Colloids and Surfaces A 539 (2018) 184–191

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Colloids and Surfaces A

journal homepage: www.elsevier.com/locate/colsurfa

Crosslinking agents effect on gelatins from carp and tilapia skins and in their T
biopolymeric films
⁎
Jaqueline P. Santosa, Vanessa M. Esquerdoa, Catarina M. Mourab, Luiz A.A. Pintoa,
a
School of Chemistry and Food, Federal University of Rio Grande–FURG, km 8 Itália Avenue, 96203–900, Rio Grande, RS, Brazil
b
Food Engineering, Federal University of Pampa–UNIPAMPA, 96400–100, Bagé, RS, Brazil

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: The extracted gelatins from carp and tilapia skins were chemically crosslinked by electrolytes (NaCl and MgSO4)
Biodegradable films and nonelectrolytes (gallic acid and citric acid) agents and, the fish gelatins films were produced by casting.
Chemical crosslinking Rheological, chemical and physicochemical properties were evaluated. All crosslinked films showed a significant
Electrolytes agents increase in tensile strength and a slight reduction in elongation compared to non–crosslinked films. The cross-
Fish skin
linked films with nonelectrolytes presented the highest reductions in water vapor permeability. Fourier
Gelatin
Nonelectrolytes agents
Transform Infrared spectra, X–Ray Diffraction and Scanning Electron Microscopy showed the interactions be-
tween protein/agents and the changes in amorphous structure of the films, which presented surfaces in chains of
twisted shape. Differential Scanning Calorimetry demonstrated that the incorporations of gallic acid and citric
acid improved the films thermal stability. The results showed that the chemical crosslinking can be used to
improve the quality of fish skin gelatin films and, thus, these films can be an alternative to food packaging, as
well to improve added value of the fish waste.

⁎
Corresponding author.
E-mail addresses: jaquelinepozzada@hotmail.com (J.P. Santos), nessafurg@gmail.com (V.M. Esquerdo), cmalimentos1@yahoo.com.br (C.M. Moura),
dqmpinto@furg.br, dqmpinto@terra.com.br (L.A.A. Pinto).

https://doi.org/10.1016/j.colsurfa.2017.12.018
Received 19 September 2017; Received in revised form 5 December 2017; Accepted 7 December 2017
Available online 12 December 2017
0927-7757/ © 2017 Elsevier B.V. All rights reserved.
J.P. Santos et al.
1. Introduction
The increasing of environmental pollution and serious ecological
problems caused by packaging derived from petroleum has led to in-
terest in the use of natural polymers to produce biodegradable materials
packaging [1]. Gelatin is considered a promising option as a raw ma-
terial for food packaging, providing low cost, film forming ability, high
availability and biodegradability [2]. Gelatin is obtained by controlled
hydrolysis of the insoluble collagen present in skins and bones of bo-
vine, pork and fish. Marine gelatin sources, especially fish skins, has
increased as alternative to mammal sources, due to the halal and kosher
markets, and the concern with pathogens transmission, such as bovine
spongiform encephalopathy [3]. Worldwide, the carp is the most
exploited fish in aquaculture, followed by tilapia, with an annual output
reaching 3.1 million tons [4,5]. In aquaculture, common carp (Cyprinus
carpio) has led to the development of numerous production systems, in
both, temperate and tropical regions, due to the combination of several
factors, such as feeding habits at a low level of the food chain, high
survival and good growth performance under its culture conditions and,
also, tolerance to high variations in water quality and diseases [6]. Nile
tilapia (Oreochromis niloticus) was widely introduced for aquaculture,
grown in extensive or semi-intensive traditional ponds, which performs
well in different breeding regimes [7]. Most of these fish are being
processed into fillets and exported, and the amount of organic waste
generated in the fishery processing plants can reach 70% by fish weight
[8]. The development of fish skin based products provides value addi-
tion and management of the waste disposed of in the environment.
Protein–based films, such as gelatin, present relatively higher water
vapor permeability and low mechanical forces, being these the main
disadvantages of gelatin films application in packaging materials [9].
Several studies have been conducted to repair this deficiency, including
the development of blends films and bilayers [10], nanoparticles ad-
dition [11] and chemical modification with crosslinkers [12]. The
chemical crosslinking provides bonds between reactive groups present
in the gelatin, resulting in improvements in mechanical properties,
thermal resistance and water permeability of the films [13]. Despite
this, a lower cost crosslinking method, efficient and non–toxic is still a
challenge for the use of gelatin in the food and pharmaceutical in-
dustries [14,15].
Electrolytes, such as sodium chloride (NaCl) and magnesium sulfate
(MgSO4), can affect the gelatin via electrostatic forces and the forma-
tion of saline bridges [16]. Nonelectrolytes, such as gallic acid (GA) and
citric acid (CA), can affect gelatin gel properties, due to the moisturizer
effect improving the gel stability [17]. These crosslinking agents (NaCl,
MgSO4, GA and CA) are interesting to improve the films characteristics
because show low cost, are not toxic to the human health and do not
harm the environment. The improvement in quality of biopolymers
films facilitates their competition with non–biodegradable packaging
and, also, could to added value to an industrial waste. However, the
changes caused by the crosslinked in the film produced by fish skin
gelatins have not been elucidated.
Thus, the aim of this study was to evaluate the addition of elec-
trolytes (NaCl and MgSO4) and nonelectrolytes (GA and CA) in the
protein matrix, to improve the mechanical properties and water vapor
permeability of films produced by crosslinked gelatins from fish skins.
The gelatins from tilapia skin and carp skin were produced, afterwards
chemically crosslinked and, characterized. The crosslinked gelatin films
were characterized by physical and mechanical properties, DSC, FT–IR,
XRD and scanning electron microscope.
2. Material and methods
2.1. Materials and chemicals
The skins of Nile tilapia (Oreochromis niloticus) and common carp
(Cyprinus carpio) were obtained from fish farmers in local units (Rio
185
Colloids and Surfaces A 539 (2018) 184–191
Grande do Sul, Brazil), and were stored at –20 °C until use. All chemi-
cals were of analytical grades. The crosslinkers NaCl and MgSO4 was
purchased from Synth (Brazil), gallic acid from Vetec (Brazil) and citric
acid from Merck (Brazil). For comparison purposes, was use mamma-
lian gelatin from bovine skin (type B, 260 g Bloom), obtained from
Sigma–Aldrich Chemical Co. (USA).
2.2. Fish gelatin extraction and modification
The pre–treatment of the fish skins and the gelatin extraction were
according to Bandeira et al. [18]. The skins were cut (1 cm²) and added
in distilled water (1: 1 w v–1) and, then, it was carried out the swelling
process with the first alkali treatment (3 mol L–1 NaOH, pH 11 and
15 min). The material was drained and, the second alkaline treatment
was performed (NaOH 3 mol L–1, pH 11 and 60 min). Finally, the acid
treatment started with the skins suspended in distilled water
(1:1 w v–1), in pH 2 (HCl 3 mol L–1) for 15 min. Gelatin extraction was
performed in a thermostatic bath (52 °C for 120 min and pH 4). The
clarification process of gelatin solution, for removing impurities, was
carried out by filtration according to Silva et al. [19]. The gelatin so-
lutions were clarified with activated charcoal (1 g kg–1 solution,
120 min at 35 °C).
The gelatin solutions from tilapia skin and carp skin were lyophi-
lized (Liotop, L108, Brazil) and, stored in sealed plastic bags at –20 °C.
The gelatins samples (100 mL of 667 g L–1) were crosslinked with
electrolytes and non-electrolytes, which were selected from the positive
effects on the properties of gels (gel strength, viscosity, melting point,
among others). The following chemicals concentrations were used:
MgSO4 at 0,8 mol L–1 (Mg) and NaCl at 0,3 mol L–1 (Na) [20], gallic acid
at 002 mol L–1 (GA) [21] and citric acid at 003 mol L–1 (CA) (de-
termined in preliminary tests). To facilitate cross–linking between the
chemical agents and the gelatin molecules, the gels of tilapia skin and
carp skin were kept under constant stirring in a water bath at 45 °C by
30 min.
2.3. Fish gelatins analysis
The gelatin solutions yield was calculated according to Eq. 1.
Cg × VS
Rg = ( ) × 100
mi (1)
where Rg is the gelatin yield (g 100 g–1
skin), Cg is the protein concentration
of the gelatin solution (g mL–1), Vs is the volume of extracted gelatin
solution (mL), and mi is the initial mass of the fish skin (g).
The hydroxyproline content in the samples was determined by
AOAC official method (990.26) [22], using L-hydroxyproline (Sig-
ma–Aldrich, USA) as standard, calculated by Eq. 2.
2.5 × h ⎞
Hyp=⎛ × 100
⎝m × V ⎠ (2)
–1
where Hyp is hydroxyproline content in the sample (g 100 g ), h is the
value read from the standard curve spectrophotometer, m is the sample
mass (g) and V is the filtrate volume (mL).
The determination of the amino acids present in the gelatin samples
was performed by high performance liquid chromatography (HPLC,
Shimadzu, Japan). The gel strength determination was according to the
AOAC official method 948.21 [23], measured using a texturometer
(TA.XTplus, Stable Micro Systems, UK). The samples viscosity was
calculated by Eq. 3 [19].
μ=t×K×ρ (3)
where μ is the gelatin viscosity (cP), t is the time (s), K is the viscosi-
meter constant and ρ is the density of gelatin solution on test tem-
perature, which was determined by picnometry (g cm–3).
The melting point of the gelatin sample was determined by BSI of-
ficial method 755 [24] using a solution of chloroform and methylene
J.P. Santos et al. Colloids and Surfaces A 539 (2018) 184–191

blue dye (3:1 v:v) as the indicator. SDS–polyacrylamide gel electro- Table 1
phoresis (SDS–PAGE) was carried out by method of Laemmli [25] with Amino acid compositions of the gelatins from tilapia skin and carp skin.
some modifications. The gelatin sample (2 mg) was dissolved in 1 mL of
Amino acids gamino acids (100 g–1protein)
10% (v:v) SDS and, then, heated at 85 °C for 1 h. The supernatant was
mixed with 1 mL of sample buffer (0.15 mol L–1 tris–HCl, pH 6.8, con- Tilapia skin gelatin Carp skin gelatin
taining 10% (v:v) SDS, 0.02% bromophenol and glycerol, 20% (v:v)
Threonine < LOQ < LOQ
2–mercaptoethanol). The samples and the standard protein marker
Serine < LOQ < LOQ
were loaded on a polyacrylamide gel made of 7.5% (v:v) separating gel Proline 12.8 11.2
and 3.8% (v:v) stacking gel, and subjected to electrophoresis at a Hydroxyproline 8.4 8.1
constant current of 25 mA and 150 V for about 3 h. The gelatin mole- Glycine 28.0 26.2
cular weight was estimated using standards of high molecular weight of Alanine 1.9 1.6
Cysteine 0.9 0.7
GE Healthcare Life Sciences (USA).
Valine 0.1 0.2
Methionine 0.6 0.8
Isoleucine 2.9 2.3
2.4. Films preparation with crosslinked gelatin Leucine 1.3 1.2
Tyrosine 1.3 1.2
Phenylalanine 1.1 1.3
The crosslinked fish gelatin films were produced by casting tech-
Lysine 1.1 2.0
nique. In gelatin solutions (50 mL) with concentration of 2% (w:v) was Arginine 3.9 5.0
added 0.20 g of glycerol, and remained under constant stirring for
30 min [18]. The filmogenic solutions of crosslinked gelatin (tilapia or LOQ: Limit of quantitation.
carp skin) were poured in acrylic plates and inserted into a drier with
forced air circulation, at 40 °C for 24 h, to complete solvent evapora- 3. Results and discussion
tion. The films were removed from the plates and placed into desiccator
at 25 °C and 75% relative humidity, for at least 48 h before the analyzes. 3.1. Characterization of crosslinked gelatins of Nile tilapia and common
carp

2.5. Characterization of crosslinked gelatin films
Tensile strength (TS) and elongation at break (EAB) of crosslinked
gelatin films were determined according to the official method
D00882–00 [26], using a texturometer (Stable Microsystems, SMD
TA.XP2i, UK). Samples thicknesses were measured using a micrometer
(Mitutoyo Manufacturing Co. Ltd., Japan), with accuracy of 0.001 mm.
The samples were measured in ten random positions around the film.
The water vapor permeability (WVP) was determined gravimetrically
using the method E96 / E96M–05 [27]. The samples were weighed at
24 h intervals by 7days. The WVP (g Pa–1 s–1 m–1) was calculated ac-
cording to Eq. 4.
mab × L ⎞
WVP=⎛
⎝ t × A × ΔP ⎠
where mab is the moisture mass absorbed (g), t the total test time (s), L
the film thickness (m), A the area of the film exposed surface (m2) and
ΔP the partial pressure difference across the film (Pa).
Thermal properties of the cross–linked gelatin films were de-
termined using differential scanning calorimetry (DSC–60, Shimadzu,
Japan), under a nitrogen atmosphere. The films samples were loaded in
a pan and sealed, and scanned over the temperature range of
20 °C–200 °C with a heating rate of 10 °C min–1; an empty pan was used
as a reference material [28]. The FT–IR spectra of the films were de-
termined in a spectrometer (Shimadzu model, Prestige 21, model
210045, Japan), using scanning over the frequency range of
4000–400 cm–1 [29]. The X–ray diffraction analysis of the crosslinked
films was performed using a Bruker D8 Advance diffractometer (Bruker
AXS, Germany) [30]. The surface morphology of the film samples was
visualized using a scanning electron microscope (SEM) (Jeol JSM
6010LV, Japan) with an accelerating voltage of 10 kV [31]. The mi-
crographs were with magnification of 2000 times.
2.6. Statistical analysis
Statistical analysis was based on analysis of variance (ANOVA) to
determine significant variables (p ≤ 0.05) in the process, and the
Tukey's test for comparison of means to determine significant differ-
ences (p ≤ 0.05) [32].
The yields of the gelatin solution obtained from tilapia and carp
skins were of 8.9 and 8.2 g 100 g–1, respectively, based on the wet
weight. These values were higher than those found by Abdelmalek et al.
[33]. for squid skin gelatin (6.82 g 100 g–1). The collagen contents in
the skins gelatins were determined by the hydroxyproline contents, and
the values found for tilapia gelatin and carp gelatin were of 8.4 g 100 g–
1
and 8.1 g 100 g– 1, respectively. The gelatin yields were similar to
values found by Silva et al. [19]. of 8.8 g 100 g–1, which studied the
extraction of cobia skin gelatin. According to Chandra [34], the ex-
traction yield depends on the collagen content and extraction method.
Table 1 shows the results of the gelatins amino acid compositions.
The predominant amino acid sequence in fish gelatin is glycine–proli-
ne–hydroxyproline. The higher levels of amino acids (proline and hy-
droxyproline) in gelatin skins may contribute to better rheological
(4)
properties, due to the formation and stabilization of the triple helix in
the gelatin molecules [35,36]. The glycine and proline contents for ti-
lapia gelatin were higher than carp gelatin, suggesting better rheolo-
gical properties. Sila et al. [37], studying barber skin gelatin, found
proline levels around 8.3 g 100 g– 1.
Gel strength, viscosity and melting point of gelatins are shown in
Table 2. Gel strength presented a greater value for tilapia gelatin
crosslinked with gallic acid (253 g), value was higher to those found in
commercial bovine gelatin (227 g). This result can be justified by the
formation of hydrogen bond between the multiple hydroxyl groups and
the carboxyl of the proteins. The modification may also have occurred
by the stabilization caused by hydrophobic interactions between the
aromatic ring of the crosslinking agent and the hydrophobic regions of
the protein [21]. On the other hand, cross–linking with NaCl in skins
gelatins of tilapia and of carp, resulted in a reduction of gel strength
when compared to the control samples. This decrease in gel strength by
the addition of NaCl can be associated with the breaking of hydrogen
bonds and the increase in ionic strength, interfering in electrostatic
interactions [38,39].
The tilapia gelatin showed higher gel strength when compared to
the carp gelatin, and this is justified by the greater concentration of
hydroxyproline in tilapia gelatin. The hydroxyproline performs an im-
portant role in the gel stability, due to its hydrogen bonding capability
through the hydroxyl group, although the proline is also important
[34].
The gel viscosity was not affected by the different fish species, but

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Table 2
Gel strength, viscosity and melting point of pure gelatins (control) and crosslinked gelatins from tilapia skin and carp skin.

Gel strength (g)* viscosity (cP)* melting point (°C)*

Bovine 227.2 ± 2.1c 5.35 ± 0.41a 26.60 ± 0.22b

d c
Nile tilapia Control 219.2 ± 2.3 3.86 ± 0.25 26.65 ± 0.25b
NaCl 215.3 ± 2.1d 3.63 ± 0.15d 26.54 ± 0.55b
MgSO4 240.4 ± 3.2b 3.84 ± 0.10c 26.72 ± 0.22b
Gallic acid 252.7 ± 4.1a 4.86 ± 0.31b 27.68 ± 0.52a
Citric acid 225.6 ± 3.2c 4.22 ± 0.24b 27.36 ± 0.35a

Common carp Control 217.5 ± 2.1d 3.43 ± 0.18d 25.64 ± 0.30c

NaCl 214.5 ± 2.3e 3.26 ± 0.14e 25.65 ± 0.33c
MgSO4 231.8 ± 4.0c 3.74 ± 0.09c 26.33 ± 0.35b
Gallic acid 241.7 ± 5.2b 4.53 ± 0.34b 27.34 ± 0.42a
Citric acid 221.7 ± 3.3d 4.06 ± 0.21bc 26.85 ± 0.63ab

* Means values ± standard deviation (n = 3). Different letters (a, b, c, d, e) in the same column for each crosslinked chemical agent show significant differences (p ≤ 0.05).

was clearly increased by the crosslinked carried out. The none-

lectrolytes crosslinking agents led to an increase in the gelatin viscosity.
The viscosity increase can be a result of changes in structure with the
elimination of water around the protein molecule [20]. The viscosities
for tilapia gelatin modified with gallic acid obtained an increase about
26% when compared to the control gelatin. This difference is related to
the gel strength values and, occurs due to the degradation of the
polypeptide chain responsible for an ordered network, which can in-
crease the viscosity. The results are a with the according to the viscosity
values in literature (2.0–7.0 cP for most gelatins). Gelatin solution with
low viscosity generally produces a low and brittle texture gel, while
gelatin solution with high viscosity produces a durable and stretchable
gel [40].
Crosslinked of carp skin gelatin led to a slight increase in melting
temperature with gallic acid followed by citric acid and MgSO4, when
compared to control. Similar trends were observed for gelatins from
tilapia skin, because when crosslinked with the nonelectrolytes pre-
sented higher melting temperatures, being higher than to the melting
temperature of bovine gelatin and of control gelatin (non–crosslinked).
The differences in gel strengths, viscosities and melting points of gela-
tins obtained in this study can be attributed to the to the different re-
action mechanisms of the chemical cross–linking agents.
The SDS–PAGE patterns of the fish gelatins chemically crosslinked
are shown in Fig. 1. All gelatins obtained showed α1 chains and the
reticulated showed β chains. However, the intensity of α1 in tilapia skin
gelatin was greater than in carp skin gelatin, indicating different mo-
lecular weights.
In Fig. 1, the intensity of the band around 100 kDa decreased with
the addition of crosslinking agents, and increased to near 220 kDa.
These results indicated that, the agents led to a crosslinked the gelatin
matrix for both species, causing an increased molecular weight. Similar
results were reported by Bae et al. [41]., where there was a decrease in
intensity of band at about 100 kDa and increased intensity of the band
at the top position of the gel when treated with MTGase. Go- Fig. 1. SDS–PAGE patterns: (A) Nile tilapia skin gelatins and (B) common carp skin ge-
latins. Legend: H = high molecular weight markers; C = control gelatin (non–-
mez–Guillen et al. [42] reported that crosslinking could cause an in-
crosslinked); Na = NaCl; Mg= MgSO4; CA = citric acid; GA = gallic acid.
crease in the molecular weight of fish gelatin, thus as, an increased in
viscosity and in the gel strength. The molecular characteristics of gels
contributed to elucidate the best functional properties obtained from about 55% compared to the control films. Lower results were found by
crosslinked gelatin of tilapia with nonelectrolytes agents. Rouhi et al. [43]. (21 MPa) when studying the increase of ZnO in fish
gelatin films and, similar to those of Uranga et al. [12] (28 MPa) when
3.2. Films characterization analyzing fish gelatin films incorporated with citric acid. For gelatin
films crosslinked with electrolytes was observed a small increase when
3.2.1. Physical and mechanical properties compared to control. The increase of tensile strength indicated that the
Table 3 shows the values of tensile strength, elongation at break, new interactions induced by the reactions between the crosslinking
water vapor permeability (WVP) and thickness of crosslinked gelatin agents, and gelatin were stronger than the interactions of the control
films. gelatin (non–crosslinked).
The tensile strength values of the films crosslinked with gallic acid Elongation at break was significantly higher (p ≤ 0.05) for the
were higher for both, tilapia gelatin and carp gelatin. The action of this control film of carp skin gelatin than for the control film of tilapia skin
crosslinking agent on the films was able to increase tensile strength gelatin. There was a reduction in the elongation at break for all

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Table 3
Tensile strength, elongation at break, water vapor permeability (WVP) and thickness of the films of pure gelatins (control) and crosslinked gelatins from tilapia skin and carp skin.

Tensile strength (MPa)* Elongation at break (%* WVP (g s−1 m−1 Pa−1) × 10−11* Thickness (mm)*

Nile tilapia Control gelatin 19.52 ± 0.43f 9.45 ± 0.32b 2.12 ± 0.04f 0.052 ± 0.002f
NaCl 23.63 ± 0.85c 5.36 ± 0.53e 2.10 ± 0.10h 0.101 ± 0.003b
MgSO4 24.72 ± 0.33bc 4.60 ± 0.43e 2.19 ± 0.08i 0.110 ± 0.004a
Gallic acid 28.85 ± 0.74a 3.62 ± 0.26f 1.78 ± 0.02e 0.063 ± 0.003d
Citric acid 26.35 ± 0.64b 4.92 ± 0.44e 1.32 ± 0.06d 0.072 ± 0.003c
Common carp Control gelatin 17.73 ± 0.75g 10.52 ± 0.53a 2.70 ± 0.17a 0.054 ± 0.002f
NaCl 21.43 ± 0.52d 7.21 ± 0.42c 2.52 ± 0.08g 0.095 ± 0.003b
MgSO4 19.62 ± 0.45e 8.55 ± 0.62b 2.64 ± 0.18e 0.113 ± 0.003a
Gallic acid 25.42 ± 0.63b 5.41 ± 0.30e 2.17 ± 0.09b 0.058 ± 0.002d
Citric acid 23.73 ± 0.55c 6.32 ± 0.42d 2.31 ± 0.08c 0.067 ± 0.002c

* Means values ± standard deviation (n = 3). Different letters (a, b, c, d, e, f, g) in the same column for each crosslinked chemical agent shw significant differences (p ≤ 0.05).

crosslinked gelatin films, indicating a decrease in the flexibility and

extensibility. The inclusion of crosslinkers between molecules of the
protein matrix resulted in an increase of intermolecular interactions,
reducing the free volume between molecules. The mobility of the pro-
teins chains and the flexibility of the films were decreased, where the
films mechanical strengths were increased with decreasing of the ex-
tensibility [3,18,44]. The addition of glycerol was the same in all
crosslinked samples, thus the extensive effect characteristic of their use
was not interacted with the results.
Regarding to water vapor permeability, the all films of tilapia skin
gelatin showed best results (in general, the lowest values in Table 3).
The WVP of tilapia skin gelatin films decreased with the addition of the
nonelectrolytes agents for the crosslinking and, similar results were also
obtained for the carp skin gelatin films. This suggests that, the free
volume of the gelatin matrix can be reduced by the addition of these
agents in the proteins structure. Mechanical properties of the obtained
films presented the same behavior of the physicochemical results of the
crosslinked gelatins, which can be inferred that the interaction between
the chemical agents and the gel matrix was successfully performed.
The thickness of the control films using carp and tilapia skin gelatins
had not significant differences (p ≤ 0.05). However, the thicknesses of
the crosslinked films were increased when the chemicals agents were
incorporated, in particular the electrolytes. The thickening occurred
due to the increased solids content in the crosslinked films. Moreover,
chemicals incorporation can affect the ordered structure of the proteins,
thereby producing a thick network [3].

3.2.2. Differential scanning calorimetry (DSC)

Fig. 2 shows the DSC thermograms of the crosslinked films from
tilapia skin gelatin (Fig. 2(A)) and common carp skin gelatin (Fig. 2(B)).
The endothermic events observed in the thermograms are due to
melting of crystals residues in the polymers matrix. They can be re-
garded as waste because they appeared at temperatures higher than
90 °C [45]. The melting temperatures (Tm) were strongly dependent on
the crosslinking degree of the polymers structure. The melting tem-
peratures in the crosslinked gelatins films were different, compared
with the control gelatins, 168 °C and 149 °C for the skin gelatins films of
tilapia and of carp, respectively. Fig. 2. DSC thermograms of pure and crosslinked gelatin films: (A) Nile tilapia films and,
The results suggest that the electrolytes increased the gelatin mo- (B) common carp films. Legend: C = control (non–crosslinked); Na = NaCl; Mg= MgSO4;
CA = citric acid; GA = gallic acid.
lecular mobility, as evidenced from the melting temperature decrease.
However, the films of crosslinked gelatin with nonelectrolyte (organic
compounds) led to increase the melting temperatures. The gallic acid 3.2.3. FT–IR spectroscopy
and citric acid within the gelatin matrix may increase the interaction The FT–IR analysis of gelatin films was performed to characterize
through functional groups acting on their reactive side groups and, the changes induced by incorporation of crosslinkers, to distinguish the
thus, increasing the rigidity of the gelatin film matrix [46]. The results vibrational changes related to chemical interactions in the matrix
of the thermograms are accordance to the found mechanical properties, (Fig. 3(A) for film of tilapia skin gelatin and Fig. 3(B) for film of carp
where nonelectrolytes agents led to an increase of films rigidity skin gelatin).
(Table 3). The spectra of all films showed similarity of characteristic bands
around 1630 cm–1 (amide–I, due to stretching C]O /hydrogen bond

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Fig. 3. Fourier transform infrared (FT–IR) spectra of pure and crosslinked gelatin films:
(A) Nile tilapia films and, (B) common carp films. Legend: C = control (non–crosslinked);
Na = NaCl; Mg= MgSO4; CA = citric acid; GA = gallic acid.

with COO), 1545 cm–1 (Amide II, due to bending NeH and stretching
CeN) and 1235 cm–1 (amide III, due to CeN and CeH stretching vi-
bration and CH2 of glycine group) [9,14]. The band that refers to
amideeA in gelatin films was observed around 3275 cm–1, representing
the NeH elongation together with hydrogen bonds. The bands formed
by the amide–B observed around 2940 cm–1, represent the elongation of
the CeH bonds [18,47]. The crosslinked between the reactive groups of
the chemical agents occurs between the free amino groups of glycine,
proline and hydroxyproline present in the gelatin. For crosslinked ge-
Fig. 4. X–ray diffraction (XRD) patterns of pure and crosslinked gelatin films: (A) Nile
latin films were observed peaks in the bands between 1745 and
tilapia films (B) common carp films. Legend: C = control (non–crosslinked); Na = NaCl;
1755 cm–1, which may be caused by a combination of deformation Mg= MgSO4; CA = citric acid; GA = gallic acid.
NH3+ groups presented as free amino acids.

3.2.4. X–ray diffraction analysis (XRD)
The dispersion of the crosslinking agents in the gelatin films was
performed using XRD. Fig. 4(A) and (B) show the results for the films of
tilapia and of carp skins gelatin, respectively.
For the reticulated gelatins with gallic acid and citric acid, the films
remained amorphous, similar to the control films (non–crosslinked
gelatin). These showed a low crystallinity, with main diffraction peaks
at around 2θ = 15 attributed to triple–helical crystalline structure of
the denatured collagen during the gelatin extraction. The peak around
of 2θ = 25.5 can be attributed to the amorphous halo proteins [48,49].
In Fig. 4, the position and intensity of the diffraction peaks not changed
after the crosslinking with nonelectrolytes agents. These results suggest
that the structure has been changed only in the amorphous phase of the
gelatin matrix. On other hand, a structural change from amorphous to
semicrystalline was observed for crosslinked gelatin films with elec-
trolytes. The crosslinking with NaCl and MgSO4 modified the con-
formation of the proteins chains, leading to a new structure, more or-
dered and stable. The diffractograms for these films showed a sharp
peak located around 2θ = 26.5, indicating a partially crystalline gelatin
film structure [3,50].

189
J.P. Santos et al.
Fig. 5. SEM micrographs of pure and crosslinked gelatin films: Nile tilapia films and common carp films. Legend: C = control (non–crosslinked); Na = NaCl; Mg= MgSO4; CA = citric
acid; GA = gallic acid.
3.2.5. Surface morphology
Fig. 5 shows the scanning electron microscopy (SEM) images for the
films of tilapia skin gelatin (Fig. 5(A)) and of carp skin gelatin
(Fig. 5(B)).
A homogeneous and compact distribution on surface was found for
the control film of tilapia skin gelatin, while small cracking was ob-
served for the control film of carp skin gelatin. This can be due to the
complex fibril structure of tilapia gelatin, which has been associated
with a high wet-ability, i.e., the ability to hold water [8]. This result
confirms the higher permeability of the carp gelatin film compared with
the tilapia gelatin film (Table 3).
The addition of NaCl and MgSO4 in the proteins matrix maintained
a homogeneous distribution for tilapia films, as well as, a frangible
structure for the carp gelatin films. However, the films crosslinked with
gallic acid and citric acid, showed a microstructure forming a network
of twisted wires. These microstructures were β–chains compounds of
proteins molecules [51]. The networks with thicker wire and compact
and smooth surfaces are related to the increase of the gelatin gel
strength. This led to the increased the mechanical properties and im-
provements in the WVP of the films. Thus, the crosslinking agents
studied played an important role in the preparation of biopolymer-
based films of fish skin gelatin.
4. Conclusion
The addition of crosslinking agents in the fish skin gelatin matrix
improved the main rheological properties of the gels and, consequently,
was directly proportional to the functional improvements of the bio-
polymer film produced from these fish skins gelatins. The results re-
vealed that the incorporation of nonelectrolytes agents into tilapia ge-
latin films, especially gallic acid, significantly influence the mechanical
properties. Also, there was an improvement in water vapor permeability
when were incorporated nonelectrolyte agents. Electrolytes reduced the
films thermal stability. Fish gelatin films prepared using the appropriate
conditions and crosslinking agents can be used as an alternative film
packaging. Therefore, it is possible use the fish waste to produce pro-
ducts with high added value, such as, crosslinked biopolymers films,
being an alternative for the substitution of synthetic materials films.
Acknowledgments
The authors would like to thank the financial support of CAPES/
Brazil and CNPq/Brazil, and to CEME–Sul/FURG/Rio Grande/RS/
Brazil by SEM images.
190
Colloids and Surfaces A 539 (2018) 184–191
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