Accepted Manuscript Title: Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite edible films Author: Mourad Jridi Sawssan Hajji Hanen Ben Ayed Imen Lassoued A¨ıcha Mbarek Maher Kammoun Nabil Souissi Moncef Nasri PII: DOI: Reference:
S0141-8130(14)00226-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.03.054 BIOMAC 4266
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
9-2-2014 22-3-2014 29-3-2014
Please cite this article as: M. Jridi, S. Hajji, H.B. Ayed, I. Lassoued, A. Mbarek, M. Kammoun, N. Souissi, M. Nasri, Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite edible films., International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.03.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite
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edible films.
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Mourad Jridi1*, Sawssan Hajji1, Hanen Ben Ayed1, Imen Lassoued1, Aïcha Mbarek2, Maher
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Kammoun1, Nabil Souissi3 and Moncef Nasri1
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1. Laboratoire de Génie Enzymatique et de Microbiologie – Université de Sfax, Ecole Nationale
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d’Ingénieurs de Sfax, B.P 1173-3038 Sfax, Tunisia.
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2. Laboratoire de Chimie Industrielle, Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, B.P.
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1173-3038 Sfax, Tunisia.
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3. Laboratoire de Biodiversité et Biotechnologie Marine. Institut National des Sciences et Technologies de
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la Mer. Centre de Sfax. BP 1035. 3018 Sfax, Tunisia.
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*Corresponding author: Tel.: 216 28-142-818; fax: 216 74-275-595;
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E-mail address: [email protected] (M. Jridi)
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Abstract Physico-chemical and mechanical properties of cuttlefish skin gelatin (G), chitosan (C)
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from shrimp (Penaeus kerathurus) and composite films (G75/C25, G50/C50, G25/C75)
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plasticized with glycerol were investigated. The results indicated that chitosan film had higher
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tensile strength and lower elongation at break when compared with the other films. Composite
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films show no significant difference in tensile strength (TS), thickness and transparency. The
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structural properties evaluated by FTIR and DSC showed total miscibility between both
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polymers. DSC scans showed that the increase of chitosan content in the composite films
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increases the transition temperature (Tg) and enthalpy (ΔHg) of films. The morphology study of
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gelatin, chitosan and composite films showed a compact and homogenous structure. In addition,
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gelatin and G75/C25 films demonstrated a high antioxidant activities monitored by β-carotene
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bleaching, DPPH radical-scavenging and reducing power activity, while films contained chitosan
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exhibited higher antimicrobial activity against Gram-positive than Gram-negative bacteria.
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Keywords: Composite gelatin-chitosan films; Microstructure; Antioxidant and antibacterial
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activity.
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1. Introduction Given the potential application of gelatin ad chitosan as eco-friendly active packaging
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materials, it is of great interest to study their use together in the preparation of edible films.
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Indeed, to our knowledge few works have been conducted on edible films based on gelatin and
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chitosan polymers [1, 2, 3, 4]. These biodegradable films improve in general the preservation of
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food, mainly by acting as barriers to water, oxygen and light. Gelatin has been reported to
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develop edible films due to its functional properties and biodegradability [5]. Bovine and porcine
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wastes are the most frequent sources to obtain gelatin of good quality. However, other sources of
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gelatin are becoming increasingly relevant, such as fish bones and skins [6].
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Fish skin, which is a major by-product of the fish-processing industry, causing waste and
64
pollution, could provide a valuable source of gelatin [7]. However, films based on fish gelatins
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have relatively poor water vapor permeability, mechanical properties and water resistance, which
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may limit their use as potential packaging materials. One of the effective strategies used to
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improve the physical performance of gelatin films is to elaborate composite films by mixing fish
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gelatin with other biopolymers exhibiting film forming properties and derived from renewable
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resources such as chitosan [4].
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Chitosan is a cationic polysaccharide with excellent film forming properties. It is obtained
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from chitin by deacetylation in the presence of alkali [8]. This polysaccharide is widely utilized
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not only for its film-forming ability but also to its antimicrobial properties [2, 9]. Indeed, the
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antimicrobial properties of chitosan and its derivated (chitosan-oligo-saccharides) have been
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largely reported in the literature [10, 11, 12, 13], thereby encouraging its use as potential
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packaging material. 3
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Composite films of chitosan and gelatin have been reported to have improved mechanical,
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transport and physical properties compared with those of single polymer based films [2, 4]. In
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addition various cross-linkers [1] (glutaraldehyde, transglutaminase, carbodiimide) have been
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used to improve the physical performance of fish gelatin films [14, 15]. The interactions between
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gelatin and chitosan have been probed by infrared spectroscopy, X-ray diffraction and pH
81
titrations [16]. In the formation of composite film it is important to study the compatibility of its
82
components and intermolecular interactions that may occur between them, since they finally
83
affect the film structure and determine the film properties.
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Despite the numerous works reported the effect of chitosan addition on chemical, physical
85
and biological properties of the fish gelatin films, information about fish gelatin-chitosan
86
composite films is limited.
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In a previous work we investigated the physical and mechanical properties of edible films
88
made from cuttlefish (Sepia officinalis) skin gelatins obtained by pretreatment with different
89
pepsin concentrations [17]. This paper is a continuation of this work and is focused on
90
investigation of physical, mechanical and antioxidant properties of cuttlefish skin gelatin and/or
91
chitosan edible films. Antimicrobial effect of edible films over Gram-positive and Gram-negative
92
bacteria was also investigated.
93
2. Materials and methods
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2.1. Gelatin preparation
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Skin from cuttlefish (S. officinalis) was obtained from the fish market of Sfax City, Tunisia.
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Cuttlefish skin was cut into small pieces (1 cm x 1cm) and then soaked in 0.05 M NaOH (1:10 4
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w/v). The mixture was stirred for 2 h at 4 °C and alkaline solution was changed every 30. The
98
alkaline-treated skins were then washed with distilled water until a neutral pH was obtained. The
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alkaline-treated skin was soaked in 100 mM glycin-HCl buffer, pH 2.0 with a solid/solvent ratio
100
of 1:10 (w/v) and subjected to hydrolysis collagen with 5 units of pepsin /g of skin, as described
101
in our previous study [18]. Cuttlefish skin gelatin (G) obtained was used for films preparation.
102
2.2. Chitosan preparation
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Chitosan was prepared from shrimp (Penaeus kerathurus) waste as described by Hajji et al.
104
[19]. Deproteinization was carried out in a thermostated stirred Pyrex reactor (300 ml).
105
Shrimp waste homogenate (15 g) was mixed with 45 ml distilled water. The pH of the
106
mixture was adjusted to 9.0. Then, the shrimp waste proteins were digested with crude enzyme
107
from Bacillus mojavensis A21 at 50 °C. The reaction was then stopped by heating the
108
solution at 90 °C during 20 min to inactivate enzymes. The solid phase was washed and
109
then pressed manually through four layers of gauze. Solid fractions obtained was treated
110
with 1.5 M HCl in 1:10 (w/v) ratio for 6 h at 50 °C under constant stirring (150 rpm).
111
The chitin product was filtered through four layers of gauze with the aid of a vacuum
112
pump and washed to neutrality with deionized water and then dried for 1 h at 60 °C. The
113
purified chitin was treated with 12.5 M NaOH in 1:10 (w/v) ratio at 140 °C for 4 h until
114
it was deacetylated to chitosan. After filtration, the residue was washed with deionized
115
water, and the crude chitosan was obtained by drying in a dry heat incubator at 50 °C
116
overnight. The deacetylation degree of resulted shrimp chitosan (C) was about 88%.
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2.3. Edible films preparation and characterization
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2.3.1. Preparation of composite films To prepare film forming solutions, cuttlefish skin gelatin powder was dissolved in distilled
121
water, and chitosan in 1% (v/v) acetic acid, to achieve the final concentration of 4% and 2%
122
(w/v), respectively. Glycerol was added as plasticizer to the gelatin and/or chitosan solutions at a
123
level of 15% (based on protein and/or polysaccharide). The film forming solutions were
124
incubated at 40 °C for 30 min with gentle stirring.
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The composite (Gelatin/Chitosan) solutions were obtained by mixing film forming
126
solutions of gelatin and chitosan at different ratio (G75:C25, G50:C50, G25:C75). The resulting
127
mixtures were gently stirred during 30 min at 40 °C.
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In all formulations, 20 mL of each solution was cast onto a rimmed silicone resin plate (6
129
cm x 6 cm), air-blown for 12 h at room temperature (25 °C) and dried at a temperature of 25 °C
130
and 50% relative humidity for 48 h. Dried films were manually peeled off and then subjected to
131
analyses. Composite films prepared were referred as G75/C25, G50/C50 and G25/C75. Films,
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prepared by using gelatin (G100) or chitosan (C100) were used as control.
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Prior to mechanical properties testing, films were conditioned for 48 h at 25 °C. For FTIR
134
and DSC studies, films were conditioned in a dessicator containing dried silica gel for 3 weeks at
135
room temperature (25 °C) to obtain the most dehydrated films.
136
2.3.2. Film thickness
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The thickness of films was measured using a micrometer (Mitutoyo, Model ID-C112PM,
138
Kawasaki-shi, Japan). Ten random locations around each film sample were used for thickness
139
determination.
140
2.3.3. Mechanical properties
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TS and EAB of film samples were determined as described by Iwata et al. [20] using the
142
Universal Testing Machine (Lloyd Instrument, Hampshire, UK). The test was performed in the
143
controlled room at 25 °C and 50±5% RH. Ten film samples (2 cm × 4 cm) with the initial grip
144
length of 3 cm were used for testing. The film samples were clamped and deformed under tensile
145
loading using a 100 N load cell with the cross head speed of 30 mm/min until the samples were
146
broken. The maximum load and the final extension at break were used for calculation of TS and
147
EAB, respectively.
148
2.3.4. Water solubility
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Film samples (3 cm x 2 cm) were weighed and placed in 50 ml-centrifuge tube containing
150
10 ml of distilled water with 0.1% (w/v) sodium azide, and stirred at room temperature for 24 h.
151
The remaining undissolved film was removed after centrifugation at 3000 g for 10 min at 25 °C,
152
and then dried at 105 °C for 24 h. The test was carried out in triplicate. Film solubility was
153
determinated according to the following equation:
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FS (%) =
155
W i - Wf
100
Wi
156
where Wi was the initial weight expressed as dry matter and Wf was the weight of the
157
undissolved film residue. 7
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2.3.5. Color, light transmission and transparency Color of film samples was determined using a ColorFlex spectrocolorimeter (Hunter
160
Associates Laboratory, Inc., Reston, VA, USA). Color of the film was expressed as L*
161
(lightness/brightness), a* (redness/greenness) and b* (yellowness/blueness) values. Total
162
difference in color (E*) was calculated according to the following equation.
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E* = (L*)2 + (a*)2 + (b*)2
164
where L*, a* and b* are the differences between the corresponding color parameter of the
165
sample and that of white standard (L* = 92.84, a* = -1.25 and b* = 0.49).
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The barrier properties of composite films against ultraviolet (UV) and visible light were
167
measured at selected wavelengths between 200 and 800 nm, using a UV–Visible Recording
168
spectrophotometer. The transparency value of the film was calculated by the following equation:
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Transparency value = - log T600/x
170
where T600 is the fractional transmittance at 600 nm and x is the film thickness (mm). The greater
171
transparency value represents the lower transparency of films.
172
2.3.6. Fourier transform infrared spectroscopy
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FTIR spectra of films prepared with gelatin and/or chitosan were determined as described
174
by Jridi, et al. [18], using a Nicolet FTIR spectrometer equipped with an attenuated total
175
reflection (ATR) accessory.
176 177 178
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179
2.3.7. Thermal properties Conditioned films were scanned using a differential scanning calorimeter (Mettler Toledo
181
Star) from 0 to 100 °C at a rate of 5 °C/min. Nitrogen was used as the purge gas at a flow rate of
182
50 ml/min.
183
2.3.8. Microstructure
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Microstructure of cryo-fractured cross-section of the composite film samples was visualized
185
using a Scanning Electron Microscope (SEM) (Cambridge Scan-360 microscope) at an
186
accelerating voltage of 3.0 kV. The film samples were cryo-fractured by immersion in liquid
187
nitrogen. Prior to visualization, film samples were mounted on brass stub and sputtered with gold
188
in order to make the sample conductive. Samples were photographed with an angle of 90° to the
189
surface to allow observation of the films cross section.
190
2.4. Antioxidant activities of films
191
2.4.1. DPPH free radical-scavenging assay
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The DPPH free radical-scavenging activity of films was determined as described by
193
Bersuder et al. [21], with some modifications. The films were cut into small pieces (m = 10 mg)
194
and immersed in 500 µl of ethanol-DPPH solution ([DPPH] = 0.02 mM) and incubated 24 hours
195
with shaking at room temperature in the dark. The reduction of DPPH radical was measured at
196
517 nm using a UV-Visible spectrophotometer (T70, UV/VIS spectrometer, PG Instruments Ltd.,
197
China). The results were expressed as mmol equivalents of Vitamin C Equivalent per g of film,
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Page 9 of 34
based on standard curves previously prepared for Vitamin C. The test was carried out in
199
triplicate.
200
2.4.2. Reducing power assay
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The ability of films to reduce iron (III) was determined according to the method of Yildirim
202
et al. [22]. Films were cut into small pieces (m = 10 mg) and immersed in 1.25 ml phosphate
203
buffer (0.2 M, pH = 6.6) and 1.25 ml of 1% (w/v) potassium ferricyanide. The mixtures were
204
incubated for 3 h at 50 °C. Then, 1 ml was collected and 500 μl of 10 % (w/v) trichloroacetic acid
205
TCA was added to the mixture which is centrifuged 10 min at 10,000 g. Finally, 1.25 ml of the
206
supernatant solution of each sample mixture was mixed with 1.25 ml of distilled water and 0.25
207
ml of 0.1% (w/v) ferric chloride. After 10 min reaction time, the absorbance of the resulting
208
solutions was measured at 700 nm. The results were expressed as mmol equivalents of Vitamin C
209
Equivalent per g of film, based on standard curves previously prepared for Vitamin C. The test
210
was carried out in triplicate. The values are presented as the means of triplicate analyses.
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2.4.3. β-carotene-linoleate bleaching assay
213
The ability of films to prevent bleaching of β-carotene was determinated. 0.5 mg β-carotene
214
in 1 ml chloroform was mixed with 25 μl of linoleic acid and 200 μl of Tween-40. The
215
chloroform was completely evaporated under vacuum in a rotator evaporator at 40 °C, then 100
216
ml of bidistilled water were added, and the resulting mixture was vigorously stirred. The
217
emulsion obtained was freshly prepared before each experiment. Films were cut into small pieces
218
(m = 10 mg) and immersed in 2.5 ml of the β-carotene-linoleic acid emulsion. The tubes were
219
immediately placed in water bath and incubated at 50 °C for 60 min. Thereafter, the absorbance 10
Page 10 of 34
of each sample was measured at 470 nm. Solution of β-carotene-linoleic acid was used as control.
221
The results were expressed as mmol equivalents of Vitamin C Equivalent per g of film, based on
222
standard curves previously prepared for Vitamin C. The test was carried out in triplicate. Values
223
presented are the mean of triplicate analyses.
224
2.5. Antimicrobial activity of films
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The microorganisms used for antimicrobial activity were Micrococcus luteus (ATCC
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4698), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella
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pneumonia (ATCC 13883), Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC
228
25923) Salmonella typhi and Salmonella enteric.
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The antimicrobial activity of the film forming solutions and the resulted films was
230
investigated. Culture suspension (100 µL) of the tested microorganism about 106 colony forming
231
units (Cfu) was spread over the Luria Bertani (LB) agar. Then, wells (7 mm depth, 6 mm
232
diameter) were cut in the agar, and 60 µL of film forming solutions were delivered into them.
233
The antagonistic zones were detected after incubating for 24 h at 37 °C. On other hand, the films
234
were placed on the plate surfaces and incubated at 37 °C for 24 h. The appearance of a clear area
235
below or around the film was deemed to be positive for antimicrobial activity.
236
2.6. Statistical analysis
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Statistical analyses were performed with SPSS ver. 17.0, professional edition using
238
ANOVA analysis. Differences were considered significant at p < 0.05. All tests were carried out
239
in triplicate.
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240
3. Results and discussion
241
3.1. Mechanical properties Adequate mechanical strength and extensibility are generally required for packaging films
243
to withstand external stress and maintain its integrity during applications in packaging [23]. TS,
244
EAB and thickness of composite films and those based on gelatin or chitosan (control films), are
245
presented in Table 1.
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Film prepared with cuttlefish skin gelatin (G100) exhibited lower tensile strength (TS)
247
(40.26 MPa) but higher elongation at break (EAB) (4.76%) values. These values are in
248
accordance with those reported with Limpisophon et al. [24]. However, chitosan film showed
249
substantially higher TS (59.4 MPa) but lower EAB (1.26%) values (p < 0.05), than those of
250
gelatin and composite films. In composite films, it was observed that the increase of chitosan
251
content increases values of tensile strength, leading to stronger films as compared with gelatin
252
film. However, the presence of chitosan decreases significantly the EAB values (p 0.05).
299
This result is in agreement with previous reports on gelatin-chitosan films [4]. Differences in
300
transparency of films obtained with different mass ratio gelatin/chitosan might be due to the
301
formation of poly-anion/cation complexes [16]. Therefore, mechanical properties, thickness and
302
transparency of composite films were influenced by interaction between gelatin and chitosan.
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303
Color of films with different mass ratios of cuttlefish skin gelatin-chitosan is shown in
304
Table 3. In general, gelatin film appeared slightly clear which could be displayed by lightness 14
Page 14 of 34
(92.82) of the gelatin film. Chitosan film had lower values of a* (redness) and L* (lightness) but
306
higher b* (yellowness) and E* (difference in color) values, than those of composite films (p
307
c>d>e; p < 0.05).
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531 532
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533 534
538 539 540 541 542 543 544 545
te
537
Ac ce p
536
d
535
546 547 548 549
25
Page 25 of 34
Table 2. Light transmission and transparency
551
280
350
400
500
600
700
800
values
G100
0.01
0.3
48.0
67.9
76.3
85.4
85.2
90.1
0.60±0.01 c
G75/C25
0.01
0.8
49.3
72.6
77.9
84.2
86.2
G50/C50
0.01
1.0
42.7
70.9
79.6
85.1
88.3
G25/C75
0.05
1.5
38.7
70.4
79.1
84.6
89.3
90.5
0.92±0.12 b
C100
0.07
1.6
42.1
65.4
70.26
80.2
84.3
89.1
1.78±0.03 a
a,b,c,
563 564 565 566
cr
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562
0.85±0.10 b
d
556
561
90.6
an
555
560
0.99±0.12 b
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554
559
90.9
Different letters in the same column indicate significant differences (a>b>c, p < 0.05).
553
558
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552
557
Transparency
Wavelength (nm)
Films
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550
567 568 569 570 571
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Page 26 of 34
Table 3. Color and thermal properties of films Film
Color parameters
Thermal properties
a*
b*
E*
Tg (°C)
ΔHg (J/g)
G100
92.82±0.78 a
2.02±0.04 e
10.38±0.43 c
7.61±0.65 a
60.26
51.33
G75/C25
89.96±0.37 b
1.33±0.01 d
9.67±0.69 d
4.98±0.14 c
64.70
66.4
G50/C50
82.31±0.3 c
0.77±0.16 c
14.83±0.03 a
4.46±0.08 c
65.30
68.69
G25/C75
82.52±2.12 cd
0.38±0.21 b
11.75±0.22 b
2.92±0.45 d
68.90
73.94
C100
79.38±0.34 d
0.15±0.12 a
11.47±0.35 b
6.11±0.11 b
77.40
94.35
cr
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L*
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572
573
a,b,c,d
574
Values were given as mean ± standard deviation. Tg and ΔHg mean transition temperature and
575
enthalpy, respectively.
an
Different letters in the same column indicate significant differences (a>b>c>d; p < 0.05).
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576 577
581 582 583 584 585 586 587 588
te
580
Ac ce p
579
d
578
589 590 591 592 593
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Page 27 of 34
594
Table 4: Antioxidant activities of films. Results were expressed as mmol equivalents of Vitamin
595
C Equivalent per g of film (mmol Vit C equivalent/g film).
597
Values were given as mean ± standard deviation.
598
M
599 600
605 606 607 608 609 610 611
te
604
Ac ce p
603
d
601 602
an
596
us
cr
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Antioxidant Films activity G100 G75/C25 G50/C50 G25/C75 C100 Radical 7.24±0.25 a 5.32±0.32 b 2.65±0.1 c 1.10±0.03 d 1.24±0.2 e scavenging (DPPH) Ferric reducing 10.21±0.02 a 6.15±0.12 b 4.10±0.15 c 2.19±0.14 d 1.36±0.10 e ability β-carotene 0.32±0.01 a 0.2±0.02 b 0.99±0.04 c 0.44±0.07 d 0.13±0.01 e bleaching method a,b,c ,d,e Different letters in the same line indicate significant differences (a>b>c>d>e, p < 0.05).
612 613 614 615 616
28
Page 28 of 34
Table. 5. Antimicrobial activity of films.
618
Indicator organism
Gram +
621 622
G75/C25
G50/C50
G25/C75
C100
S. aureus
ND
ND
15±0.2 c
17±0.2 b
20±0.2 a
B. cereus
ND
14±0.1 c
14±0.5 c
16±0.2 b
25±0.1 a
M. luteus
ND
10±0.1 d
12±0.4 c
16±0.3 b
18±0.2 a
S. enterica
ND
12±0.3 c
13±0.3 b
18±1.1 a
17±0.3 a
K. pneumoniae
ND
13.2 c
14±0.4 c
15±0.3 b
17±0.4 a
E. coli
ND
14±0.6 b
10±0.6 c
15±0.7 a
16±0.6 a
S. typhimurium
ND
10±1.1 c
14±0.3 ab
13±1.1 b
15±1.1 a
an
Gram -
623 624 625 626 627 628 629
G100
ip t
620
Inhibition (mm)
cr
619
us
617
630
a,b,c,d
631
Values were given as mean ± standard deviation. ND: Not Detected.
Different letters in the same line indicate significant differences (a>b>c>d; p < 0.05).
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632
636 637 638 639 640 641 642 643
te
635
Ac ce p
634
d
633
644 645 646 647 648
29
Page 29 of 34
Fig. 1. Fourier transform infrared spectra of gelatin, chitosan and composite films.
649
Fig. 2. DSC profiles of gelatin, chitosan and composite films.
650
Fig. 3. SEM micrographs (cryo-fractured cross-section) of gelatin, chitosan and composite films.
ip t
648
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cr
651
652
an
653
M
654
658 659 660 661 662 663
te
657
Ac ce p
656
d
655
664 665 666 667 668
30
Page 30 of 34
669 670
Fig. 1. Fourier transform infrared spectra of chitosan, gelatin and composite films. G
Amide II
671
676
cr
675
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674
Absorbance
673
Amide I
Amide B
672
678 679
M
680 681
685 686 687 688 689 690 691
C50/G50 C75/G25
10
3500
3000
2500
d
4000
2000
C100
-1
1500
1000
500
Wavenumber (cm )
te
684
C25/G75
Ac ce p
683
G100
an
677
682
Amide III
ip t
Amide A
692 693 694 695 696
31
Page 31 of 34
697 698 699
Fig. 2. DSC profiles of chitosan, gelatin and composite films.
705 706 707
cr
704
us
703
708
713 714 715 716 717 718 719 720
40
50
d
712
0.1 W/g 60
te
711
C75/G25 C100
70
80
90
100
Temperature (°C)
Ac ce p
710
C50/G50
M
709
G100
C25/G75
an
702
Exothermal Heat Flux
701
ip t
700
721 722 723 724 725
32
Page 32 of 34
726 727 728 729
Fig. 3. SEM micrographs (cryo-fractured cross-section) of gelatin, chitosan and composite films.
731
C100
ip t
730
C50/G50
C75/G25
cr
732 733
us
734 735 736
C25/G75
G100
an
737 738
M
739 740
744 745 746 747 748 749 750 751
te
743
Ac ce p
742
d
741
752 753 754 755
33
Page 33 of 34
755
Combining gelatin and chitosan improves the properties of fish gelatin films.
757
Homogenous microstructure was observed for all films.
758
DSC analyses were demonstrated the good miscibility of the chitosan and gelatin
759
Film containing chitosan can be an effective antibacterial film-forming material
760
Antioxidant activities of film increased with increase solubility of film.
cr
ip t
756
us
761
Ac ce p
te
d
M
an
762
34
Page 34 of 34
S0141-8130(14)00226-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.03.054 BIOMAC 4266
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
9-2-2014 22-3-2014 29-3-2014
Please cite this article as: M. Jridi, S. Hajji, H.B. Ayed, I. Lassoued, A. Mbarek, M. Kammoun, N. Souissi, M. Nasri, Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite edible films., International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.03.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
Physical, structural, antioxidant and antimicrobial properties of gelatin-chitosan composite
3
edible films.
ip t
4
Mourad Jridi1*, Sawssan Hajji1, Hanen Ben Ayed1, Imen Lassoued1, Aïcha Mbarek2, Maher
6
Kammoun1, Nabil Souissi3 and Moncef Nasri1
cr
5
7
1. Laboratoire de Génie Enzymatique et de Microbiologie – Université de Sfax, Ecole Nationale
9
d’Ingénieurs de Sfax, B.P 1173-3038 Sfax, Tunisia.
us
8
2. Laboratoire de Chimie Industrielle, Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, B.P.
11
1173-3038 Sfax, Tunisia.
12
3. Laboratoire de Biodiversité et Biotechnologie Marine. Institut National des Sciences et Technologies de
13
la Mer. Centre de Sfax. BP 1035. 3018 Sfax, Tunisia.
M
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19 20 21 22 23 24 25
te
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*Corresponding author: Tel.: 216 28-142-818; fax: 216 74-275-595;
Ac ce p
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an
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E-mail address: [email protected] (M. Jridi)
26 27 28 29
1
Page 1 of 34
30 31
Abstract Physico-chemical and mechanical properties of cuttlefish skin gelatin (G), chitosan (C)
33
from shrimp (Penaeus kerathurus) and composite films (G75/C25, G50/C50, G25/C75)
34
plasticized with glycerol were investigated. The results indicated that chitosan film had higher
35
tensile strength and lower elongation at break when compared with the other films. Composite
36
films show no significant difference in tensile strength (TS), thickness and transparency. The
37
structural properties evaluated by FTIR and DSC showed total miscibility between both
38
polymers. DSC scans showed that the increase of chitosan content in the composite films
39
increases the transition temperature (Tg) and enthalpy (ΔHg) of films. The morphology study of
40
gelatin, chitosan and composite films showed a compact and homogenous structure. In addition,
41
gelatin and G75/C25 films demonstrated a high antioxidant activities monitored by β-carotene
42
bleaching, DPPH radical-scavenging and reducing power activity, while films contained chitosan
43
exhibited higher antimicrobial activity against Gram-positive than Gram-negative bacteria.
44
Ac ce p
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an
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32
45
Keywords: Composite gelatin-chitosan films; Microstructure; Antioxidant and antibacterial
46
activity.
47 48 49 50 51 52
2
Page 2 of 34
53 54
1. Introduction Given the potential application of gelatin ad chitosan as eco-friendly active packaging
56
materials, it is of great interest to study their use together in the preparation of edible films.
57
Indeed, to our knowledge few works have been conducted on edible films based on gelatin and
58
chitosan polymers [1, 2, 3, 4]. These biodegradable films improve in general the preservation of
59
food, mainly by acting as barriers to water, oxygen and light. Gelatin has been reported to
60
develop edible films due to its functional properties and biodegradability [5]. Bovine and porcine
61
wastes are the most frequent sources to obtain gelatin of good quality. However, other sources of
62
gelatin are becoming increasingly relevant, such as fish bones and skins [6].
M
an
us
cr
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55
Fish skin, which is a major by-product of the fish-processing industry, causing waste and
64
pollution, could provide a valuable source of gelatin [7]. However, films based on fish gelatins
65
have relatively poor water vapor permeability, mechanical properties and water resistance, which
66
may limit their use as potential packaging materials. One of the effective strategies used to
67
improve the physical performance of gelatin films is to elaborate composite films by mixing fish
68
gelatin with other biopolymers exhibiting film forming properties and derived from renewable
69
resources such as chitosan [4].
Ac ce p
te
d
63
70
Chitosan is a cationic polysaccharide with excellent film forming properties. It is obtained
71
from chitin by deacetylation in the presence of alkali [8]. This polysaccharide is widely utilized
72
not only for its film-forming ability but also to its antimicrobial properties [2, 9]. Indeed, the
73
antimicrobial properties of chitosan and its derivated (chitosan-oligo-saccharides) have been
74
largely reported in the literature [10, 11, 12, 13], thereby encouraging its use as potential
75
packaging material. 3
Page 3 of 34
Composite films of chitosan and gelatin have been reported to have improved mechanical,
77
transport and physical properties compared with those of single polymer based films [2, 4]. In
78
addition various cross-linkers [1] (glutaraldehyde, transglutaminase, carbodiimide) have been
79
used to improve the physical performance of fish gelatin films [14, 15]. The interactions between
80
gelatin and chitosan have been probed by infrared spectroscopy, X-ray diffraction and pH
81
titrations [16]. In the formation of composite film it is important to study the compatibility of its
82
components and intermolecular interactions that may occur between them, since they finally
83
affect the film structure and determine the film properties.
us
cr
ip t
76
Despite the numerous works reported the effect of chitosan addition on chemical, physical
85
and biological properties of the fish gelatin films, information about fish gelatin-chitosan
86
composite films is limited.
M
an
84
In a previous work we investigated the physical and mechanical properties of edible films
88
made from cuttlefish (Sepia officinalis) skin gelatins obtained by pretreatment with different
89
pepsin concentrations [17]. This paper is a continuation of this work and is focused on
90
investigation of physical, mechanical and antioxidant properties of cuttlefish skin gelatin and/or
91
chitosan edible films. Antimicrobial effect of edible films over Gram-positive and Gram-negative
92
bacteria was also investigated.
93
2. Materials and methods
94
2.1. Gelatin preparation
Ac ce p
te
d
87
95
Skin from cuttlefish (S. officinalis) was obtained from the fish market of Sfax City, Tunisia.
96
Cuttlefish skin was cut into small pieces (1 cm x 1cm) and then soaked in 0.05 M NaOH (1:10 4
Page 4 of 34
w/v). The mixture was stirred for 2 h at 4 °C and alkaline solution was changed every 30. The
98
alkaline-treated skins were then washed with distilled water until a neutral pH was obtained. The
99
alkaline-treated skin was soaked in 100 mM glycin-HCl buffer, pH 2.0 with a solid/solvent ratio
100
of 1:10 (w/v) and subjected to hydrolysis collagen with 5 units of pepsin /g of skin, as described
101
in our previous study [18]. Cuttlefish skin gelatin (G) obtained was used for films preparation.
102
2.2. Chitosan preparation
us
cr
ip t
97
Chitosan was prepared from shrimp (Penaeus kerathurus) waste as described by Hajji et al.
104
[19]. Deproteinization was carried out in a thermostated stirred Pyrex reactor (300 ml).
105
Shrimp waste homogenate (15 g) was mixed with 45 ml distilled water. The pH of the
106
mixture was adjusted to 9.0. Then, the shrimp waste proteins were digested with crude enzyme
107
from Bacillus mojavensis A21 at 50 °C. The reaction was then stopped by heating the
108
solution at 90 °C during 20 min to inactivate enzymes. The solid phase was washed and
109
then pressed manually through four layers of gauze. Solid fractions obtained was treated
110
with 1.5 M HCl in 1:10 (w/v) ratio for 6 h at 50 °C under constant stirring (150 rpm).
111
The chitin product was filtered through four layers of gauze with the aid of a vacuum
112
pump and washed to neutrality with deionized water and then dried for 1 h at 60 °C. The
113
purified chitin was treated with 12.5 M NaOH in 1:10 (w/v) ratio at 140 °C for 4 h until
114
it was deacetylated to chitosan. After filtration, the residue was washed with deionized
115
water, and the crude chitosan was obtained by drying in a dry heat incubator at 50 °C
116
overnight. The deacetylation degree of resulted shrimp chitosan (C) was about 88%.
Ac ce p
te
d
M
an
103
117
5
Page 5 of 34
118
2.3. Edible films preparation and characterization
119
2.3.1. Preparation of composite films To prepare film forming solutions, cuttlefish skin gelatin powder was dissolved in distilled
121
water, and chitosan in 1% (v/v) acetic acid, to achieve the final concentration of 4% and 2%
122
(w/v), respectively. Glycerol was added as plasticizer to the gelatin and/or chitosan solutions at a
123
level of 15% (based on protein and/or polysaccharide). The film forming solutions were
124
incubated at 40 °C for 30 min with gentle stirring.
us
cr
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120
The composite (Gelatin/Chitosan) solutions were obtained by mixing film forming
126
solutions of gelatin and chitosan at different ratio (G75:C25, G50:C50, G25:C75). The resulting
127
mixtures were gently stirred during 30 min at 40 °C.
M
an
125
In all formulations, 20 mL of each solution was cast onto a rimmed silicone resin plate (6
129
cm x 6 cm), air-blown for 12 h at room temperature (25 °C) and dried at a temperature of 25 °C
130
and 50% relative humidity for 48 h. Dried films were manually peeled off and then subjected to
131
analyses. Composite films prepared were referred as G75/C25, G50/C50 and G25/C75. Films,
132
prepared by using gelatin (G100) or chitosan (C100) were used as control.
Ac ce p
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d
128
133
Prior to mechanical properties testing, films were conditioned for 48 h at 25 °C. For FTIR
134
and DSC studies, films were conditioned in a dessicator containing dried silica gel for 3 weeks at
135
room temperature (25 °C) to obtain the most dehydrated films.
136
2.3.2. Film thickness
6
Page 6 of 34
The thickness of films was measured using a micrometer (Mitutoyo, Model ID-C112PM,
138
Kawasaki-shi, Japan). Ten random locations around each film sample were used for thickness
139
determination.
140
2.3.3. Mechanical properties
ip t
137
TS and EAB of film samples were determined as described by Iwata et al. [20] using the
142
Universal Testing Machine (Lloyd Instrument, Hampshire, UK). The test was performed in the
143
controlled room at 25 °C and 50±5% RH. Ten film samples (2 cm × 4 cm) with the initial grip
144
length of 3 cm were used for testing. The film samples were clamped and deformed under tensile
145
loading using a 100 N load cell with the cross head speed of 30 mm/min until the samples were
146
broken. The maximum load and the final extension at break were used for calculation of TS and
147
EAB, respectively.
148
2.3.4. Water solubility
te
d
M
an
us
cr
141
Film samples (3 cm x 2 cm) were weighed and placed in 50 ml-centrifuge tube containing
150
10 ml of distilled water with 0.1% (w/v) sodium azide, and stirred at room temperature for 24 h.
151
The remaining undissolved film was removed after centrifugation at 3000 g for 10 min at 25 °C,
152
and then dried at 105 °C for 24 h. The test was carried out in triplicate. Film solubility was
153
determinated according to the following equation:
Ac ce p
149
154
FS (%) =
155
W i - Wf
100
Wi
156
where Wi was the initial weight expressed as dry matter and Wf was the weight of the
157
undissolved film residue. 7
Page 7 of 34
158
2.3.5. Color, light transmission and transparency Color of film samples was determined using a ColorFlex spectrocolorimeter (Hunter
160
Associates Laboratory, Inc., Reston, VA, USA). Color of the film was expressed as L*
161
(lightness/brightness), a* (redness/greenness) and b* (yellowness/blueness) values. Total
162
difference in color (E*) was calculated according to the following equation.
cr
ip t
159
E* = (L*)2 + (a*)2 + (b*)2
164
where L*, a* and b* are the differences between the corresponding color parameter of the
165
sample and that of white standard (L* = 92.84, a* = -1.25 and b* = 0.49).
an
us
163
The barrier properties of composite films against ultraviolet (UV) and visible light were
167
measured at selected wavelengths between 200 and 800 nm, using a UV–Visible Recording
168
spectrophotometer. The transparency value of the film was calculated by the following equation:
M
166
Transparency value = - log T600/x
170
where T600 is the fractional transmittance at 600 nm and x is the film thickness (mm). The greater
171
transparency value represents the lower transparency of films.
172
2.3.6. Fourier transform infrared spectroscopy
Ac ce p
te
d
169
173
FTIR spectra of films prepared with gelatin and/or chitosan were determined as described
174
by Jridi, et al. [18], using a Nicolet FTIR spectrometer equipped with an attenuated total
175
reflection (ATR) accessory.
176 177 178
8
Page 8 of 34
179
2.3.7. Thermal properties Conditioned films were scanned using a differential scanning calorimeter (Mettler Toledo
181
Star) from 0 to 100 °C at a rate of 5 °C/min. Nitrogen was used as the purge gas at a flow rate of
182
50 ml/min.
183
2.3.8. Microstructure
cr
ip t
180
Microstructure of cryo-fractured cross-section of the composite film samples was visualized
185
using a Scanning Electron Microscope (SEM) (Cambridge Scan-360 microscope) at an
186
accelerating voltage of 3.0 kV. The film samples were cryo-fractured by immersion in liquid
187
nitrogen. Prior to visualization, film samples were mounted on brass stub and sputtered with gold
188
in order to make the sample conductive. Samples were photographed with an angle of 90° to the
189
surface to allow observation of the films cross section.
190
2.4. Antioxidant activities of films
191
2.4.1. DPPH free radical-scavenging assay
Ac ce p
te
d
M
an
us
184
192
The DPPH free radical-scavenging activity of films was determined as described by
193
Bersuder et al. [21], with some modifications. The films were cut into small pieces (m = 10 mg)
194
and immersed in 500 µl of ethanol-DPPH solution ([DPPH] = 0.02 mM) and incubated 24 hours
195
with shaking at room temperature in the dark. The reduction of DPPH radical was measured at
196
517 nm using a UV-Visible spectrophotometer (T70, UV/VIS spectrometer, PG Instruments Ltd.,
197
China). The results were expressed as mmol equivalents of Vitamin C Equivalent per g of film,
9
Page 9 of 34
based on standard curves previously prepared for Vitamin C. The test was carried out in
199
triplicate.
200
2.4.2. Reducing power assay
ip t
198
The ability of films to reduce iron (III) was determined according to the method of Yildirim
202
et al. [22]. Films were cut into small pieces (m = 10 mg) and immersed in 1.25 ml phosphate
203
buffer (0.2 M, pH = 6.6) and 1.25 ml of 1% (w/v) potassium ferricyanide. The mixtures were
204
incubated for 3 h at 50 °C. Then, 1 ml was collected and 500 μl of 10 % (w/v) trichloroacetic acid
205
TCA was added to the mixture which is centrifuged 10 min at 10,000 g. Finally, 1.25 ml of the
206
supernatant solution of each sample mixture was mixed with 1.25 ml of distilled water and 0.25
207
ml of 0.1% (w/v) ferric chloride. After 10 min reaction time, the absorbance of the resulting
208
solutions was measured at 700 nm. The results were expressed as mmol equivalents of Vitamin C
209
Equivalent per g of film, based on standard curves previously prepared for Vitamin C. The test
210
was carried out in triplicate. The values are presented as the means of triplicate analyses.
212
us
an
M
d
te
Ac ce p
211
cr
201
2.4.3. β-carotene-linoleate bleaching assay
213
The ability of films to prevent bleaching of β-carotene was determinated. 0.5 mg β-carotene
214
in 1 ml chloroform was mixed with 25 μl of linoleic acid and 200 μl of Tween-40. The
215
chloroform was completely evaporated under vacuum in a rotator evaporator at 40 °C, then 100
216
ml of bidistilled water were added, and the resulting mixture was vigorously stirred. The
217
emulsion obtained was freshly prepared before each experiment. Films were cut into small pieces
218
(m = 10 mg) and immersed in 2.5 ml of the β-carotene-linoleic acid emulsion. The tubes were
219
immediately placed in water bath and incubated at 50 °C for 60 min. Thereafter, the absorbance 10
Page 10 of 34
of each sample was measured at 470 nm. Solution of β-carotene-linoleic acid was used as control.
221
The results were expressed as mmol equivalents of Vitamin C Equivalent per g of film, based on
222
standard curves previously prepared for Vitamin C. The test was carried out in triplicate. Values
223
presented are the mean of triplicate analyses.
224
2.5. Antimicrobial activity of films
cr
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220
The microorganisms used for antimicrobial activity were Micrococcus luteus (ATCC
226
4698), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella
227
pneumonia (ATCC 13883), Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC
228
25923) Salmonella typhi and Salmonella enteric.
an
us
225
The antimicrobial activity of the film forming solutions and the resulted films was
230
investigated. Culture suspension (100 µL) of the tested microorganism about 106 colony forming
231
units (Cfu) was spread over the Luria Bertani (LB) agar. Then, wells (7 mm depth, 6 mm
232
diameter) were cut in the agar, and 60 µL of film forming solutions were delivered into them.
233
The antagonistic zones were detected after incubating for 24 h at 37 °C. On other hand, the films
234
were placed on the plate surfaces and incubated at 37 °C for 24 h. The appearance of a clear area
235
below or around the film was deemed to be positive for antimicrobial activity.
236
2.6. Statistical analysis
Ac ce p
te
d
M
229
237
Statistical analyses were performed with SPSS ver. 17.0, professional edition using
238
ANOVA analysis. Differences were considered significant at p < 0.05. All tests were carried out
239
in triplicate.
11
Page 11 of 34
240
3. Results and discussion
241
3.1. Mechanical properties Adequate mechanical strength and extensibility are generally required for packaging films
243
to withstand external stress and maintain its integrity during applications in packaging [23]. TS,
244
EAB and thickness of composite films and those based on gelatin or chitosan (control films), are
245
presented in Table 1.
us
cr
ip t
242
Film prepared with cuttlefish skin gelatin (G100) exhibited lower tensile strength (TS)
247
(40.26 MPa) but higher elongation at break (EAB) (4.76%) values. These values are in
248
accordance with those reported with Limpisophon et al. [24]. However, chitosan film showed
249
substantially higher TS (59.4 MPa) but lower EAB (1.26%) values (p < 0.05), than those of
250
gelatin and composite films. In composite films, it was observed that the increase of chitosan
251
content increases values of tensile strength, leading to stronger films as compared with gelatin
252
film. However, the presence of chitosan decreases significantly the EAB values (p 0.05).
299
This result is in agreement with previous reports on gelatin-chitosan films [4]. Differences in
300
transparency of films obtained with different mass ratio gelatin/chitosan might be due to the
301
formation of poly-anion/cation complexes [16]. Therefore, mechanical properties, thickness and
302
transparency of composite films were influenced by interaction between gelatin and chitosan.
Ac ce p
295
303
Color of films with different mass ratios of cuttlefish skin gelatin-chitosan is shown in
304
Table 3. In general, gelatin film appeared slightly clear which could be displayed by lightness 14
Page 14 of 34
(92.82) of the gelatin film. Chitosan film had lower values of a* (redness) and L* (lightness) but
306
higher b* (yellowness) and E* (difference in color) values, than those of composite films (p
307
c>d>e; p < 0.05).
an
531 532
M
533 534
538 539 540 541 542 543 544 545
te
537
Ac ce p
536
d
535
546 547 548 549
25
Page 25 of 34
Table 2. Light transmission and transparency
551
280
350
400
500
600
700
800
values
G100
0.01
0.3
48.0
67.9
76.3
85.4
85.2
90.1
0.60±0.01 c
G75/C25
0.01
0.8
49.3
72.6
77.9
84.2
86.2
G50/C50
0.01
1.0
42.7
70.9
79.6
85.1
88.3
G25/C75
0.05
1.5
38.7
70.4
79.1
84.6
89.3
90.5
0.92±0.12 b
C100
0.07
1.6
42.1
65.4
70.26
80.2
84.3
89.1
1.78±0.03 a
a,b,c,
563 564 565 566
cr
te Ac ce p
562
0.85±0.10 b
d
556
561
90.6
an
555
560
0.99±0.12 b
M
554
559
90.9
Different letters in the same column indicate significant differences (a>b>c, p < 0.05).
553
558
ip t
200
552
557
Transparency
Wavelength (nm)
Films
us
550
567 568 569 570 571
26
Page 26 of 34
Table 3. Color and thermal properties of films Film
Color parameters
Thermal properties
a*
b*
E*
Tg (°C)
ΔHg (J/g)
G100
92.82±0.78 a
2.02±0.04 e
10.38±0.43 c
7.61±0.65 a
60.26
51.33
G75/C25
89.96±0.37 b
1.33±0.01 d
9.67±0.69 d
4.98±0.14 c
64.70
66.4
G50/C50
82.31±0.3 c
0.77±0.16 c
14.83±0.03 a
4.46±0.08 c
65.30
68.69
G25/C75
82.52±2.12 cd
0.38±0.21 b
11.75±0.22 b
2.92±0.45 d
68.90
73.94
C100
79.38±0.34 d
0.15±0.12 a
11.47±0.35 b
6.11±0.11 b
77.40
94.35
cr
ip t
L*
us
572
573
a,b,c,d
574
Values were given as mean ± standard deviation. Tg and ΔHg mean transition temperature and
575
enthalpy, respectively.
an
Different letters in the same column indicate significant differences (a>b>c>d; p < 0.05).
M
576 577
581 582 583 584 585 586 587 588
te
580
Ac ce p
579
d
578
589 590 591 592 593
27
Page 27 of 34
594
Table 4: Antioxidant activities of films. Results were expressed as mmol equivalents of Vitamin
595
C Equivalent per g of film (mmol Vit C equivalent/g film).
597
Values were given as mean ± standard deviation.
598
M
599 600
605 606 607 608 609 610 611
te
604
Ac ce p
603
d
601 602
an
596
us
cr
ip t
Antioxidant Films activity G100 G75/C25 G50/C50 G25/C75 C100 Radical 7.24±0.25 a 5.32±0.32 b 2.65±0.1 c 1.10±0.03 d 1.24±0.2 e scavenging (DPPH) Ferric reducing 10.21±0.02 a 6.15±0.12 b 4.10±0.15 c 2.19±0.14 d 1.36±0.10 e ability β-carotene 0.32±0.01 a 0.2±0.02 b 0.99±0.04 c 0.44±0.07 d 0.13±0.01 e bleaching method a,b,c ,d,e Different letters in the same line indicate significant differences (a>b>c>d>e, p < 0.05).
612 613 614 615 616
28
Page 28 of 34
Table. 5. Antimicrobial activity of films.
618
Indicator organism
Gram +
621 622
G75/C25
G50/C50
G25/C75
C100
S. aureus
ND
ND
15±0.2 c
17±0.2 b
20±0.2 a
B. cereus
ND
14±0.1 c
14±0.5 c
16±0.2 b
25±0.1 a
M. luteus
ND
10±0.1 d
12±0.4 c
16±0.3 b
18±0.2 a
S. enterica
ND
12±0.3 c
13±0.3 b
18±1.1 a
17±0.3 a
K. pneumoniae
ND
13.2 c
14±0.4 c
15±0.3 b
17±0.4 a
E. coli
ND
14±0.6 b
10±0.6 c
15±0.7 a
16±0.6 a
S. typhimurium
ND
10±1.1 c
14±0.3 ab
13±1.1 b
15±1.1 a
an
Gram -
623 624 625 626 627 628 629
G100
ip t
620
Inhibition (mm)
cr
619
us
617
630
a,b,c,d
631
Values were given as mean ± standard deviation. ND: Not Detected.
Different letters in the same line indicate significant differences (a>b>c>d; p < 0.05).
M
632
636 637 638 639 640 641 642 643
te
635
Ac ce p
634
d
633
644 645 646 647 648
29
Page 29 of 34
Fig. 1. Fourier transform infrared spectra of gelatin, chitosan and composite films.
649
Fig. 2. DSC profiles of gelatin, chitosan and composite films.
650
Fig. 3. SEM micrographs (cryo-fractured cross-section) of gelatin, chitosan and composite films.
ip t
648
us
cr
651
652
an
653
M
654
658 659 660 661 662 663
te
657
Ac ce p
656
d
655
664 665 666 667 668
30
Page 30 of 34
669 670
Fig. 1. Fourier transform infrared spectra of chitosan, gelatin and composite films. G
Amide II
671
676
cr
675
us
674
Absorbance
673
Amide I
Amide B
672
678 679
M
680 681
685 686 687 688 689 690 691
C50/G50 C75/G25
10
3500
3000
2500
d
4000
2000
C100
-1
1500
1000
500
Wavenumber (cm )
te
684
C25/G75
Ac ce p
683
G100
an
677
682
Amide III
ip t
Amide A
692 693 694 695 696
31
Page 31 of 34
697 698 699
Fig. 2. DSC profiles of chitosan, gelatin and composite films.
705 706 707
cr
704
us
703
708
713 714 715 716 717 718 719 720
40
50
d
712
0.1 W/g 60
te
711
C75/G25 C100
70
80
90
100
Temperature (°C)
Ac ce p
710
C50/G50
M
709
G100
C25/G75
an
702
Exothermal Heat Flux
701
ip t
700
721 722 723 724 725
32
Page 32 of 34
726 727 728 729
Fig. 3. SEM micrographs (cryo-fractured cross-section) of gelatin, chitosan and composite films.
731
C100
ip t
730
C50/G50
C75/G25
cr
732 733
us
734 735 736
C25/G75
G100
an
737 738
M
739 740
744 745 746 747 748 749 750 751
te
743
Ac ce p
742
d
741
752 753 754 755
33
Page 33 of 34
755
Combining gelatin and chitosan improves the properties of fish gelatin films.
757
Homogenous microstructure was observed for all films.
758
DSC analyses were demonstrated the good miscibility of the chitosan and gelatin
759
Film containing chitosan can be an effective antibacterial film-forming material
760
Antioxidant activities of film increased with increase solubility of film.
cr
ip t
756
us
761
Ac ce p
te
d
M
an
762
34
Page 34 of 34
