles in the stabilization of a protein film matrix (Denavi et al.,
2009). Therefore, films prepared by blending proteins of different structures could complement
the advantages of each protein, forming synergetic effects that can achieve the targeted functional
properties (Galus & Kadzińska, 2015). Blending compatible protein polymers to form composite
films has been regarded as an effective approach to modify the properties of protein-based films
(Wang et al., 2009). Improved mechanical and water barrier properties of films were reported when
cod skin gelatin was blended with soy protein isolate (Denavi et al., 2009). An increase in
elongation at break, improvement in thermal stability and a decrease in water vapor permeability
and solubility were observed for films made from cuttlefish skin gelatin blended with mungbean
protein isolate (Hoque et al., 2011).
Zein, the major storage protein in corn endosperm, has been actively studied for its film forming
ability (Gu & Wang, 2013; Lawton, 2004; Panchapakesan et al., 2012; Xu et al., 2012). Zein is
regarded as one of the most hydrophobic proteins due to its high content in hydrophobic amino
acids, such as leucine, proline, alanine, and phenylalanine (Holding & Larkins, 2009). Generally,
zein films exhibit good water barrier properties (Cho et al., 2002). Therefore, the addition of zein
in a gelatin matrix could improve the water vapor barrier properties of the blended product.
In general, protein films require the addition of plasticizers to overcome film brittleness. Among
plasticizers, glycerol has been widely used in the formulation of both gelatin and zein films (Bergo
103
& Sobral, 2007; Guo et al., 2008). However, glycerol is a hydrophilic molecule which contributes
to increase water permeability of films (Andreuccetti et al., 2009). To improve the hydrophobicity
of films, lipids can be used as plasticizer alongside with glycerol. Several studies investigated the
effect of the inclusion of hydrophobic plasticizer, such as essential oils (Ahmad et al., 2012),
sunflower oil (Pérez-Mateos et al., 2009), and palm oil (Xiao et al., 2016), in gelatin based films,
as well as oleic acid (Xu et al., 2012) and olive oil (Ghanbarzadeh & Oromiehi, 2009) in zein films.
Sunflower lecithin was added to all composite films as an emulsifier to improve the homogeneity
of the films (Dickinson, 2003).
To date, there is no information reported on the properties of composite films prepared using blend
of gelatin from salmon skin and corn zein. In this study, gelatin extracted from Atlantic salmon
(Salmo salar) skin was blended with zein to study the mechanical properties and water resistance
of the composite films. Thereafter, the composite films were plasticized with glycerol and canola
oil in different ratios to investigate the effects of plasticizers on the mechanical, water resistance,
light barrier and morphological properties of the films.
6.3 Materials and methods
6.3.1 Materials
Atlantic salmon (Salmo salar) skins were obtained from a local fish market (Jean-Talon, Montreal,
Canada). Zein was purchased from Sigma Chemical Co. (St. Louis, MO, USA); porcine pancreas
trypsin (EC 3.4.21.4; powdered; 90.97 U/mg) was obtained from ICN Biomedicals Inc. (Ohio,
USA); a commercial brand of canola oil was purchased from a local store (Loblaws Inc, Toronto,
Canada); non-genetically modified sunflower liquid lecithin was purchased from Now Foods
Company (Bloomingdale, IL, USA); sodium chloride (NaCl) was purchased from BDH Inc.
(Toronto, Ontario, Canada); anhydrous ethanol was purchased from Commercial Alcohol Inc.
(Brampton, Ontario, Canada); anhydrous calcium chloride, glycerol, potassium carbonate and Tris
base were purchased from Fisher Scientific (Fair Lawn, NJ, USA).
6.3.2 Extraction of salmon skin gelatin
Fish skins were obtained from the fish market and washed with tap water. The skins were cut into
1.5 x 1.5 cm2 pieces with scissors and degreased by tumbling in warm (35 °C) water (Muyonga et
al., 2004). The skins were treated with 0.45 M NaCl at 4 °C for 3 min, and washed with distilled
104
water (Rahman et al., 2008). Our previous study demonstrated that the optimum yield of gelatin
was obtained with the following extraction parameters: the skins were soaked in 50mM tris-HCl
buffer (pH 8.0) in the presence of trypsin at 1.49 U/g, and stirred continuously at room temperature
(22-25 °C) for 5 h, then filtered with a Whatman No. 4 filter paper and washed with distilled water.
Gelatin was extracted by gently stirring the mixture of pretreated skins and distilled water using a
shaking water bath (model 25, Precision Scientific, USA) at 45 °C for 6 h 15 min. The skin/solution
ratio was 1:6 (w/v) throughout the process. The protein solutions were then centrifuged (7000 g,
15 °C, 30 min) and the supernatant was lyophilized using a freeze dryer (Modulyod-115,
ThermoSavant, Holbrook, NY, USA) at -50 °C and 120 mBar for 48 h. The lyophilized proteins
were referred as ‘gelatin powder’ and were stored at -20 °C.
6.3.3 Preparation of gelatin-zein composite films
Preliminary experiments showed that both gelatin and zein dissolved without any precipitation in
aqueous ethanol solution (1:1, v/v). Gelatin solution (5% of protein) was prepared by dissolving 5
g (of protein) in 100 ml aqueous ethanol under continuous stirring for 30 min at room temperature
(22-25 °C). Similarly, zein solution (1%, 3%, 5% of protein) was dissolved in aqueous ethanol by
stirring for 30 min at room temperature. The gelatin-zein film forming solutions (FFS) (20 ml)
were prepared by mixing gelatin solution (5%) with zein solution (1%, 3%, 5%) in a proportion of
1:1 (v/v). Glycerol was added as plasticizer (30% of protein of FFS) to all the gelatin-zein FFS
and maintained under magnetic stirring for 30 min. A vacuum pump was used to reduce air bubbles
formation. The gelatin-zein FFS obtained were dispersed into polystyrene petri dishes and were
placed on a leveled surface in a fume hood and dried for a period of 48 h at room temperature. The
films were conditioned in a desiccator at 23 ± 2 °C containing saturated solutions of potassium
carbonate (50 ± 2% relative humidity, RH) for at least 40 h before mechanical properties and water
solubility testing.
6.3.4 Preparation of gelatin-zein composite films synthesized with canola oil and lecithin
Gelatin solution (5% of protein) and zein solution (5% of protein) were prepared as previously
described. The gelatin-zein FFS (20 ml) were prepared by mixing gelatin and zein solutions in a
proportion of 1:1 (v/v). Glycerol and canola oil were used as plasticizers at a final concentration
of 30% (w/w of protein), at different glycerol:canola oil ratios (30:0; 20:10; 15:15; 10:20; 0:30%,
105
w/w). Lecithin was added to canola oil (30%, w/w of canola oil) as emulsifier. Films prepared
without canola oil were used as controls.
After mixing the gelatin and zein solutions, glycerol was added to the gelatin-zein FFS and
maintained under magnetic stirring for 30 min. Thereafter, the blend of canola oil and lecithin was
incorporated into the FFS. Each mixture was then homogenized at 20,000 rpm for 2 min using a
homogenizer (Polytron PT-MR 300, Brinkmann Instruments, NY, USA), and vacuum-filtered to
reduce air bubbles formation. All homogenized mixtures obtained were dispersed into polystyrene
petri dishes and were placed on a leveled surface in a fume hood where they dried for a period of
48 h at room temperature. The films were conditioned in a desiccator 23 ± 2 °C containing
saturated solutions of potassium carbonate (50 ± 2% relative humidity, RH) for at least 40 h before
characterization.
6.3.5 Film characterization
6.3.5.1 Film thickness
Film thickness was measured with a hand-held digital micrometer (Marathon Part No. 030025,
Marathon Watch Company Ltd., Ontario, Canada) with an accuracy of 0.002 mm. Six
measurements were taken at random positions for each film, and the averages were taken as the
results.
6.3.5.2 Mechanical properties
The TS and EAB values were determined according to ASTM method D 882-10 using an Instron
Universal Testing Machine (model 4500, Instron Corporation, Canton, MA, USA). The specimen
was fixed on the grips of the device with an initial grip separation of 30 mm, and pulled apart at a
mechanical crosshead speed of 10 mm/min and preload of 2 N. At least five replicates were tested
for each film formulation and the averages were taken as the results. TS (MPa) and EAB (%) were
calculated by the following equations:
TS (MPa) = F max / A
(9)
where F max = maximum load (N) needed at the moment of rupture, A = cross-sectional area (m2)
of the samples.
106
𝐸𝐸
(10)
EAB (%) = �30� 𝑥𝑥 100
where E = film elongation (mm) at the moment of rupture, 30 = initial grip length (mm) of samples.
6.3.5.3 Film solubility
The water solubility of the films was determined according to the method of Shakila et al. (2012).
Films of surface area of 4 cm2 were cut and weighed (± 0.0001 g) to determine the initial weight
(Wo). Each film portion was immersed separately in 15 ml of distilled water, gently shaken at room
temperature (22-25 °C) for 15 h and then filtered through a Whatman No. 1 filter paper. The
unsolubilized film fraction collected on the filter paper was dried in a hot air oven at 105 °C for
24 h and weighted (Wf). Three replicates were tested for each film and the averages were taken as
the results. The solubility of the film was calculated by the following equation:
Solubility (%) = �
𝑊𝑊𝑜𝑜 − 𝑊𝑊𝑓𝑓
𝑊𝑊𝑜𝑜
(11)
� 𝑥𝑥 100
where Wo = initial weight of the film specimen, Wf = weight of unsolubilized film fraction.
6.3.5.4 Water vapor permeability (WVP)
WVP of films was measured gravimetrically in accordance with the ASTM E96/E96M with
modification as described by Pérez-Mateos et al. (2009). A circular portion of the films was cut
and sealed on the open mouth of a plastic cup containing silica gel with silicone sealant (High
Vacuum Grease, Dow Corning, Midland, Michigan, USA). The cup was placed in desiccators
filled with distilled water at the bottom. The cup was weighed every hour for 8 h. At least three
replicates of each film type were tested for WVP. WVP of film was calculated by the following
equation:
𝑊𝑊𝑊𝑊𝑊𝑊 =
𝑤𝑤 𝑥𝑥
(12)
𝑡𝑡𝑡𝑡 ∆𝑃𝑃
where w = weight gain of the cup (g), x = film thickness (mm), t = time of gain (h), A = permeation
area (cm2), and ∆P = difference of partial vapor pressure of the atmosphere with silica gel and pure
water (2642 Pa, at 22 °C). Results were expressed as g mm h-1 cm-2 Pa-1.
107
6.3.5.5 Light transmission
The barrier properties of films against ultraviolet (UV) and visible light were measured at selected
wavelengths (200 – 800 nm) using an UV/Vis spectrophotometer, according to the method of Fang
et al. (2002). The films were cut into rectangular pieces (12 x 43 mm), directly placed into a quartz
cuvette and measured. An empty cuvette was used as the blank. The test was performed in triplicate
for each film and the averages were taken as the results. Light transmission (T) was recorded using
transmittance (%) measured at each wavelength for each film.
6.3.5.6 Fourier transform infrared (FT-IR) spectra analysis
Infrared spectra of gelatin-zein composite films were recorded using a Nicolet iS5 FT-IR
spectrometer (Thermo, Madison, WI, USA). Films were placed onto the crystal cell and the cell
was clamped into the mount of a FT-IR spectrometer. The spectra were recorded in duplicate at
32 scans and 4 cm-1 resolutions in the range of 4000-400 cm-1. The spectra were analyzed using
the OMNIC 8.2 software package (Thermo Fisher Scientific Inc., USA).
6.3.5.7 Polarized light microscopy
The morphology of the films was observed with a Nikon Eclipse LV100POL polarized light
microscope (PLM). The images were captured with a DS-Fi1camera (Nikon, Tokyo, Japan). The
observations were made at a magnification of 5x.
6.3.5.8 Statistical analysis
The linear regressions used for the calculation of WVP (R2 > 0.98) were performed using Excel
2016 software (Microsoft, Seattle, WA). Data were statistically analysed using the General Linear
Models procedure of SAS (Release 9.4, SAS Institute Inc., Cary, NC, USA) software. Mean
comparisons were carried out by Duncan’s multiple range test (p < 0.05) (Steel & Torrie, 1980).
6.4 Results and discussion
6.4.1 Effect of zein concentration on the properties of gelatin-zein composite films
6.4.1.1 Thickness
Film thickness of the gelatin-zein composite films increased from 0.11 to 0.16 mm with increasing
zein concentrations, as shown in Table 6.1. The thickness values of the films containing 5% zein
108
were significantly higher (p < 0.05) than the other films. Gelatin films without zein had the lowest
thickness value. The addition of zein increased the thickness of the films, indicating the higher
degree of disruption occurring on the alignment of the protein molecules in gelatin or between
gelatin and zein. Similar conclusion was drawn for films prepared from cuttlefish skin gelatin
containing higher proportion of mungbean protein isolate (Hoque et al., 2011).
Table 6.1 Thickness, tensile strength (TS), elongation at break (EAB) and water solubility of
salmon skin gelatin films blended with zein at different concentrations.1,2
Film samples
Corn
zein Thickness (mm) TS (MPa)
EAB (%)
concentration
(% of protein)
Control
0
3.65 ± 0.49a 208.4 ± 33.8a
0.11 ± 0.02c
Composite film
1
3.32 ± 0.35a 78.3 ± 11.1b
0.12 ± 0.02bc
3
3.31 ± 0.77a 58.9 ± 13.7b
0.14 ± 0.01b
5
1.81 ± 0.05b 26.5 ± 9.1b
0.16 ± 0.01a
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
Water
(%)
solubility
92.6 ± 1.2a
88.3 ± 3.6a
74.6 ± 4.2b
65.5 ± 6.1c
6.4.1.2 Mechanical properties
As shown in Table 6.1, the TS and EAB values decreased from 3.65 to 1.81 MPa and from 208.4
to 26.5%, respectively, as the zein concentration increased from 0 to 5%. The gelatin-zein
composite film containing 5% zein exhibited the lowest TS and EAB values (p < 0.05). Zein films
are known for their brittleness and poor TS and EAB due to the strong molecular forces between
the zein protein molecules (Xu et al., 2012). Zein film formation involves interaction between zein
molecules through hydrophobic, hydrogen and sulfide bonds (Singh et al., 2012). Fish gelatin
forms highly extensible films with good mechanical properties (Cao et al., 2007). During the film
forming process, gelatin can renature and re-acquire part of the triple helix structure of the collagen
by hydrogen bonds (Cao et al., 2007; Galea et al., 2000). The results from this study indicate that
blending zein into gelatin films probably hindered the renaturation of the gelatin. This led to the
formation of film networks with a reduced degree of organization, resulting in decreased
mechanical properties as compared to the film made from gelatin alone. The results were consistent
with the findings reported for the incorporation of soy protein isolate into bovine gelatin films
(Cao et al., 2007) and casein into bovine skin gelatin films (Chambi & Grosso, 2006).
109
6.4.1.3 Film solubility
Table 6.1 shows that gelatin films prepared without zein had the highest solubility (92.6%) in
water. Gelatin is known for its poor water resistance due to its high amount of hydrophilic amino
acids (Denavi et al., 2009; Hoque et al., 2010; Jongjareonrak et al., 2006). The solubility decreased
from 88.3% to 65.5% (p < 0.05) at increasing concentrations of zein in the composite films. Zein
is regarded as a hydrophobic protein as it contains a high proportion of non-polar amino acid
residues (Holding & Larkins, 2009). Thus, the lower solubility of films with increasing
concentrations of zein in the composite films indicated that interactions between zein and gelatin
proteins occur in the film matrix. Similar findings were also reported from Hoque et al. (2011) for
cuttlefish skin gelatin film blended with mungbean protein isolate of higher hydrophobicity.
6.4.2 Effect of glycerol/canola oil ratio on the properties of gelatin-zein composite films
6.4.2.1 Thickness
The thickness of gelatin-zein films increased from 0.16 to 0.23 mm as the concentration of canola
oil increased (Table 6.2). The films plasticized with 30% canola oil had the highest thickness (0.23
mm, p < 0.05). Lipids contribute to the solid content of the films and can interfere with the
interaction and alignment of protein molecules in the matrix. Hence, the compact and ordered film
network can be disrupted and expanded, leading to increased thickness (Tongnuanchan et al.,
2015).
Table 6.2 Thickness, tensile strength (TS) and elongation at break (EAB) of gelatin-zein composite
films incorporated with canola oil at various concentrations.1,2
Film samples
Thickness (mm)
TS (MPa)
EAB (%)
(% canola oil)
1.81 ± 0.05a
26.5 ± 9.1a
0.16 ± 0.01d
Control
1.50 ± 0.91ab
24.9 ± 5.0a
0.19 ± 0.01c
10
bc
ab
0.20 ± 0.01
1.33 ± 0.28
12.8 ± 8.6b
15
0.20 ± 0.01b
1.30 ± 0.51ab
1.1 ± 0.6c
20
0.23 ± 0.02a
0.84 ± 0.29b
0.3 ± 0.2c
30
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
110
6.4.2.2 Mechanical properties
As the concentration of canola oil increased, the TS and EAB values decreased (Table 6.2). The
lowest TS and EAB values were obtained for films plasticized with 30% canola oil (p < 0.05). The
dispersion of oil could have interfered with the protein-protein interactions, leading to the
discontinuity of the film matrix and a reduced cohesive structure of the film network, resulting in
a decrease in the strength of the films (Prodpran et al., 2007; Tongnuanchan et al., 2015). Moreover,
the increase in film thickness might have decreased the extensibility of the films to some degree
(Tongnuanchan et al., 2015). In a study on cod skin gelatin plasticized with sunflower oil, an
increased amount of oil in the film matrix had induced a decrease in the puncture force and
percentage puncture deformation of the resulting films (Pérez-Mateos et al., 2009). A decrease in
TS with an increase in EAB values were reported with the addition of palm oil to certain
concentrations in gelatin films, according to the studies by Tongnuanchan et al. (2015) and Xiao
et al. (2016). Nevertheless, these studies also suggested that increased concentration of palm oil
could be responsible for the decreased in EAB due to an increase in the film thickness.
According to this study, glycerol has better plasticizing effect attributed to its lower molecular
mass, as compared to oil which has a higher molecular weight (i.e. long chain fatty acids). As a
result, the incorporation of oil probably weakened the film structure and produced films with lower
mechanical properties, as evidenced by the lower TS and EAB values (Table 6.2) of films.
6.4.2.3 Film solubility
As shown in Table 6.3, gelatin-zein composite films plasticized with canola oil were less soluble
in water than the films prepared without canola oil. As expected, films plasticized with glycerol
had the highest solubility (65.5%, p < 0.05), mainly attributable to the hydrophilic character of
glycerol. The films solubility decreased from 55.3 to 47.4% with increasing concentrations of
canola oil and decreasing concentrations of glycerol (Table 6.3). The lowest solubility (47.4%, p
< 0.05) was obtained from the films with the highest concentration of canola oil. This indicated
that the incorporation of canola oil could increase the hydrophobicity of the films and to lower
water absorption, resulting in lower water solubility. This is consistent with the conclusions drawn
by Ahmad et al. (2012) and Ghasemlou et al. (2013), where the interaction of hydrophobic
substances with hydrophobic protein domain in film matrices decreased the hydrophilicity of the
111
resulting films. Additionally, this might be due to the emulsifying effect of lecithin added in the
films, since lecithin could stabilize and facilitate uniform dispersion of the oil droplets in the film
network (Dickinson, 2003), benefiting the water-resistant properties of the resulting films.
Table 6.3 Water vapor permeability (WVP) and water solubility of gelatin-zein composite films
incorporated with canola oil at various concentrations.1,2
Water solubility
Water vapor permeability
(g mm h-1 cm-2 Pa-1)
(%)
0.451 ± 0.006a
65.5 ± 6.1a
Control
55.3 ± 4.5bc
0.445 ± 0.030a
10
0.443 ± 0.012a
57.5 ± 4.7ab
15
0.437 ± 0.019a
55.9 ± 1.0b
20
0.397 ± 0.010b
47.4 ± 4.2c
30
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
Film samples
(% canola oil)
6.4.2.4 Water vapor permeability
The highest WVP value was obtained for the gelatin-zein composite films (0.451 g mm h-1 cm-2
Pa-1), as shown in Table 6.3. This is due to the presence of glycerol which induced a decrease in
intermolecular force and an increased in the mobility of the protein chains. This led to an increase
in free volume within the film matrix, resulting in greater migration of water vapor molecules
through the film (Rodríguez et al., 2006). Films with canola oil had lower WVP values than the
film without canola oil. WVP decreased from 0.451 to 0.437 g mm h-1 cm-2 Pa-1 with increasing
concentrations of canola oil and decreasing concentrations of glycerol (Table 6.3). The lowest
WVP value (0.397 g mm h-1 cm-2 Pa-1, p < 0.05) was obtained from the film plasticized with canola
oil only. The incorporation of nonpolar or hydrophobic oils into a film matrix can increase the
film’s hydrophobicity, thus reducing permeation and adsorption of water vapor (Tongnuanchan et
al., 2015). Furthermore, the significant reduction of WVP of the films might be due to the uniform
distribution of oil droplets with the presence of lecithin as surfactant (Tongnuanchan et al., 2014).
6.4.2.5 Light transmission
Regardless of the concentration of canola oil, all gelatin-zein films exhibited very low light
transmission at UV light ranging from 200 to 280 nm, and increased light transmission at visible
112
light ranging from 350 to 800 nm (Fig. 6.1). Among all films, films without canola oil (control
film) showed greater light transmission for both UV and visible light. As the canola oil
concentration increased from 10 to 30%, the light transmitted through the resulting films decreased
(wavelength from 200 to 800 nm). The lowest transmission was recorded for films with the highest
canola oil concentration (30%) at both UV and visible light ranges. Films with lipids were more
opaque or turbid due to the light scattering effect from the distribution of the oil droplets in the
film matrix and the disrupted film network (Tongnuanchan et al., 2015; Yang & Paulson, 2000).
Thus, oil-containing films exhibited high opacity, depending on concentration and distribution of
oil in the films’ matrix, as well as the interaction between the films’ constituents.
10.0
Control
9.0
10%
Light transmission (%)
8.0
15%
20%
7.0
30%
6.0
5.0
4.0
3.0
2.0
1.0
0.0
200
280
350
400
500
Wavelength (nm)
600
700
800
Fig. 6.1 Light transmission of gelatin-zein composite films incorporated with canola oil at various
concentrations.
113
6.4.2.6 FT-IR spectroscopy
As shown in Fig. 6.2, similar spectra were recorded for the control (gelatin-zein) film and films
containing canola oil, but the amplitude of the peaks varied based on the concentration of canola
oil added. All films displayed major absorption bands in the range of 1800-600 cm-1, covering
amide-I at around 1639 cm-1 (amide-I, representing C=O stretching/hydrogen bonding coupled
with COO), amide-II at around 1536 cm-1 (amide-II, attributed to the bending vibration of N-H
groups and stretching vibrations of C-N groups), and amide-III bands at around 1237 cm-1 (amideIII, attributed to the vibrations in plane of C-N and N-H groups of bound amide or vibrations of
CH2 groups of glycine) (Aewsiri et al., 2009; Muyonga et al., 2004). Compared to the control film,
all the oil-containing films presented lower amplitude of amide-I, II and III peaks. Moreover, a
decrease in amplitude was observed when increasing the concentration of canola oil from 10 to
30% (Fig. 6.2). This was due to the lower protein content in the matrix (Tongnuanchan et al., 2015).
Control
10%
15%
20%
30%
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1)
Fig. 6.2 FT-IR spectra of gelatin-zein composite films incorporated with canola oil at various
concentrations.
114
Based on the FT-IR spectra (Fig. 6.2), the absorption band at wavenumber of 1038-1043 cm-1
corresponds to the asymmetric stretching vibrations of -OH group of glycerol (Bergo & Sobral,
2007). The absorption bands at the wavenumbers of 2923 and 2853 cm-1 represents the methylene
asymmetrical and symmetrical stretching vibration of the aliphatic C-H in CH2 and CH3 groups,
respectively, indicating the addition of lipids (Guillen & Cabo, 1997). Additionally, a band at
wavenumber 1742 cm-1 was observed in films containing canola oil, but not for the control film,
suggesting the presence of C=O stretching vibration of aldehyde or ester carbonyl groups in canola
oil (Tongnuanchan et al., 2014). The amplitude of the band corresponding to the glycerol was
lower and the amplitude of the bands for canola oil was higher, confirming the replacement of
glycerol with increasing concentrations of canola oil in the matrix (Ahmad et al., 2012; Bergo &
Sobral, 2007). This mainly resulted in the loss of film elasticity as evidenced by a decrease in TS
and EAB values (Table 6.2), and the increase in hydrophobicity of the films as shown by a decrease
in films’ solubility and WVP (Table 6.3).
As shown in Fig. 6.2, amide-A (arising from the stretching vibration of N-H group) and amide-B
(corresponding to stretching vibrations of CH and NH3+ groups) bands were observed at
wavenumber of 3287-3289 cm-1 and 3064-3089 cm-1 respectively, for all film samples (Kong &
Yu, 2007). Interactions between the functional groups of proteins and the lipids could be observed
through the changes in wavenumber and amplitudes of amide-A and amide B peaks (Bahram et
al., 2014). Lower wavenumber of amide-A peak indicated higher hydrogen bonding between the
protein molecules (Xie et al., 2006). From the results, however, the amide-A peaks were gradually
shifted to higher wavenumbers, from control film at wavenumber of 3287 cm-1 to 3088, 3088, 3085,
3089 cm-1 for films having 10, 15, 20, and 30% canola oil, respectively. The amplitude of amideA bands decreased as the concentration of canola oil increased (Fig. 6.2). The higher wavenumbers
and lower amplitudes of amide-A peak indicated the disruption of the protein-protein interactions
in network as the concentration of canola oil increased (Tongnuanchan et al., 2014). Similarly,
amplitudes of the amide-B peaks were lower as the concentration of canola oil increased (Fig. 6.2),
indicating an increase in hydrophobic interactions between the -CH of the protein molecules and
canola oil (Tongnuanchan et al., 2015). Compared to the control film, when canola oil was added
to the films, the wavenumber at amide-B region shifted to lower values. The amide-B peak of the
control film was found at wavenumber of 3089 cm-1, and shifted from 3086 to 3064 cm-1 as the
concentration of canola oil increased (Fig. 6.2). Similar changes on the amide-B region were
115
reported by Ahmad et al. (2012) for fish gelatin-based films plasticized with essential oils,
suggesting the interaction between peptide chains.
6.4.2.7 Polarized light microscopy
Comparing the PLM images with the control film (Fig. 6.3a), the films synthesized with canola oil
showed dispersed droplets within the proteins (Fig. 6.3b). The dispersion of canola oil within the
film matrix indicated that lecithin could help in the distribution and stabilization of the oil
(Tongnuanchan et al., 2014). However, as the concentration of canola oil increased to 30%, the
oil migrated to the surface of the films (Fig. 6.3c). Similar phenomenon was reported by
Tongnuanchan et al. (2015) for gelatin films having increasing concentrations of palm oil. As a
result, the presence of canola oil on top layer of films increased the hydrophobicity of the resulting
films, and served as barrier to water vapor (Table 6.3).
116
a
Canola oil droplets
b
c
Canola oil migration
Fig. 6.3 Polarized light microscopy (at 5x magnification) of gelatin-zein composite films
incorporated with canola oil at various concentrations: (a) control film (without canola oil), (b)
15%, and (c) 30%.
117
6.5 Conclusion
This study showed that as the zein concentration increased in gelatin-zein composite films, the
mechanical properties of the films decreased (lower TS and EAB values). Nevertheless, since zein
proteins are hydrophobic, their addition contributed to the lower solubility of the films.
The addition of canola oil in replacement of glycerol as plasticizer affected the properties of
gelatin-zein composite films. At increasing concentrations of canola oil, the TS and EAB values
decreased, suggesting that the dispersion of the oil droplets had reduced the cohesiveness of the
structure. Nevertheless, the addition of canola oil increased the hydrophobicity as evidenced by
the lower solubility in water and the WVP values. The FT-IR spectra and PLM images revealed
that protein-lipid interactions were possibly responsible for the poorer mechanical properties but
improved water and light barrier properties of the films. Therefore, the use of zein and canola oil
effectively enhanced the water and light barrier properties of salmon gelatin films, but further
investigation is needed to improve the mechanical performance of these composite films.
118
CONNECTING STATEMENT 5
Incorporation of corn zein and canola oil increased the water resistance and water vapor barrier
capabilities of salmon skin gelatin films; however, the mechanical strength and elasticity of the
resultant films were reduced, particularly with increased content of canola oil (Chapter VI). Based
on the results alluded to above, there was interest to explore the cross-linking induced by
glutaraldehyde for improving the mechanical properties and water resistance of gelatin-zein
composite films. Chapter VII is focused on the formation of glutaraldehyde-crosslinked gelatinzein composite films using a 2-factors 5-levels central composite design (CCD) by response
surface methodology (RSM). The effects of zein and glutaraldehyde content are discussed, and the
optimum conditions that maximize the tensile strength and elongation at break, while minimizing
the water solubility of the films were determined.
The results of this study have been submitted to the International Journal of Biological
Macromolecules as:
Fan, H.Y., Dumont, M.J. & Simpson, B.K. (2018). Salmon skin gelatin-corn zein composite films
produced via crosslinking with glutaraldehyde: optimization using response surface methodology
and characterization. International Journal of Biological Macromolecules, 120, 263-273.
119
CHAPTER VII. SALMON SKIN GELATIN-CORN ZEIN COMPOSITE FILMS
PRODUCED VIA CROSSLINKING WITH GLUTARALDEHYDE:
OPTIMIZATION USING RESPONSE SURFACE METHODOLOGY
120
7.1 Abstract
Composite films comprised of salmon (Salmo salar) skin gelatin and zein were prepared via
crosslinking with glutaraldehyde. Response surface methodology (RSM) was used to optimize
film composition to maximize tensile strength (TS) and elongation at break (EAB), and to
minimize water solubility (WS) of the films. The significant (p < 0.05) variables affecting film
properties were: glutaraldehyde for TS, and zein and glutaraldehyde for both EAB and WS. The
optimum concentrations (g/ml) to maximize TS and EAB and to minimize WS were 3% zein and
0.02% glutaraldehyde, which yielded films TS of 3.11 ± 0.01 MPa, EAB of 22.43 ± 1.57%, and
WS of 38.82 ± 1.71%.
Keywords: salmon skin gelatin, zein, crosslinking, composite film, response surface methodology
121
7.2 Introduction
In recent years, attention has been focused on biodegradable films produced from processing
discards as packaging materials for food and related applications. Common bio-based materials
being studied include carbohydrates, proteins, and lipids (Falguera et al., 2011). Protein-based
films generally are more superior than polysaccharide-based films owing to their structure with
specificities (based on 20 different amino acids) and abilities in forming stronger intermolecular
covalent bonds (Cuq et al., 1995). Proteins are known for their good film forming ability, high
transparency, extensibility and high gas barrier properties (Chiou et al., 2008). However, they also
exhibit poor water barrier and inferior mechanical properties when compared with synthetic
polymers (such as polystyrene and polyethylene terephthalate), which limit their potential
applications (Falguera et al., 2011; Siracusa et al., 2008).
Gelatin from fish skin protein (collagen) has received attention in film production due to its
abundance and excellent film-forming properties (Karim & Bhat, 2009). Several strategies have
been employed to improve fish gelatin film properties. One of the most common strategies is by
blending it with other biopolymers to form composite films that incorporate the advantages of the
constituent biopolymers, and minimize their individual disadvantages (Galus & Kadzińska, 2015).
Studies have shown that improved mechanical and water barrier properties of films were obtained
when cod skin gelatin was blended with soy protein isolate (Denavi et al., 2009), while increased
elongation and thermal stability, and decreased water vapor permeability and solubility were
observed for films made from cuttlefish skin gelatin blended with mungbean protein isolate
(Hoque et al., 2011). To date, there have been only few studies reporting on improving properties
of salmon skin gelatin-based films. A large amount of Atlantic salmon (Salmo salar) skin are
produced annually from the high demand fillet production (Blanco et al., 2007), which can serve
as fish gelatin source for the preparation of biodegradable films.
Zein, the major storage protein in corn endosperm, is a hydrophobic protein (Holding & Larkins,
2009) and has been actively studied for its film forming ability (Gu & Wang, 2013; Panchapakesan
et al., 2012; Xu et al., 2012). Zein films exhibit excellent water barrier properties and have been
used in hydrophobic film production (Cho et al., 2002). As such, this makes it potentially useful
as a hydrophobic component to be incorporated into biodegradable films for the improvement of
film water resistance. The effect of blending zein with other biopolymers on water barrier
122
properties was reported for zein-whey protein composite films (Ghanbarzadeh & Oromiehi, 2008)
and zein-gliadin composite films (Gu & Wang, 2013). It is known that gelatin is a water-soluble
protein, while zein is soluble in aqueous ethanol (Chiou et al., 2008; Gu & Wang, 2013).
Nevertheless, gelatin and zein are both soluble and miscible in aqueous ethanol, e.g. 50-90%
ethanol (Shukla & Cheryan, 2001), while gelatin dissolved well in aqueous ethanol up to 50%
(Farrugia & Groves, 1999).
To further improve properties of fish gelatin film, another strategy that produces stronger and
higher water resistance protein film is by incorporating cross-linkers (Wihodo & Moraru, 2013).
Among chemical cross-linkers, glutaraldehyde is by far the most widely used due to its ability to
react with protein and stabilize collagenous materials with high efficiency (Bigi et al., 2001). The
crosslinking reaction involves covalent intermolecular and intramolecular bonding between the
aldehyde groups of glutaraldehyde and free amino groups of lysine or hydroxylysine amino acid
residues of polypeptide chains (Damink et al., 1995). Several studies exploring the use of
glutaraldehyde to improve various physical and barrier properties of gelatin films have been
reported (Bigi et al., 2000; Bigi et al., 2001; Chiou et al., 2008; Matsuda et al., 1999). Meanwhile,
the drawback of crosslinking with glutaraldehyde is the possibility of cytotoxicity on the resulting
films; however, the cytotoxicity can be reduced effectively by applying glycine or glutamic acid
during post-treatment (Gough et al., 2002), washing with saline solution (Cooke et al., 1983), or
incorporating at concentration not more than 1% (w/v) (Jayakrishnan & Jameela, 1996), or at trace
amounts (Li et al., 2013).
In order to attain the desired composite film properties, it is crucial to determine the optimum
concentrations of film compositions since they can alter the properties of the resulting films. To
pursuit this aim, central composite design (CCD) using response surface methodology (RSM) has
been generally employed in the optimization of film compositions. Moreover, the relationships
between film compositions and film properties can be revealed through the mathematical models
generated by RSM (Granato & de Araújo Calado, 2014). Studies that have recently employed RSM
to obtain optimal formulation in achieving various properties of protein films include the use of
sesame protein isolate films (Sharma & Singh, 2016), soy protein films (Nandane & Jain, 2015),
and salmon gelatin added with boldine films (López et al., 2017). Therefore, CCD from RSM was
used to optimize the film composition comprising gelatin extracted from salmon skin, zein and
123
glutaraldehyde as crosslinker, with the aim to maximize TS and EAB, and minimize WS of the
optimized film.
7.3 Materials and methods
7.3.1 Materials
Atlantic salmon (Salmo salar) skins were obtained from a local fish market (Jean-Talon, Montreal,
Canada). Zein and glutaraldehyde were purchased from Sigma Chemical Co. (St. Louis, MO,
USA); porcine pancreas trypsin (EC 3.4.21.4; powdered; 90.97 U/mg) was obtained from ICN
Biomedicals Inc. (Ohio, USA); sodium chloride was purchased from BDH Inc. (Toronto, Ontario,
Canada); anhydrous ethanol was purchased from Commercial Alcohol Inc. (Brampton, Ontario,
Canada); anhydrous calcium chloride, glycerol, potassium carbonate and Tris base were all
purchased from Fisher Scientific (Fair Lawn, NJ, USA).
7.3.2 Extraction of salmon skin gelatin
Fish skins were washed with tap water. The skins were cut into 1.5 x 1.5 cm2 pieces with scissors
and degreased by tumbling in warm water (35 °C) (Muyonga et al., 2004). The skins were treated
with 0.45 M NaCl at 4 °C for 3 min, and washed with distilled water (Rahman et al., 2008). A
previous study produced optimum yields of gelatin with the following extraction parameters: the
skins were soaked in 50mM tris-HCl buffer (pH 8.0) in the presence of trypsin at 1.49 U/g, and
stirred continuously at room temperature (22-25 °C) for 5 h, then filtered through a Whatman No.
4 filter paper and washed with distilled water. Gelatin was extracted by gently stirring the mixture
of pretreated fish skins and distilled water using a shaking water bath (model 25, Precision
Scientific, USA) at 45 °C for 6 h 15 min. The skin/solution ratio was 1:6 (w/v) throughout the
process. The protein solutions were then centrifuged (7000 g, 15 °C, 30 min) and the supernatant
was lyophilized using a freeze dryer (Modulyod-115, ThermoSavant, Holbrook, NY, USA) at -50
°C and 120 mBar for 48 h. The lyophilized proteins were referred as ‘gelatin powder’ and were
stored at -20 °C.
7.3.3 Experimental design
A five-level-two-factors, central composite design (CCD) from RSM (Design expert 7.0.0, Stat
Ease, Inc., The United States) was adopted in this optimization study of gelatin-zein composite
124
film crosslinked with glutaraldehyde. Zein and glutaraldehyde concentrations were varied in this
study to determine their optimal levels to achieve the highest mechanical strength and water
resistance capability when incorporated into gelatin film. Zein (X1, 3-5%, w/v) and glutaraldehyde
(X2, 0.005-0.025%, w/v) that were used as the independent variables were assessed at five coded
levels (-1.414, -1, 0, +1, and +1.414), with the central values of variables coded as zero. Thirteen
experiments augmented with five replications were carried out at the center points to evaluate the
pure error. Tensile strength or TS (Y1), elongation at break or EAB (Y2) and water solubility or WS
(Y3), obtained from the average values of at least the triplicates were taken as the responses of the
design experiments.
Values obtained from experimental runs of RSM on gelatin extraction were subjected to analysis
of variance (ANOVA), using the Design Expert (Version 7.0.0) software. The responses were
fitted to a second-order polynomial model to correlate the response variable to the independent
variable (Sen & Das, 2017), using the following regression equation:
𝑌𝑌 = 𝐵𝐵0 + 𝐵𝐵1 𝑋𝑋1 + 𝐵𝐵2 𝑋𝑋2 + 𝐵𝐵11 𝑋𝑋12 + 𝐵𝐵22 𝑋𝑋22 + 𝐵𝐵12 𝑋𝑋1 𝑋𝑋2
(13)
where Y is the response, i.e. TS, EAB and WS (dependent variables); B0, B1, B2, B11, B22, B12 were
the regression coefficients for the model for the intercept, linear, quadratic and interaction effect,
respectively; and X1 and X2 were the coded values of the zein and glutaraldehyde concentrations
(independent variables), respectively.
RSM was employed further to optimize the independent variables to identify the mechanical
properties (TS and EAB) and WS of the optimized film. The combination of independent variables
that produced the highest overall desirable properties was selected as the optimized levels. To
estimate the optimal levels, independent variables were set as ‘in range’ for goal. Both TS and
EAB responses were set as ‘maximize’ for goal and assigned with equal importance (+++), while
WS was set as ‘minimize’ for goal and assigned with the highest importance (+++++). Film
samples were prepared using the generated optimized levels of independent variables, and their
TS, EAB and WS were measured. Experimental values measured were then compared with the
predicted values from RSM for the model validation.
125
7.3.4 Preparation of the films
Preliminary experiments showed that both gelatin and zein, respectively, dissolved well without
any precipitation and formed films successfully in aqueous ethanol solution consisted of ethanolwater mixture (1:1, v/v). Gelatin solution (5% of protein) was prepared into film forming solutions
(FFS) by dissolving the gelatin (5 g) in 100 ml aqueous ethanol under continuous magnetic stirring
for 30 min at room temperature (22-25 °C). Similarly, zein solution at different protein
concentrations (%, w/v) were prepared separately by dissolving the zein (g) in 100 ml aqueous
ethanol under continuous magnetic stirring for 30 min at room temperature (22-25 °C). The
gelatin-zein composite films were prepared by mixing gelatin solution (5%) and zein solution (at
different concentrations) into 20 ml FFS at a proportion of 1:1 (v/v), and added with glycerol as
plasticizer (30% of protein in FFS). The filmogenic solutions were mixed by magnetic stirring for
30 min. Cross-linked gelatin-zein films were prepared by adding glutaraldehyde as crosslinker at
different concentrations (% of FFS, w/v) into the gelatin-zein FFS and maintained under magnetic
stirring for another 30 min. After using a vacuum pump to reduce air bubbles formed, all gelatinzein FFS obtained were dispensed into polystyrene petri dishes and placed on a leveled surface in
a fume hood to evaporate the solvent for a period of 48 h at room temperature. After drying, the
films were repeatedly washed with 0.9% (w/v) NaCl (Cooke et al., 1983), air dried, and then
conditioned in a desiccator at 23 ± 2 °C containing saturated solutions of potassium carbonate (50
± 2% relative humidity, RH) for at least 40 h before mechanical and WS testing. Film thickness
was measured at 6 random positions with a hand-held digital micrometer (Marathon Part No.
030025, Marathon Watch Company Ltd., Ontario, Canada) with an accuracy of 0.002 mm.
7.3.5
Measurement of properties
7.3.5.1 Mechanical properties
The mechanical properties consisting of the TS and EAB values were determined according to
ASTM method D 882-10 using an Instron Universal Testing Machine (model 4500, Instron
Corporation, Canton, MA, USA). The specimen was fixed on the grips of the device with an initial
grip separation of 30 mm, and pulled apart at a mechanical crosshead speed of 10 mm/min and
preload of 2 N. At least five replicates were tested for each film and the averages were taken as
the results. TS (MPa) and EAB (%) were calculated by the following equations:
126
TS (MPa) = F max / A
(14)
where F max = maximum load (N) needed at the moment of rupture, A = cross-sectional area (m2)
of the samples.
𝐸𝐸
(15)
𝐸𝐸𝐸𝐸𝐸𝐸 (%) = �30� 𝑥𝑥 100
where E = film elongation (mm) at the moment of rupture, 30 = initial grip length (mm) of samples.
7.3.5.2 Film solubility
The WS of the films was determined according to the method of Shakila et al., (2012). Films of
surface area of 4 cm2 were cut and weighed (± 0.0001 g) to determine the initial weight (Wo). Each
film portion was immersed separately in 15 ml of distilled water, gently shaken at room
temperature (22-25 °C) for 15 h and then filtered through a Whatman No. 1 filter paper. The
unsolubilized film fraction collected on the filter paper was dried in a hot air oven at 105 °C for
24 h and weighed (Wf). Three replicates were tested for each film and the average values were
taken as the results. The solubility of the film was calculated by the following equation:
𝑊𝑊𝑜𝑜 − 𝑊𝑊𝑓𝑓
Solubility (%) = �
𝑊𝑊𝑜𝑜
(16)
� 𝑥𝑥 100
where Wo = initial weight of the film specimen, Wf = weight of unsolubilized film fraction.
7.3.5.3 Statistical analysis
Data were statistically analysed using the General Linear Models procedure of SAS (Release 9.4,
SAS Institute Inc., Cary, NC, USA) software, where mean comparisons were carried out by LSD
t-test (p < 0.05). p
127
7.4 Results and discussion
7.4.1 Statistical analysis
The coded and uncoded (actual) levels of the independent variables, as well as the corresponding
response values are shown in the Table 7.1. The response values were correlated with the two
independent variables using the second-order-polynomial equation (Eq. 13). The regression model
equation coefficients were calculated and three models for the TS (Y1), EAB (Y2) and WS (Y3)
were determined using Design Expert 7.0.0 software from Stat Ease Inc. These responses are
expressed in the coded form as the following regression equations, where positive and negative
signs in front of the terms (X1 and X2) indicate synergistic and antagonistic effect, and higher values
in front of the terms indicate higher impact of the coefficient, respectively:
𝑌𝑌1 = 2.17 − 0.07𝑋𝑋1 + 0.42𝑋𝑋2 + 0.17𝑋𝑋12 + 0.15𝑋𝑋22 − 0.12𝑋𝑋1 𝑋𝑋2
(17)
𝑌𝑌3 = 36.68 − 4.80𝑋𝑋1 − 7.37𝑋𝑋2 + 0.54𝑋𝑋12 + 4.68𝑋𝑋22 + 0.087𝑋𝑋1 𝑋𝑋2
(19)
𝑌𝑌2 = 17.05 − 7.83𝑋𝑋1 − 4.18𝑋𝑋2 + 2.16𝑋𝑋12 + 1.00𝑋𝑋22 + 1.13𝑋𝑋1 𝑋𝑋2
(18)
Table 7.1 Independent variables and their actual and coded values (in brackets) used for
optimization of gelatin-zein composite films crosslinked with glutaraldehyde.
Standard
order
Run
order
Zein (%),
X1
Glutaraldehyde (%),
X2
TS (MPa),
Y1
EAB (%),
Y2
WS (%),
Y3
1
2
3
4
1
13
11
7
3 (-1)
5 (1)
3 (-1)
5 (1)
0.005 (-1)
0.005 (-1)
0.025 (1)
0.025 (1)
1.97
1.93
3.15
2.64
33.83
15.44
23.30
9.41
50.96
42.18
40.04
31.61
5
6
4
5
2.586 (-1.414)
5.414 (1.414)
0.015 (0)
0.015 (0)
2.58
2.59
31.83
10.35
45.96
30.96
7
8
10
8
4 (0)
4 (0)
0.001 (-1.414)
0.029 (1.414)
2.02
3.07
24.74
12.78
60.00
33.50
9
10
12
2
4 (0)
4 (0)
0.015 (0)
0.015 (0)
2.20
2.08
17.15
16.91
36.02
35.48
11
12
3
6
4 (0)
4 (0)
0.015 (0)
0.015 (0)
2.11
2.31
17.54
16.85
37.90
38.96
13
9
4 (0)
0.015 (0)
2.13
16.78
35.04
128
By using RSM, the coefficient of the response surface models (Eq. 13) and the significant model
terms from ANOVA analysis for all responses are presented in Table 7.2. Based on a 95%
confidence level, the F-values for TS, EAB and WS (19.21, 627.08 and 21.98, respectively) with
the low p-values (0.0006, < 0.0001 and 0.0004, respectively) indicated that all three models were
highly significant (p < 0.05). In addition, the non-significant lack of fit values for these three
models of more than 0.05 (p > 0.05) also showed that the quadratic models were valid for this
present study. Likewise, the coefficient of variation (CV) for TS, EAB and WS were relatively
low (5.86, 2.44 and 6.64, respectively), suggesting the models were highly reproducible when the
CV is not greater than 10% (Beg et al., 2003). These statistical tests indicated that the models are
adequate for predicting the properties of film measured within the range of the variables studied.
Table 7.2 Coefficients and their significance in best fitted regression models of different responses.
Nature of best fit model
F-value
P-value
Coefficients
B0
Linear
B1
B2
Quadratic
B11
B22
Interaction
B12
R2
adj. R2
Lack of Fit
C.V.
*: significant at p < 0.05
TS (MPa)
Quadratic
19.21
0.0006*
EAB (%)
Quadratic
627.08
<0.0001*
WS (%)
Quadratic
21.98
0.0004*
+2.17
+17.05
+36.68
-0.07
+0.42*
-7.83*
-4.18*
-4.80*
-7.37*
+0.17*
+0.15*
+2.16*
+1.00*
+0.54
+4.68*
-0.12
0.9321
0.8835
0.1073
5.86
+1.13*
0.9978
0.9962
0.1109
2.44
+0.09
0.9401
0.8973
0.0904
6.64
129
In terms of the model fitting, the high values of R2 for TS, EAB and WS (0.9321, 0.9978 and
0.9401, respectively) and adjusted R-squared (0.8835, 0.9962 and 0.8973, respectively) indicated
the developed models were a good fit with the experimental values (a value > 0.75 indicates fitness
of the model) (Reddy et al., 2008). Hence, there were only 6.79%, 0.22% and 5.99% of the total
variation for TS, EAB and WS, respectively, that were not explained by the models. The regression
models were successfully developed and could accurately represent the variables studied within
the experimental ranges.
7.4.2 Effect on tensile strength
TS values corresponding to all experimental runs ranged from 1.93 to 3.15 MPa (Table 7.1) and
the predicted versus actual experimental values of TS of this study are presented in Fig. 7.1a. The
effect of zein and glutaraldehyde is further elucidated through the response surface plot (Fig. 7.1b)
which shows that TS increased significantly with increase in glutaraldehyde concentration as
evident from the upward trend, whereas TS remained almost same with different concentrations
of zein. As shown in Table 7.2, coefficients of X2, X12, X22 are significant (p < 0.05), indicating
glutaraldehyde (X2) is the significant variable (p < 0.05) with higher impact (higher value of
coefficient) on TS than zein.
An increase in zein concentration (X1) shows decreasing effect on TS (negative sign of coefficient,
B1) (Table 7.2), suggesting that the blending of increasing concentration of zein into gelatin films
had probably hindered the renaturation of the gelatin during formation of film network. Zein films
are known for their brittleness and poor mechanical properties, due to the strong intermolecular
forces between the zein protein molecules (Xu et al., 2012). Therefore, the interaction of zein with
gelatin had possibly resulted in the formation of film network with a reduced degree of
organization, resulting in decreased TS values. Blending at increasing concentration of soy protein
isolate into cod skin gelatin films (Cao et al., 2007) and casein into bovine skin gelatin films
(Chambi & Grosso, 2006) had shown similar effect on TS values as well.
Nevertheless, the effect of zein on TS was not significant statistically in this present study, as
compared to glutaraldehyde. Increasing glutaraldehyde concentration (X2), in contrast with zein,
significantly increased (p < 0.05) the TS of resulting films (positive sign of coefficient, B2), as
shown in Table 7.2. This can be explained by the fact that glutaraldehyde induced the forming of
covalent intermolecular and intramolecular cross-linking, increased adhesion and reduced the
130
interstitial spaces between protein polymers, resulting in the formation of rigid film network with
higher strength (Park et al., 2000). An increase in TS of film due to the addition of glutaraldehyde
was also reported for soy protein isolate film (Park et al., 2000), whey protein isolate film (Ustunol
& Mert, 2004), as well as pectin and fish skin gelatin or soy protein flour composite film (Liu et
al., 2007).
Predicted vs. Actual
3.20
Tensile strength (MPa)
3.2
Predicted
2.85
2.50
2.85
2.5
2.15
1.8
2.15
0.025
0.020
0.015
0.010
X2: glutaraldehyde conc.
1.80
1.88
2.20
2.51
2.83
5.000
4.500
4.000
X1: zein conc.
3.500
0.005 3.000
3.15
Actual
(a)
(b)
Fig. 7.1 (a) Predicted versus actual experimental values for TS. (b) Three-dimensional (3D)
response surface contour plot indicating the effect of interaction between zein and glutaraldehyde
concentrations for TS of the resulting films.
7.4.3 Effect on elongation at break
As shown in Table 7.1, EAB values corresponding to all experimental runs ranged from 9.41 to
33.83%, with the predicted versus actual experimental values of EAB presented in Fig. 7.2a. The
comprehensive effect of zein and glutaraldehyde is elucidated further with the response surface
plot as shown in Fig. 7.2b. The ascent of the surface increased more obviously with decrease in
zein concentration as compared to glutaraldehyde. This is consistent with the results in Table 7.2,
131
where coefficients of all terms of X1, X2, X12, X22, X12 were found to be significant (p < 0.05) in
affecting the EAB of the resulting films, with zein (X1) being the most significant variable (higher
coefficient value). Zein (X1) and glutaraldehyde (X2) were both found as negative linear terms (B1
and B2), indicating that the increase of both zein and glutaraldehyde concentrations decreased the
EAB of films.
Predicted vs. Actual
34.00
Elongation at break (%)
34
Predicted
27.75
21.50
27.75
21.5
15.25
9
15.25
0.025
0.020
0.015
0.010
X2: glutaraldehyde conc.
9.00
9.32
15.45
21.57
27.70
5.000
4.500
4.000
X1: zein conc.
3.500
0.005 3.000
33.83
Actual
(a)
(b)
Fig. 7.2 (a) Predicted versus actual experimental values for EAB. (b) Three-dimensional (3D)
response surface contour plot indicating the effect of interaction between zein and glutaraldehyde
concentrations for EAB of the resulting films.
Fish gelatin forms highly extensible films with good mechanical properties (Cao et al., 2007). Zein
protein molecules, however, are less flexible than gelatin due to its strong molecular interaction
forces through hydrophobic, hydrogen and limited sulfide bonds (Singh et al., 2012; Xu et al.,
2012). As such, incorporation of zein could highly disrupt the film network and produce film with
less flexibility, thus, explaining why an increase in zein concentrations decreased the EAB values
of films (negative sign of coefficient, B1) (Table 7.2). In addition, incorporation of glutaraldehyde
132
induces cross-linking and promotes restriction in segmental mobility, leading to reduced
elongation of films (Ramaraj, 2007). This was in agreement with the decreased EAB values as the
glutaraldehyde concentration increased (negative sign of coefficient, B2) (Table 7.2). Lower
elongation values were reported after addition of glutaraldehyde in the preparation of whey protein
isolate films (Ustunol & Mert, 2004), porcine gelatin films (Bigi et al., 2001), as well as pectin
and fish skin gelatin or soy protein flour composite films (Liu et al., 2007). Nevertheless,
comparable elongation values were observed for salmon skin gelatin films when incorporated with
0.25 to 0.75% (w/w) of glutaraldehyde (Chiou et al., 2008), or increased elongation values were
shown for soy protein isolate films after addition of 0.1 to 0.4% (w/w) of glutaraldehyde (Park et
al., 2000). A wider range of behaviour was found on the elongation of gelatin films with addition
of cross-linker (Chiou et al., 2008), which possibly depends on the type and extend of the
interactions occurring between the protein and crosslinkers.
7.4.4 Effect on water solubility
Table 7.1 shows that the WS values corresponding to all experimental runs ranged from 30.96 to
60.00%, with the predicted versus actual experimental values of WS shown in Fig. 7.3a. The effect
of zein (X1) and glutaraldehyde (X2) concentration is illustrated further with the response surface
plot shown in Fig. 7.3b. The response surface increased when both zein and glutaraldehyde
concentrations decreased (Fig. 7.3b). However, the decrease of WS was attributed to the increased
concentration of zein and glutaraldehyde incorporated into the gelatin films. From Table 7.2,
coefficients of terms of X1, X2, X22 are found to be significant (p < 0.05) in affecting the WS of the
resulting films, with glutaraldehyde exhibiting higher impact (higher coefficient value) than zein.
The incorporation of zein (X1) at increasing concentrations significantly decreased the WS of
gelatin films (negative sign of coefficient, B1) (Table 7.2). Zein is a hydrophobic protein as it
contains high proportion of non-polar amino acid residues (Holding et al., 2009). Thus, increased
incorporation of zein proteins reduced the hydrophilic sites for water absorption by gelatin films,
that could form composite films with higher water resistance (lower WS values). Films with
reduced WS were previously observed from an increased proportion of hydrophobic soy protein
isolate incorporation into cod skin gelatin films (Denavi et al., 2009), and hydrophobic mungbean
protein isolate into cuttlefish skin gelatin film (Hoque et al., 2011).
133
Predicted vs. Actual
60.00
55
Water solubility (%)
Predicted
52.25
44.50
48.5
42
35.5
29
36.75
0.025
29.00
0.020
0.015
0.010
X2: glutaraldehyde conc.
29.82
37.36
44.91
52.45
5.000
4.500
4.000
X1: zein conc.
3.500
0.005 3.000
60.00
Actual
(a)
(b)
Fig. 7.3 (a) Predicted versus actual experimental values for WS. (b) Three-dimensional (3D)
response surface contour plot indicating the effect of interaction between zein and glutaraldehyde
concentrations for WS of the resulting films.
As compared with zein, glutaraldehyde exhibited similar effect on WS of resulting films, where
the solubility tended to decrease at increasing concentrations of glutaraldehyde (X2) (negative sign
of coefficient, B2) (Table 7.2). When glutaraldehyde is incorporated into a film matrix, it restricts
the polymer chain mobility by forming crosslinking between polymer molecules, thus limiting the
availability of free hydroxyl groups and hydrophilic sites for water solubility (Li et al., 2013; Sen
et al., 2017). Previous studies had reported that the incorporation of glutaraldehyde not only
hindered the solubility of porcine-derived gelatin films (Bigi et al., 2001) and soy protein isolate
films (Park et al., 2000), but also polysaccharide-based pullulan films (Chen et al., 2017) and
chitosan-starch composite films (Li et al., 2013).
134
7.4.5 Validation of the predicted model of optimized compositions
The optimum compositions of zein and glutaraldehyde were obtained using numerical
optimization of RSM, to attain the maximum mechanical properties (TS and EAB) and minimum
WS of the films. As shown in Table 7.3, the optimum concentrations for zein and glutaraldehyde
were 3% and 0.02%, respectively. Film samples were prepared experimentally using the optimized
concentrations of compositions and their TS, EAB and WS values were compared with the
predicted values (Table 7.3) generated by the software for the model validation. The experimental
values of TS (3.11 ± 0.01 MPa), EAB (22.43 ± 1.57%) and WS (38.82 ± 1.71%) found no
significant differences (p < 0.05) when compared with the predicted values (3.10 MPa, 22.73%,
39.25%, respectively). This demonstrated the validity of the model, thereby making the formation
of gelatin-zein composite film crosslinked with glutaraldehyde as achieved by RSM practical.
Table 7.3 Predicted and experimental response values under the optimum compositions.
Optimum compositions
Responses
TS (MPa)
3.10a
Zein: 3%
Predicted value
Glutaraldehyde: 0.02%
Experimental value 3.11 ± 0.01a
EAB (%)
WS (%)
22.73a
39.25a
22.43 ± 1.57a
38.82 ± 1.71a
Values within each column with the same letter are not significantly different (p > 0.05).
7.5 Conclusion
Gelatin-zein composite film crosslinked with glutaraldehyde was prepared, and the effects of
composition on film properties were evaluated through the developed mathematical models
generated via RSM. The results were further used in optimizing the compositions needed to
maximize the TS and EAB values, but minimize WS values of the films. The optimum
compositions of 3% zein and 0.02% glutaraldehyde successfully yielded films with TS of 3.11 ±
0.01 MPa, EAB of 22.43 ± 1.57%, and WS of 38.82 ± 1.71%, with no significant differences as
compared to the predicted values generated by RSM. Thus, this study revealed the possibility of
producing gelatin-zein composite film crosslinked with glutaraldehyde as biodegradable
packaging film material.
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CONNECTING STATEMENT 6
The formulated glutaraldehyde-crosslinked salmon gelatin-zein film with improved water
resistance, mechanical strength, and elongation (Chapter VII) was evaluated for film properties,
then compared with the gelatin film and gelatin-zein composite film in Chapter VIII. These films
were compared for their mechanical, water and light barrier properties as well as thermal stability.
In-depth characterization of their structural and morphological properties was also conducted to
provide an understanding of the properties of these films, for the possible application in food
packaging.
The results of this study have been prepared for publication as:
Fan, H.Y., Duquette, D., Dumont, M.J. & Simpson, B.K. (2018). Characterization of salmon skin
gelatin-corn zein composite films crosslinked with glutaraldehyde.
136
CHAPTER VIII. CHARACTERIZATION OF SALMON SKIN GELATIN-CORN ZEIN
COMPOSITE FILMS CROSSLINKED WITH GLUTARALDEHYDE
137
8.1 Abstract
The objective of this study was to evaluate the properties of composite films made of salmon
(Salmo salar) skin gelatin (5% of protein) and zein (3% of protein), and crosslinked with
glutaraldehyde (0.02%, w/v). Before crosslinking, the morphological studies showed that the
gelatin-zein composite films had poor mechanical strengths, but were high in opacity and water
barrier properties, as compared to films made of gelatin only. Subsequent addition of
glutaraldehyde formed crosslinks in the gelatin-zein composite film successfully, as shown in
infrared and morphological studies. The crosslinked composite films of gelatin-zein had improved
thermal stability, light transparency, water resistance, and mechanical strength even when exposed
to high relative humidity.
Keywords: salmon skin gelatin, zein, glutaraldehyde, crosslinking, film
138
8.2 Introduction
Fish gelatin can be made abundantly available owing to the large volume of bones and skins
discarded as wastes from the fish processing industry (Badii & Howell, 2006). Gelatin is obtained
from pretreated collagenous skins and bones via thermal hydrolysis (Denavi et al., 2009). Fish
gelatin is a biocompatible polymer which has been studied for the development of biodegradable
films to protect food products (Gómez-Guillén et al., 2009). These films are known to be
transparent, extensible and have good gas barrier properties against oxygen and carbon dioxide
(Arvanitoyannis, 2002; Chiou et al., 2008). However, these films are usually brittle and have a
very poor water resistance which restricts their potential for food packaging applications (Chambi
& Grosso, 2006; Chiou et al., 2008; Denavi et al., 2009).
To improve the properties of gelatin films, different methods have been explored. One common
method is by blending gelatin with other biopolymers to form composite films (Galus & Kadzińska,
2015). Gelatin has amino acids with hydrophobic groups, suggesting possible interactions with
other hydrophobic biopolymers (Farris et al., 2010). Zein, one of the most hydrophobic protein
found in corn endosperm, has been used in the synthesis of hydrophobic films (Holding & Larkins,
2009). Therefore, blending gelatin with zein could decrease the hydrophilicity of gelatin films and
further improve the water barrier properties. It has been shown that higher mechanical performance
and improved water barrier properties could be achieved when cod skin gelatin was blended with
soy protein isolate, which is hydrophobic (Denavi et al., 2009). Moreover, an increase in
elongation at break and an improved thermal stability, and a decrease in water vapor permeability
were observed for films composed of gelatin from cuttlefish skin and mungbean protein isolate,
another hydrophobic protein (Hoque et al., 2011).
To further improve the properties of the blends, the proteins can be physically, chemically or
enzymatically crosslinked (Wihodo & Moraru, 2013). Chemical crosslinking allows improvement
of several properties such as the thermal stability, mechanical performance and the water resistance
of gelatin films (Farris et al., 2010). Among chemical crosslinkers, aldehydes can effectively
improve the functional properties of proteins, due to its capability to bond with proteins quickly
(Donohue et al., 1983). Glyoxal (Carvalho & Grosso, 2004), formaldehyde (Carvalho & Grosso,
2004), and glutaraldehyde (Bigi et al., 2001; Chiellini et al., 2001; Chiou et al., 2008) are examples
of aldehydes that have been used to modify gelatin based films. The use of glutaraldehyde is
139
advantageous since it has a low cost and high efficiency in collagenous materials stabilization and
biocompatibility, and its cytotoxicity can be overcome by washing with saline solution (Bigi et al.,
2001; Cooke et al., 1983). Glutaraldehyde crosslinks gelatin by forming new interchange imine
linkages between amino groups of lysine or hydroxylysine of protein chains and the aldehyde
groups of glutaraldehyde via Schiff base reactions (Damink et al., 1995).
Recent studies showed that crosslinking induced by glutaraldehyde successfully improved the
properties of fish gelatin films. Chiou et al. (2008) observed that the use of glutaraldehyde
improved significantly the water barrier properties of Alaska pink salmon skin gelatin films, but
no improvement was observed on the oxygen barrier and mechanical properties of the films. Liu
et al. (2007) reported that a pectin-fish skin gelatin composite film crosslinked with glutaraldehyde
displayed improved mechanical properties and water resistance than gelatin films.
In this study, composites films synthesized with gelatin extracted from Atlantic salmon skin and
zein were produced and further crosslinked with glutaraldehyde, and their structural, thermal
stability, barrier, mechanical, and morphological properties were studied to evaluate the potential
of these films as coating or packaging material for the food industry. The films studied were
gelatin-only film (G), gelatin-zein composite film (GZ), and glutaraldehyde-crosslinked gelatinzein film (GZ-gla). Atlantic salmon skin was chosen as the source of gelatin due to the high
availability of skins generated from high demand fillet production (Blanco et al., 2007). Hence,
for the first time, this study exploited the advantageous of the zein incorporation and
glutaraldehyde crosslinking on the salmon skin gelatin film properties.
8.3 Materials and methods
8.3.1 Materials
Atlantic salmon skins were obtained from a local fish market (Jean-Talon, Montreal, Canada).
Zein and glutaraldehyde were purchased from Sigma Chemical Co. (St. Louis, MO, USA); porcine
pancreas trypsin (EC 3.4.21.4; powdered; 90.97 U/mg) was obtained from ICN Biomedicals Inc.
(Ohio, USA); sodium chloride (NaCl) was purchased from BDH Inc. (Toronto, Ontario, Canada);
anhydrous ethanol was purchased from Commercial Alcohol Inc. (Brampton, Ontario, Canada);
glycerol, potassium carbonate and Tris base were purchased from Fisher Scientific (Fair Lawn, NJ,
USA).
140
8.3.2 Extraction of salmon skin gelatin
Fish skins were obtained from the fish market and washed with tap water. The skins were cut into
1.5 x 1.5 cm2 pieces with scissors and degreased by tumbling in warm (35 °C) water (Muyonga et
al., 2004). The skins were treated with 0.45 M NaCl at 4 °C for 3 min, and washed with distilled
water (Rahman et al., 2008). Based on Fan et al. (2017), the optimum yield of gelatins was obtained
with the following extraction parameters: the skins were soaked in 50mM tris-HCl buffer (pH 8.0)
in the presence of trypsin at 1.49 U/g, and stirred continuously at room temperature (22-25 °C) for
5 h, then filtered with a Whatman No. 4 filter paper and washed with distilled water. Gelatin was
extracted by gently stirring the mixture of pretreated skins and distilled water using a shaking water
bath (model 25, Precision Scientific, USA) at 45 °C for 6 h 15 min. The skin/solution ratio was
1:6 (w/v) throughout the process. The protein solutions were then centrifuged (7000 g, 15 °C, 30
min) and the supernatant was lyophilized using a freeze dryer (Modulyod-115, ThermoSavant,
Holbrook, NY, USA) at -50 °C and 120 mBar for 48 h. The lyophilized proteins were referred as
‘gelatin powder’ and were stored at -20 °C.
8.3.3 Preparation of the films
Preliminary experiments showed that both gelatin and zein dissolved well in aqueous ethanol
solution consisting of ethanol-water mixture (1:1, v/v). Thus, the gelatin film forming solution
(FFS) (5%, w/v) was prepared by dissolving the gelatin (5 g of protein) in 100 ml aqueous ethanol
under continuous magnetic stirring for 30 min at room temperature (22-25 °C). Similarly, zein FFS
(3%, w/v) was prepared by dissolving the zein (3 g of protein) in 100 ml aqueous ethanol under
continuous magnetic stirring for 30 min at room temperature (22-25 °C).
For the preparation of G film, gelatin FFS and glycerol (30% of protein) were mixed under
magnetic stirring for 30 min. For the GZ composite film, gelatin FFS and zein FFS was mixed at
a proportion of 1:1 (v/v), added with glycerol (30% of protein) and then mixed under magnetic
stirring for 30 min. Lastly, for the GZ-gla film, the mixture of gelatin-zein FFS (1:1, v/v) and
glycerol (30% of protein) was added with glutaraldehyde (0.02%, w/v), then maintained under
magnetic stirring for another 30 min. All filmogenic solutions were filtered using a vacuum pump
to reduce the presence of air bubbles, and were then dispersed on polystyrene petri dishes and
placed in a fume hood for 48 h at room temperature to allow the solvent to evaporate. After drying,
141
the films were repeatedly washed with 0.9% (w/v) NaCl (Cooke et al., 1983), air dried, and then
conditioned for a minimum of 40 h prior to perform the mechanical and water solubility
experiments. The conditioning was done in a desiccator at 23 ± 2 °C containing saturated solutions
of potassium carbonate (50 ± 2% relative humidity, RH).
8.3.4
Film characterization
8.3.4.1 Infrared analysis
Infrared spectra of films were recorded using a Nicolet iS5 FT-IR spectrometer (Thermo, Madison,
WI, USA). The spectra were collected in 32 scans with a resolution of 4 cm-1 over the range of
4000-400 cm-1, and the spectra were analyzed using the OMNIC 8.2 software package (Thermo
Fisher Scientific Inc., USA).
8.3.4.2 Thermal analysis
The thermal properties of films were determined using a differential scanning calorimetry (DSCQ2000, TA instrument, Inc., New Castle, DE, USA). Approximately 10 mg of film sample was
placed in hermetically-sealed aluminium pan, and then heated from 20 to 110 °C with a heating
rate of 10 °C/min under a nitrogen flow of 50 ml/min (Chiou et al., 2008). The glass transition
temperature (Tg) was determined as the inflexion point of the base line caused by the specific heat
of the sample. The melting temperature (Tm) was estimated from the endotherm peak (Farris et al.,
2011).
The decomposition temperatures of the films were determined according to ASTM method D
3850-94 using a thermogravimetric analyzer (TGA, Q50, TA instrument, Inc., New Castle, DE,
USA). Five to 10 mg of film were placed in a platinum pan, and heated from room temperature to
600 °C at a constant rate of 10 °C/min under a nitrogen flow of 60 ml/min.
8.3.4.3 Light barrier properties
The barrier properties of the films against ultraviolet (UV) and visible light were measured at
selected wavelengths (200 – 800 nm) using an UV/Vis spectrophotometer (Fang et al., 2002). The
films were cut into rectangular pieces (12 x 43 mm), directly placed into a quartz cuvette and
measured. Light transmission (T) was recorded as transmittance (%) using an empty cuvette as the
blank.
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8.3.4.4 Mechanical properties
The tensile strength (TS) and elongation at break (EAB) values were determined according to
ASTM method D 882-10 using an Instron Universal Testing Machine (model 4500, Instron
Corporation, Canton, MA, USA). The samples were fixed on the grips of the Instron. The initial
grip separation was 30 mm, and the experiments were conducted at a mechanical crosshead speed
of 10 mm/min and preload of 2 N. TS (MPa) and EAB (%) were calculated by the following
equations:
TS (MPa) = F max / A
(20)
where F max = maximum load (N) needed at the moment of rupture, A = cross-sectional area (m2)
of the samples.
𝐸𝐸
(21)
EAB (%) = �30� 𝑥𝑥 100
where E = film elongation (mm) at the moment of rupture, 30 = initial grip length (mm) of samples.
8.3.4.5 Water barrier properties
The water solubility of the films was determined according to the method of Shakila et al. (2012).
Films of surface area of 4 cm2 were cut and weighed (± 0.0001 g) to determine the initial weight
(Wo). Each film portion was immersed separately in 15 ml of distilled water, gently shaken at room
temperature (22-25 °C) for 15 h and then filtered through a Whatman No. 1 filter paper. The
insolubilized film fraction collected on the filter paper was dried in a hot air oven at 105 °C for 24
h and weighted (Wf). The solubility of the film was calculated by the following equation:
Solubility (%) = �
𝑊𝑊𝑜𝑜 − 𝑊𝑊𝑓𝑓
𝑊𝑊𝑜𝑜
(22)
� 𝑥𝑥 100
where Wo = initial weight of the film specimen, Wf = weight of insolubilized film fraction.
The water vapor permeability (WVP) of films was measured gravimetrically in accordance with
the ASTM E96/E96M-10 standard with modification as described by Pérez-Mateos et al. (2009).
A round portion of film was cut and sealed on a plastic cup containing silica gel with silicone
sealant (High Vacuum Grease, Dow Corning, Midland, Michigan, USA). The cup was placed in
143
desiccators filled with distilled water at the bottom. The cup was weighed every hour for 8 h. WVP
of the films were calculated by the following equation:
𝑊𝑊𝑊𝑊𝑊𝑊 =
𝑤𝑤 𝑥𝑥
(23)
𝑡𝑡𝑡𝑡 ∆𝑃𝑃
where w = weight gain of the cup (g), x = film thickness (mm), t = time of weight gain (h), A =
permeation area (cm2), and ∆P = difference of partial vapor pressure of the atmosphere with silica
gel and pure water (2642 Pa, at 22 °C). Results were expressed as g mm h-1 cm-2 Pa-1.
8.3.4.6 Dynamic mechanical analysis (DMA)
The storage modulus (E’) and loss modulus (E’’) of films were studied using a DMA Q800 (TA
instrument, Inc., New Castle, DE, USA) connected with a relative humidity (RH) chamber. The
films were cut into strips (7 mm x 18 mm), and loaded in the DMA at 1 Hz frequency and at
amplitude of 5 µm. The test was performed at 25 °C with the humidity ramp set at 1% RH per 10
min up to 90% RH (Escalante et al., 2012).
8.3.4.7 Morphological properties
The morphology of the films was studied using a field emission gun scanning electron microscope
(FEG-SEM) (JSM-7600TFE, JEOL, Tokyo, Japan). The samples were mounted on specimen stubs
using double sided adhesive tape, and made conductive by sputter-coating with gold-palladium.
This step was repeated twice for 15 s using a sputter-coater under vacuum for 30 s under a current
of 15 mA. After coating, the samples were observed at an accelerating voltage of 2 kV using a low
secondary electron image (LEI) detector at low current, at a magnification of 1500x.
The films were observed with a Nikon Eclipse LV100POL polarized light microscope (PLM), and
the images were captured with the fitted DS-Fi1camera (Nikon, Tokyo, Japan). The observations
were made at a magnification of 10x at room temperature.
8.3.4.8 Statistical analysis
The linear regressions used for the calculation of WVP (R2 > 0.98) were performed using Excel
2016 software (Microsoft, Seattle, WA). Data were statistically analyzed using the General Linear
Models procedure of SAS (Release 9.4, SAS Institute Inc., Cary, NC, USA) software. Mean
comparisons were carried out by Duncan’s multiple range test (p < 0.05) (Steel & Torrie, 1980).
144
8.4 Results and discussion
8.4.1 Infrared analysis
Figure 8.1 shows the FT-IR spectra recorded for G, GZ, GZ-gla films. FT-IR spectral data were
used to investigate the possible interaction and crosslinking among gelatin, zein and
glutaraldehyde. The FT-IR spectrum of G showed that the amide-A (arising from the stretching
vibration of the N-H group) band was observed at wavenumber of 3289 cm-1 (Kong & Yu, 2007).
The amide-I band appeared at around 1636 cm-1 (representing C=O stretching/hydrogen bonding
coupled with COO), amide-II at around 1537 cm-1 (attributed to the bending vibration of N-H
groups and stretching vibrations of C-N groups), and amide-III bands at around 1239 cm-1
(attributed to the vibrations in plane of C-N and N-H groups of bound amide or vibrations of CH2
groups of glycine) (Aewsiri et al., 2009; Muyonga et al., 2004).
For amide-A peak, the GZ film displayed a shift to lower wavenumber (from 3289 cm-1 to 3288
cm-1) and lower amplitude, as compared with G film (Fig. 8.1). The lower wavenumber and lower
amplitude of the amide-A peak indicated an increase in interactions between gelatin and zein in
film mainly through hydrogen bonding, which is coherent with the results reported by Arfat et al.
(2014). Similar changes on the amide-A band were observed for films of cuttlefish skin gelatin
blended with mungbean protein isolate (Hoque et al., 2011), and fish protein isolate blended with
fish skin gelatin (Arfat et al., 2014).
For the GZ-gla film, the amide-A peak shifted to higher wavenumber of 3290 cm-1 (Fig. 8.1). The
shift to a higher wavenumber of the amide-A peak confirmed the chemical crosslinking by
glutaraldehyde as reported on gelatins (Sutaphanit et al., 2014) and blends of gelatin and
carboxymethylcellulose (Asma et al., 2014). Moreover, the amide-I band of the spectrum of GZgla film exhibited an increase in amplitude when compared to GZ film. This was possibly due to
the carbonyl group of glutaraldehyde forming covalent imine bonds with the amino groups via
Schiff base reaction (Damink et al., 1995). Therefore, a crosslinking between protein chains
occurred using glutaraldehyde, which was indicated by an increase in the amide C=O stretching
peak that contributed to the increase in amplitude of the amide-I peak (Asma et al., 2014). In
addition, Liu et al. (2014) suggested that the shift of the amide-I band to a higher frequency was
another indication of the crosslinking between collagen molecules. In this present study, the
145
spectrum of GZ-gla film also demonstrated the shift of the amide-I band from 1638 to 1645 cm-1,
when compared to GZ film.
Amide I
Amide A
1645.86
Amide B
3290.49
Amide II
Amide III
GZ-gla
3288.05
1638.28
GZ
3289.06
1635.87
G
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1)
Fig. 8.1 FT-IR spectra of gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked with
glutaraldehyde (GZ-gla) films.
8.4.2
Thermal analysis
The DSC thermograms obtained for G, GZ, and GZ-gla films are presented in Fig. 8.2A. The
thermogram of G showed a glass transition temperature (Tg) at 59.48 °C, followed by an
endothermic peak at melting temperature (Tm) at 68.94 °C. The endothermic peak is associated
with the helix-to-coil transition of gelatin, which involves the rupture of hydrogen bonds and
disruption of the ordered structure in the protein film matrix (Achet & He, 1995). The GZ sample
146
displayed a Tg at 58.64 °C, followed by a Tm at 69.56 °C. The slight differences between Tg and
Tm for GZ films as compared to G films might be due to the different molecular arrangement and
interactions that occurred between gelatin and zein, resulting in changes in the organization and
flexibility of the polymer matrix. Farris et al. (2011) also reported a slight increase of Tg with a
slight decrease of Tm for gelatin-pectin blend films as compared to pure gelatin films.
For the GZ-gla films, the thermogram (Fig. 8.2A) showed a higher Tg value (60.77 °C) and Tm
value (70.92 °C). The higher Tg of GZ-gla films might be attributed to a higher crosslinking degree
than G and GZ films. The Tm of GZ-gla film increased as well with the addition of glutaraldehyde,
suggesting an increase in the thermal stability of the resulting film. The improved thermal stability
is attributed to the greater heat resistance of film matrix after crosslinking (Wang et al., 2016).
This result is in agreement with that reported by Bigi et al. (2001), who observed an increased in
Tm for pigskin gelatin after crosslinking with glutaraldehyde, as well as an increase in the Tg and
Tm for gelatin based composite film after crosslinking with glutaraldehyde (Asma et al., 2014).
The TGA thermograms of all G, GZ, and GZ-gla films are presented in Fig. 8.2B and their
corresponding degradation temperatures and weight loss are presented in Table 8.1. Three
degradation stages were observed for all films. The first stage of weight loss occurred (∆w1) over
the temperature (Td1) from 59 to 77 °C with an associated weight loss of 4.7 to 6%. This was due
to the loss of free and bound water absorbed in the film. The second stage of degradation occurred
at temperature (Td2) ranging from 242 to 250 °C with associated weight loss (∆w2) ranging from
30 to 33%. This degradation stage is due to the degradation of lower molecular weight protein
fractions or glycerol compounds in films. The third stage of degradation occurred at temperature
(Td3) ranging from 292 to 297 °C. The weight loss (∆w3) ranged from 50 to 53%. The degradation
stage was attributed to the degradation of highly interacted protein fractions in film matrix.
147
A
0
Temperature (°C)
0
20
40
60
80
100
120
Exothermal Heat Flow →
-0.1
-0.2
-0.3
-0.4
G
GZ
GZ-gla
-0.5
B
100
G
GZ
GZ-gla
90
80
Weight (%)
70
60
50
40
30
20
10
0
0
100
200
300
400
500
600
Temperature (° C)
Fig. 8.2. DSC thermograms (A) and TGA curves (B) of gelatin (G), gelatin-zein (GZ), and gelatinzein crosslinked with glutaraldehyde (GZ-gla) films.
148
Table 8.1. Thermal degradation temperature (Td, °C) and weight loss (∆w, %) of G, GZ, and GZgla films.
Film
samples
∆1
Td1
G
GZ
GZ-gla
63.0
59.1
77.0
∆w1
∆2
Td2
6.1
4.7
5.0
241.7
243.6
250.4
∆w2
∆3
Td3
∆w3
33.6
30.5
31.5
292.0
294.9
297.3
52.7
51.3
50.5
Ash
content
(%)
16.0
16.8
17.4
Among the films, GZ-gla films had a higher degradation temperature (Td) as compared to G and
GZ films (Table 8.1). This is attributed probably to the higher crosslinking degree of these matrices.
Bonds resulted from the interaction between gelatin and zein, as well as the presence of imine
linkages resulting from the crosslinking induced by glutaraldehyde. This was in agreement with
the increased transition temperatures showed in the DSC scans. Higher heat stability was reported
for blend films based on cuttlefish skin gelatin and mungbean protein isolate (Hoque et al., 2011),
and glutaraldehyde-crosslinked chitosan/corn cob biocomposite film (Chan et al., 2013). Moreover,
the ash content of the GZ-gla film (17.4%) at 600 °C was higher than the G and GZ films (16.0
and 16.8%, respectively) (Table 8.1), indicating the presence of stronger interaction between
protein-protein in the film matrix after crosslinking glutaraldehyde, resulting in a greater thermal
stability (Arfat et al., 2014; Chan et al., 2013).
8.4.3 Light barrier properties
Transmission of ultraviolet (UV) and visible light at wavelength ranging from 200-800 nm for G,
GZ, and GZ-gla films are shown in Table 8.2. Among all films, GZ films exhibited the lowest
transmission in visible light (400-800 nm) as compared to G and GZ-gla films (p < 0.05). This is
evidenced by the increase in the opacity or turbidity of the GZ film sample (Fig. 8.3). Zein-only
film exhibits low transparency attributed to the strong interactions between zein proteins that
formed a denser network structure (Gu et al., 2013). Moreover, the lowest visible light
transmission of GZ films suggested poor interaction of gelatin, which has hydrophilic nature, and
zein, which has hydrophobic nature. Low compatibility of the molecules in the film decreased the
transmission of light ascribed to the light reflection and scattering effect from the distribution of
molecules in the film network (Bai et al., 2012; Li et al., 2013). This is consistent with the lowered
149
transmission of light observed for cuttlefish gelatin films when added with mungbean protein
isolate (Hoque et al., 2011), and soy protein films when incorporated in gelatin (Bai et al., 2012).
Table 8.2 Light transmission (%T) of gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked
with glutaraldehyde (GZ-gla) films.1,2
Film
200 nm 280 nm
350 nm
400 nm
500 nm
600 nm
700 nm
samples
0.1 ± 0.0a
0.7 ± 0.0a 62.6 ± 0.2a 68.2 ± 0.2a 75.9 ± 0.1a
78.4 ± 0.1a
80.3 ± 0.1a
G
0.1 ± 0.1a
0.1 ± 0.1c
3.6 ± 0.0b 11.1 ± 0.0c 17.0 ± 0.1c
19.3 ± 0.0c
20.4 ± 0.0c
GZ
a
b
c
b
b
b
0.1 ± 0.0
1.6 ± 0.1 19.1 ± 0.8
39.0 ± 1.0
57.8 ± 1.0
65.6 ± 0.4b
GZ-gla 0.1 ± 0.1
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
G
800 nm
81.9 ± 0.1a
21.4 ± 0.0c
68.5 ± 0.2b
GZ-gla
GZ
Fig. 8.3 Samples of gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked with
glutaraldehyde (GZ-gla) films.
GZ-gla films had the lowest transmission in UV light range (p < 0.05) (Table 8.2). Mu et al. (2012)
stated that the UV barrier properties of crosslinked gelatin films were probably due to the abundant
C=N groups occurring between the amino groups of gelatins and the aldehyde groups in the
crosslinker via Schiff base reactions. As a consequence, crosslinked gelatin films had excellent
barrier properties against UV which is a great quality for food packaging materials as it prohibits
lipid oxidation in the food system (Garavand et al., 2017). In the visible light range, GZ-gla films
150
exhibited lower light transmission as compared to G films, but higher light transmission when
compared to GZ films (Table 8.2). As discussed, the high opacity of GZ films was probably due
to the formation of less organized networks, resulting from poor interaction between gelatin and
zein; while GZ-gla films showed lower light transmission and yellowish than G films, as a
consequence of the crosslinking effect by the use of glutaraldehyde. GZ-gla films were clearer as
compared to other films (Fig. 8.3), which could increase the consumer acceptance as a food
packaging film (Garavand et al., 2017).
8.4.4 Mechanical properties
The mechanical properties of G, GZ, and GZ-gla films are presented in Table 8.3. As shown in
Table 8.3, GZ films had lower TS and EAB values than the other films. Zein films are known for
their brittleness, poor TS and EAB (Xu et al., 2012), while gelatin films have better mechanical
properties as they have a more organized network (Cao et al., 2007). Films formed with highly
organized polymer chains have optimum molecular packing and mechanical properties (Hoque et
al., 2011), which reflected by the highest TS and EAB of by G films (Table 8.3). The lower
mechanical properties of GZ films suggested the formation of less organized film matrices,
possibly due to the different nature of gelatin and zein proteins which are hydrophilic and
hydrophobic, respectively, which is consistent with the lower light transmission observed (Table
8.2). Chambi and Grosso (2006) reported that lower TS was attributed to the less organized matrix
formed when casein was blended with gelatin to form films. In addition, lower TS and EAB were
observed when soy protein isolate was added to cod skin gelatin films (Denavi et al., 2009) and
bovine gelatin films (Cao et al., 2007).
Table 8.3 Mechanical properties (TS and EAB) and film solubility (water solubility and WVP) of
gelatin (G), gelatin-zein (GZ), and gelatin-zein crosslinked with glutaraldehyde (GZ-gla) films.1,2
Film samples
TS (MPa)
Water solubility
(%)
92.61 ± 1.20a
74.59 ± 4.16b
38.82 ± 1.71c
EAB (%)
WVP
(g mm h-1 cm-2 Pa-1)
0.997 ± 0.038a
0.458 ± 0.023b
0.276 ± 0.017c
G
3.65 ± 0.49a
208.35 ± 33.79a
GZ
3.31 ± 0.77a
58.90 ± 13.70b
GZ-gla
3.11 ± 0.01a
22.43 ± 1.57b
1
Data are expressed as mean ± standard deviation.
2
Different superscripts in the same column indicate statistical differences (p < 0.05).
151
Crosslinking with glutaraldehyde had further decreased the mechanical strength of GZ film, as
evidenced by the lowest TS and EAB (Table 8.3). Li et al. (2013) explained that glutaraldehyde
promotes the formation of brittle films. Previous studies proposed that dialdehyde can penetrates
and degrades the molecular structure of biomaterials when glutaraldehyde was added at a particular
concentration, resulting in an increased brittleness and decreased in mechanical strength
(Schiffman and Schauer, 2007). Presumably, this might explain the higher transparency of the GZgla film (Table 8.2), as a result of the addition of glutaraldehyde, which increased the compatibility
between gelatin and zein via formation of crosslinked networks, resulting in a more organized film
matrix that allowed higher transmission of light. As such, the degree of the interaction between
protein chains and the extent of crosslinking within the films had greatly influence the properties
of the resulting films.
8.4.5
Water barrier properties
The water solubility and WVP were measured to evaluate the water resistance properties of G, GZ
and GZ-gla films. As shown in Table 8.3, G films showed the highest values for both water
solubility (92.61%) and WVP (0.997 g mm h-1 cm-2 Pa-1) (p < 0.05). The highest solubility revealed
the water binding capacity ascribed to the hydrophilic nature of gelatin and glycerol (Denavi et al.,
2009), while the highest WVP probably indicated the highest degree of the polymer chain mobility,
as evidenced by its highest EAB value (Table 8.3). An increase in the chain mobility allows higher
diffusion of water molecule which facilitates higher water vapor permeability (Chambi & Grosso,
2006).
The introduction of zein into the gelatin matrix (GZ films) decreased the water solubility (74.59%)
and WVP (0.458 g mm h-1 cm-2 Pa-1) (p < 0.05) as compared to G film. This was expected as zein
is hydrophobic in nature (Holding & Larkins, 2009). The lowest water solubility and WVP values
were obtained for the GZ-gla film (38.82% and 0.276 g mm h-1 cm-2 Pa-1, p <0.05), as shown in
Table 8.3. The addition of glutaraldehyde formed chemical crosslinked which decreased the
mobility of the polymer chains and reduced free volume of the matrix. This obstructed the
migration of water molecules within the matrix, and resulted in an improved water resistance of
the films (Garavand et al., 2017). In addition, the highest water resistance of GZ-gla films were
consistent with its lowest EAB value (Table 8.3), which revealed the effect of the reduced chains
mobility in the film matrix after crosslinking.
152
8.4.6 Dynamic mechanical analysis
Dynamic mechanical analysis was performed on GZ and GZ-gla films to evaluate the crosslinking
effect on the mechanical properties of gelatin-zein composite films as a function of humidity.
Because water acts as a plasticizer, it is expected that the stiffness of the films will decrease, which
is reflected through the storage modulus and loss modulus curves (Escalante et al., 2012). The
storage modulus curves (E’) are presented in Fig. 8.4A, and the loss modulus curves (E”) are
presented in Fig. 8.4B. As relative humidity increased, the E’ and E” curves decreased for both
films, indicating the water absorption and plasticization effects on the films. Bonnaillie and
Tomasula (2015) explained that the reconfiguration of the proteins within the films can be
triggered when exposed at a critical relative humidity value, which leads to the formation of looser
structure that promotes higher water absorption. Compared with the GZ film, the GZ-gla film
exhibited higher storage modulus (Fig. 8.4A) and loss modulus curves (Fig. 8.4B), which is due
to the addition of glutaraldehyde as a crosslinking agent.
153
A
Storage Modulus. E’ (MPa)
2000
····
–– –– –
GZ-gla
GZ
1500
1000
500
0
0
10
20
30
40
50
60
Relative Humidity (%)
70
80
90
B
Loss Modulus,
E”(MPa)
(
)
250
····
––––
GZ-gla
GZ
200
150
100
50
0
0
10
20
30
40
50
60
70
80
90
Relative Humidity (%)
Fig. 8.4 Storage modulus (A) and loss modulus (B) of gelatin-zein (GZ) and gelatin-zein
crosslinked with glutaraldehyde (GZ-gla) films.
154
8.4.7 Morphological properties
The morphology of GZ and GZ-gla films were examined using SEM (Fig. 8.5A) and PLM (Fig.
8.5B). From Figs. 8.5A and 8.5B, (Fig. 8.5), it can be seen that the zein proteins did agglomerate
without dispersing in the gelatin matrix, which is attributed to their differences in nature. These
observations supported the reduced mechanical strength and light transmission of GZ films as
compared to the crosslinked matrices.
The addition of glutaraldehyde improved the dispersion of zein (Fig. 8.5). The addition of
glutaraldehyde to zein proteins induced the penetration of the dialdehyde that resulted in a
shrinkage of the zein agglomerates, and further formed more organized matrices (Akin & Hasirci,
1995; Schiffman & Schauer, 2007). The SEM and PLM images showed the crosslinking effect of
glutaraldehyde on the homogeneity of the matrices (Fig. 8.5).
A
B
GZ
GZ
GZ-gla
GZ-gla
Fig. 8.5 SEM, at 1500x magnification (A), and polarized light microscopy, at 10x magnification
(B) of gelatin-zein (GZ) and gelatin-zein crosslinked with glutaraldehyde (GZ-gla) films.
155
8.5 Conclusion
The addition of hydrophobic zein proteins into salmon skin gelatin significantly increased the
water barrier properties of the resulting composite films. However, the incompatibility between
gelatin and zein proteins, as shown from the morphological study, produced a less organized film
network that contributed to reduced mechanical strength, but higher opacity and water barrier
properties. The addition of glutaraldehyde had yielded crosslinked gelatin-zein films with
improved thermal stability, light transparency, water resistance, and mechanical strength under
high relative humidity conditions, as shown in infrared and morphological studies. The results
demonstrated the possibility of producing salmon skin gelatin films with improved water
resistance, but its mechanical properties were greatly influenced by the organization and the
crosslinking of the polymer chains in the film network.
156
CHAPTER IX. GENERAL CONCLUSIONS, CONTRIBUTIONS TO KNOWLEDGE
AND RECOMMENDATIONS FOR FUTURE WORK
157
9.1 General Conclusions
This research was focused on the recovery of gelatin with high molecular weight from cold-water
fish skins for the purpose of biopolymer film formation. Selected gelatin extraction methods were
compared, which used saline, saline in combination with alkaline, and trypsin, respectively, to
pretreat salmon fish skins prior to gelatin extraction. Trypsin-aided extraction process was found
to be the most effective method of obtaining salmon skin gelatin with a higher yield. Further
investigation on the effect of different extraction conditions revealed the efficiency of trypsinaided extraction process in facilitating the recovery of gelatin from salmon, skate and dogfish skins.
Particularly, the yields and protein electrophoretic patterns of gelatins yielded from all fish skins
elucidated the distinct effects of the trypsin concentrations used in pre-treatment. A high trypsin
concentration produced higher gelatin yield, but also degraded gelatin structure, as evidenced by
the concomitant disappearance of high molecular weight protein chains. Meanwhile, a very low
trypsin concentration successfully minimized protein chain degradation, however, decreased the
gelatin yields from all fish skins. Nevertheless, among these gelatins, a higher yield of gelatin with
high molecular weight was recovered from salmon skins as compared with other fish skins, which
confirmed the viability of trypsin in assisting the extraction of fish gelatin for the potential use in
biopolymer film formation.
The present study reported the trypsin-aided process for the extraction of salmon skin gelatins with
high molecular weight protein chains. The conditions of trypsin-aided extraction process showed
that the concentration of trypsin used for salmon skin pre-treatment as well as the temperature and
time used for gelatin extraction had significantly affected the gelatin yield and the content of high
molecular weight protein chains of the resultant gelatins. By targeting maximizing the yield of
salmon skin gelatins with high molecular weight protein chains, the optimized process conditions
were established as: trypsin concentration at 1.49 U/g; extraction temperature at 45 °C; and
extraction time at 6 h 15 min. Two-fold higher yields of gelatin were successfully achieved when
compared to the process without trypsin.
In order to test the film forming capability and to evaluate the properties of the film formed with
the extracted salmon skin gelatins, different gelatin (protein) and glycerol concentrations were
explored for the formation of the film using the wet process. The properties of the resultant films
were determined in terms of their mechanical, water and light barrier properties, and were further
158
correlated with their protein patterns, structural and morphological properties. The films’
assessment showed that the increased content of high molecular weight chains resulting from the
higher gelatin concentration had contributed to an increase in mechanical strength and light barrier
properties, while the increased plasticization effect attributed to the higher glycerol concentration
had increased the elasticity of the films. Among the films, salmon skin gelatin film prepared at 5%
protein and 30% glycerol was identified to have good tensile strength and elasticity; however,
there was no difference in its water resistance as compared with other films at varying protein and
glycerol concentrations.
Taking advantage of the high hydrophobicity of the corn zein protein, the development of salmon
skin gelatin film blended with selected levels of zein was investigated aiming for the improvement
of water barrier properties. A notable increment in water barrier capability was obtained from the
resultant gelatin-zein composite films and attributed to the increased levels of zein incorporated.
Unfortunately, the mechanical properties of the composite films were impaired owing to the
disruption of the polymer chains organization in the presence of zein. Further investigation with
canola oil as replacement for glycerol as plasticizer did not improve the mechanical properties of
the gelatin-zein composites films but only contributed to lowering the water solubility, water vapor
permeability and light transmission of the films. These varying mechanical and barrier properties
were confirmed by the structural and morphological analyses that reflected the involvement of
canola oil in the protein-lipid interactions. Thus, the incorporation of canola oil seemed to have a
greater negative effect on the mechanical properties of the gelatin-zein composite films.
The effectiveness of glutaraldehyde as cross-linker in enhancing the mechanical properties of the
biopolymer films was investigated for the improvement of mechanical strength and elasticity as
well as water resistance. This was conducted using a CCD design from RSM with 5-levels and 2factors, namely the zein and glutaraldehyde concentrations. Results showed that increasing content
of glutaraldehyde produced a higher impact by significantly increasing the resultant films’
mechanical strength and water insolubility, while increasing content of zein had a greater effect in
significantly decreasing the films’ elasticity. An optimized formulation of the glutaraldehydecrosslinked gelatin-zein composite film with maximized mechanical strength, elasticity and water
resistance was generated and experimentally validated. Remarkably, the optimized film exhibited
a pronounced improvement in water resistance, while retaining mechanical strength with a slight
159
reduction in elasticity. In addition, higher mechanical integrity and thermal stability, as well as
transparency were achieved with the optimized formulation. The glutaraldehyde-induced crosslinks enhanced the gelatin-zein film’s mechanical properties, which was supported by the
structural and morphological analyses.
Taken together, this research widens the scientific knowledge of biopolymer film formation using
fish skin gelatin extracted from the trypsin-aided process, and the improvement of film properties,
particularly water resistance, by incorporating hydrophobic zein and cross-linking with
glutaraldehyde.
9.2 Contributions to Knowledge
1. For the first time, the use of a novel trypsin-aided process for the recovery of gelatin with
maximal yield and minimal protein chains degradation was carried out and reported.
2. The formation of films using the salmon skin gelatins extracted under the aid of trypsin
was reported for the first time.
3. The blending of hydrophobic zein with hydrophilic fish gelatin for the development of a
composite film was studied for the first time. This is also the first study that reported the
cross-linking effect of glutaraldehyde in reinforcing the properties of the resultant films.
4. The in-depth characterization of the properties of the glutaraldehyde-crosslinked gelatinzein film was reported for the first time.
9.3 Recommendations for Future Work
•
Investigation on the charge density by manipulating the pH of the film-forming solutions
to study the extent of the associations between gelatin and zein molecules and
corresponding film properties.
•
Assessment of the nanostructure using atomic force microscopy under high resolution and
real-time to understand the aggregation of the polymer molecules during drying of the
glutaraldehyde-crosslinked gelatin-zein film and to establish the relationships between the
nanostructure and resulting film properties.
160
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