Crosslinking agents effect on gelatins from carp and tilapia skins and in their biopolymeric films

Accepted Manuscript Title: Crosslinking agents effect on gelatins from carp and tilapia skins and in their biopolymeric films Authors: Jaqueline P. Santos, Vanessa M. Esquerdo, Catarina M. Moura, Luiz A.A. Pinto PII: DOI: Reference:

S0927-7757(17)31106-8 https://doi.org/10.1016/j.colsurfa.2017.12.018 COLSUA 22142

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

19-9-2017 5-12-2017 7-12-2017

Please cite this article as: Santos JP, Esquerdo VM, Moura CM, Pinto LAA, Crosslinking agents effect on gelatins from carp and tilapia skins and in their biopolymeric films, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2010), https://doi.org/10.1016/j.colsurfa.2017.12.018 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.

Crosslinking agents effect on gelatins from carp and tilapia skins and in their biopolymeric films

Jaqueline P. Santos1, Vanessa M. Esquerdo1, Catarina M. Moura2, Luiz A. A. Pinto1*

School of Chemistry and Food, Federal University of Rio Grande–FURG, km 8 Itália Avenue,

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96203–900, Rio Grande, RS, Brazil.

Food Engineering, Federal University of Pampa–UNIPAMPA, 96400–100, Bagé, RS, Brazil

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*

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Corresponding author: e–mail address: [email protected]; Tel/Fax: +55 53 3233 6969. http://orcid.org/0000-0002-4477-0686

e–mail address: [email protected]

Vanessa Mendonça Esquerdo

e–mail address: [email protected]

Catarina Motta de Moura

e–mail: [email protected]

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Jaqueline Pozzada dos Santos

e–mail address: [email protected]

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Luiz Antonio de Almeida Pinto

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Graphical abstract

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Abstract The extracted gelatins from carp and tilapia skins were chemically crosslinked by electrolytes (NaCl and MgSO4) and nonelectrolytes (gallic acid and citric acid) agents and, the fish gelatins films were produced by casting. Rheological, chemical and physicochemical properties were evaluated. All crosslinked films showed a significant increase in tensile strength and a slight

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reduction in elongation compared to non–crosslinked films. The crosslinked films with nonelectrolytes presented the highest reductions in water vapor permeability. Fourier

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Transform Infrared spectra, X–Ray Diffraction and Scanning Electron Microscopy showed the

interactions between protein/agents and the changes in amorphous structure of the films, which presented surfaces in chains of twisted shape. Differential Scanning Calorimetry demonstrated

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that the incorporations of gallic acid and citric acid improved the films thermal stability. The

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results showed that the chemical crosslinking can be used to improve the quality of fish skin

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added value of the fish waste.

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gelatin films and, thus, these films can be an alternative to food packaging, as well to improve

Keywords: Biodegradable films; Chemical crosslinking; Electrolytes agents; Fish skin;

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1. Introduction

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Gelatin; Nonelectrolytes agents.

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The increasing of environmental pollution and serious ecological problems caused by

packaging derived from petroleum has led to interest in the use of natural polymers to produce biodegradable materials packaging [1]. Gelatin is considered a promising option as a raw material for food packaging, providing low cost, film forming ability, high availability and biodegradability [2]. Gelatin is obtained by controlled hydrolysis of the insoluble collagen

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present in skins and bones of bovine, 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)

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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

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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

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traditional ponds, which performs well in different breeding regimes [7]. Most of these fish are

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being processed into fillets and exported, and the amount of organic waste generated in the

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fishery processing plants can reach 70% by fish weight [8]. The development of fish skin based

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products provides value addition and management of the waste disposed of in the environment.

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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

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packaging materials [9]. Several studies have been conducted to repair this deficiency, including the development of blends films and bilayers [10], nanoparticles addition [11] and

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chemical modification with crosslinkers [12]. The chemical crosslinking provides bonds between reactive groups present in the gelatin, resulting in improvements in mechanical

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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 industries [14,15]. Electrolytes, such as sodium chloride (NaCl) and magnesium sulfate (MgSO4), can affect the gelatin via electrostatic forces and the formation of saline bridges [16].

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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

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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.

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Thus, the aim of this study was to evaluate the addition of electrolytes (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

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skins. The gelatins from tilapia skin and carp skin were produced, afterwards chemically

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crosslinked and, characterized. The crosslinked gelatin films were characterized by physical

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2. Material and methods

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and mechanical properties, DSC, FT–IR, XRD and scanning electron microscope.

2.1 Materials and Chemicals

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The skins of Nile tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio) were obtained from fish farmers in local units (Rio Grande do Sul, Brazil), and were stored at

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–20 °C until use. All chemicals were of analytical grades. The crosslinkers NaCl and MgSO 4 was purchased from Synth (Brazil), gallic acid from Vetec (Brazil) and citric acid from Merck

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(Brazil). For comparison purposes, was use mammalian gelatin from bovine skin (type B, 260 g Bloom), obtained from Sigma–Aldrich Chemical Co. (USA).

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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

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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

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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 solutions were clarified with activated charcoal (1g kg–1 solution, 120 min at 35 °C).

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The gelatin solutions from tilapia skin and carp skin were lyophilized (Liotop, L108,

) were crosslinked with electrolytes and non-electrolytes, which were selected from the

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Brazil) and, stored in sealed plastic bags at –20 °C. The gelatins samples (100 mL of 66,7 g L–

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positive effects on the properties of gels (gel strength, viscosity, melting point, among others).

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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 0,02 mol L–1 (GA) [21] and citric acid at 0,03 mol L–1 (CA)

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(determined 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

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a water bath at 45 °C by 30 min.

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2.3 Fish gelatins analysis

Rg = (

The gelatin solutions yield was calculated according to Equation 1. C g  VS mi

)  100

(1)

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where Rg is the gelatin yield (g 100g–1skin), 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 (Sigma–Aldrich, USA) as standard, calculated by

2.5 × h × 100 m ×V

(2)

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Hyp =

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Equation 2.

where Hyp is hydroxyproline content in the sample (g 100g–1), h is the value read from the standard curve spectrophotometer, m is the sample mass (g) and V is the filtrate volume (mL).

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The determination of the amino acids present in the gelatin samples was performed by

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high performance liquid chromatography (HPLC, Shimadzu, Japan). The gel strength

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determination was according to the AOAC official method 948.21 [23], measured using a

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texturometer (TA.XTplus, Stable Micro Systems, UK). The samples viscosity was calculated

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by Equation 3 [19].

μ=t × K × ρ

(3)

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where μ is the gelatin viscosity (cP), t is the time (s), K is the viscosimeter constant and  is the

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density of gelatin solution on test temperature, which was determined by picnometry (g cm–3). The melting point of the gelatin sample was determined by BSI official method 755 [24]

using a solution of chloroform and methylene blue dye (3:1 v:v) as the indicator. SDS–

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polyacrylamide gel electrophoresis (SDS–PAGE) was carried out by method of Laemmli [25] with some modifications. The gelatin sample (2 mg) was dissolved in 1 mL of 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, containing 10 % (v:v) SDS, 0.02 % bromophenol and glycerol, 20 % (v:v) 2–mercaptoethanol). The samples and the standard protein marker were loaded on a 6

polyacrylamide gel made of 7.5% (v:v) separating gel and 3.8% (v:v) stacking gel, and subjected to electrophoresis at a constant current of 25 mA and 150 V for about 3 h. The gelatin molecular weight was estimated using standards of high molecular weight of GE Healthcare Life Sciences (USA). 2.4 Films preparation with crosslinked gelatin

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The crosslinked fish gelatin films were produced by casting technique. In gelatin

solutions (50 mL) with concentration of 2% (w:v) was added 0.20 g of glycerol, and remained

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under constant stirring for 30 min [18]. The filmogenic solutions of crosslinked gelatin (tilapia

or 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 evaporation. The films were removed from the plates and

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2.5 Characterization of crosslinked gelatin films

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placed into desiccator at 25 ° C and 75 % relative humidity, for at least 48 h before the analyzes.

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Tensile strength (TS) and elongation at break (EAB) of crosslinked gelatin films were

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determined according to the official method D00882–00 [26], using a texturometer (Stable

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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

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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

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at 24 h intervals by 7 days. The WVP (g Pa–1 s–1 m–1) was calculated according to Equation 4. mab × L

(4)

t × A × ΔP

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WVP =

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).

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Thermal properties of the cross–linked gelatin films were determined 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 to 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 determined in a spectrometer (Shimadzu model, Prestige

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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

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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 micrographs were with magnification of 2000

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times.

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2.6 Statistical analysis

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Statistical analysis was based on analysis of variance (ANOVA) to determine significant

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significant differences (p ≤ 0.05) [32].

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variables (p ≤ 0.05) in the process, and the Tukey's test for comparison of means to determine

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3. Results and discussion

3.1 Characterization of crosslinked gelatins of Nile tilapia and common carp

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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

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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 100g– 1 and 8.1 g 100g– 1, respectively. The gelatin yields were similar to values found by Silva et al. [19] of 8.8 g 100g–1, which studied the extraction

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of cobia skin gelatin. According to Chandra [34], the extraction 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–proline–hydroxyproline. The higher levels of amino acids (proline and hydroxyproline) in gelatin skins may contribute to better rheological

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properties, due to the formation and stabilization of the triple helix in the gelatin molecules [35, 36]. The glycine and proline contents for tilapia gelatin were higher than carp gelatin,

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suggesting better rheological properties. Sila et al. [37], studying barber skin gelatin, found proline levels around 8.3 g 100g– 1.

Gel strength, viscosity and melting point of gelatins are shown in Table 2. Gel strength

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presented a greater value for tilapia gelatin crosslinked with gallic acid (253 g), value was

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higher to those found in commercial bovine gelatin (227 g). This result can be justified by the

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formation of hydrogen bond between the multiple hydroxyl groups and the carboxyl of the

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proteins. The modification may also have occurred by the stabilization caused by hydrophobic

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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

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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

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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

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this is justified by the greater concentration of hydroxyproline in tilapia gelatin. The hydroxyproline performs an important 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 was clearly increased by the crosslinked carried out. The nonelectrolytes crosslinking agents led to an increase in the

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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 increase the viscosity. The

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results are a with the according to the viscosity values in literature (2.0 to 7.0 cP for most

gelatins). Gelatin solution with low viscosity generally produces a low and brittle texture gel,

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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

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observed for gelatins from tilapia skin, because when crosslinked with the nonelectrolytes

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presented higher melting temperatures, being higher than to the melting temperature of bovine

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gelatin and of control gelatin (non–crosslinked). The differences in gel strengths, viscosities

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and melting points of gelatins obtained in this study can be attributed to the to the different

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reaction mechanisms of the chemical cross–linking agents. The SDS–PAGE patterns of the fish gelatins chemically crosslinked are shown in Fig.

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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

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molecular weights.

In Fig. 1, the intensity of the band around 100 kDa decreased with the addition of

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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. Gomez–Guillen et al. [42] reported that crosslinking could cause an increase in

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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 crosslinked gelatin of tilapia with nonelectrolytes agents. 3.2 Films Characterization 3.2.1 Physical and mechanical properties

permeability (WVP) and thickness of crosslinked gelatin films.

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Table 3 shows the values of tensile strength, elongation at break, water vapor

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The tensile strength values of the films crosslinked with gallic acid were higher for both,

tilapia gelatin and carp gelatin. The action of this crosslinking agent on the films was able to

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increase tensile strength about 55% compared to the control films. Lower results were found

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by Rouhi et al. [43] (21 MPa) when studying the increase of ZnO in fish gelatin films and,

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similar to those of Uranga et al. [12] (28 MPa) when analyzing fish gelatin films incorporated

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with citric acid. For gelatin films crosslinked with electrolytes was observed a small increase when compared to control. The increase of tensile strength indicated that the new interactions

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induced by the reactions between the crosslinking agents, and gelatin were stronger than the interactions of the control gelatin (non–crosslinked).

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Elongation at break was significantly higher (p ≤ 0.05) for the control film of carp skin

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gelatin than for the control film of tilapia skin gelatin. There was a reduction in the elongation at break for all 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

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of intermolecular interactions, reducing the free volume between molecules. The mobility of the proteins chains and the flexibility of the films were decreased, where the films mechanical strengths were increased with decreasing of the extensibility [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.

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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.

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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

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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

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the chemicals agents were incorporated, in particular the electrolytes. The thickening occurred

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due to the increased solids content in the crosslinked films. Moreover, chemicals incorporation

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can affect the ordered structure of the proteins, thereby producing a thick network [3]. 3.2.2 Differential scanning calorimetry (DSC)

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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

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thermograms are due to melting of crystals residues in the polymers matrix. They can be

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regarded 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 temperatures in the crosslinked gelatins films were different, compared

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with the control gelatins, 168 °C and 149 °C for the skin gelatins films of tilapia and of carp, respectively. The results suggest that the electrolytes increased the gelatin molecular 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

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acid and citric acid within the gelatin matrix may increase the interaction through functional groups acting on their reactive side groups and, thus, increasing the rigidity of the gelatin film matrix [46]. The results of the thermograms are accordance to the found mechanical properties, where nonelectrolytes agents led to an increase of films rigidity (Table 3). 3.2.3 FT–IR Spectroscopy

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The FT–IR analysis of gelatin films was performed to characterize the changes induced by incorporation of crosslinkers, to distinguish the vibrational changes related to chemical

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interactions in the matrix (Fig. 3(A) for film of tilapia skin gelatin and Fig. 3(B) for film of carp skin gelatin).

The spectra of all films showed similarity of characteristic bands around 1630 cm –1

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(amide–I, due to stretching C=O /hydrogen bond with COO), 1545 cm–1 (Amide II, due to

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bending N–H and stretching C–N) and 1235 cm–1 (amide III, due to C–N and C–H stretching vibration and CH2 of glycine group) [9,14]. The band that refers to amide–A in gelatin films

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was observed around 3275 cm–1, representing the N–H elongation together with hydrogen

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bonds. The bands formed by the amide–B observed around 2940 cm–1, represent the elongation of the C–H bonds [18,47]. The crosslinked between the reactive groups of the chemical agents

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occurs between the free amino groups of glycine, proline and hydroxyproline present in the gelatin. For crosslinked gelatin films were observed peaks in the bands between 1745 and 1755

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cm–1, which may be caused by a combination of deformation NH3+ groups presented as free amino acids.

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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 4(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

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crystallinity, with main diffraction peaks at around 2 = 15 attributed to triple–helical crystalline structure of the denatured collagen during the gelatin extraction. The peak around of 2 = 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

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gelatin matrix. On other hand, a structural change from amorphous to semicrystalline was

observed for crosslinked gelatin films with electrolytes. The crosslinking with NaCl and MgSO4

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modified the conformation of the proteins chains, leading to a new structure, more ordered and

stable. The diffractograms for these films showed a sharp peak located around 2 = 26.5,

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indicating a partially crystalline gelatin film structure [3,50].

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3.2.5 Surface morphology

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Fig. 5 shows the scanning electron microscopy (SEM) images for the films of tilapia

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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

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tilapia skin gelatin, while small cracking was observed 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

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a high wet-ability, i.e., the ability to hold water [8]. This result confirms the higher permeability

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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,

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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

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and improvements 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

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rheological properties of the gels and, consequently, was directly proportional to the functional improvements of the biopolymer film produced from these fish skins gelatins. The results

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revealed that the incorporation of nonelectrolytes agents into tilapia gelatin films, especially

gallic acid, significantly influence the mechanical properties. Also, there was an improvement in water vapor permeability when were incorporated nonelectrolyte agents. Electrolytes

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reduced the films thermal stability. Fish gelatin films prepared using the appropriate conditions

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and crosslinking agents can be used as an alternative film packaging. Therefore, it is possible

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use the fish waste to produce products with high added value, such as, crosslinked biopolymers

Acknowledgment

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films, being an alternative for the substitution of synthetic materials films.

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The authors would like to thank the financial support of CAPES/Brazil and CNPq/Brazil, and

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to CEME–Sul/FURG/Rio Grande/RS/Brazil by SEM images.

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Figure Captions

A

Figure 1. SDS–PAGE patterns: (A) Nile tilapia skin gelatins and (B) common carp skin gelatins. Legend: H= high molecular weight markers; C= control gelatin (non–crosslinked); Na= NaCl; Mg= MgSO4; CA= citric acid; GA= gallic acid.

21

Figure 2. DSC thermograms 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. Figure 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=

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NaCl; Mg= MgSO4; CA= citric acid; GA= gallic acid.

Figure 4. X–ray diffraction (XRD) patterns of pure and crosslinked gelatin films: (A) Nile

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tilapia films (B) common carp films. Legend: C= control (non–crosslinked); Na= NaCl; Mg= MgSO4; CA= citric acid; GA= gallic acid.

Figure 5. SEM micrographs of pure and crosslinked gelatin films: Nile tilapia films and

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common carp films. Legend: C= control (non–crosslinked); Na= NaCl; Mg= MgSO4; CA=

A

CC E

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ED

M

A

N

citric acid; GA= gallic acid.

22

A ED

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Fig. 1.

23

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N

A

M

A ED

PT

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Fig. 2.

24

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U

N

A

M

A ED

PT

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Fig. 3.

25

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U

N

A

M

Fig. 4.

26

A ED

PT

CC E

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U

N

A

M

A ED

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A

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Fig. 5.

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Table 1. Amino acid compositions of the gelatins from tilapia skin and carp skin. gamino acids (100 g–1protein)

Amino acids

Tilapia skin gelatin Carp skin gelatin < LOQ

< LOQ

Serine

< LOQ

< LOQ

Proline

12.8

11.2

Hydroxyproline

8.4

8.1

Glycine

28.0

26.2

Alanine

1.9

1.6

Cysteine

0.9

0.7

Valine

0.1

0.2

Methionine

0.6

0.8

Isoleucine

2.9

2.3

Leucine

1.3

Tyrosine

1.3

Phenylalanine

1.1

Lysine

1.1

Arginine

3.9

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1.2

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A

1.2

A

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LOQ: Limit of quantitation

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Threonine

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1.3 2.0 5.0

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)* 5.35 ± 0.41a

26.60 ± 0.22b

Control

219.2 ± 2.3d

3.86± 0.25c

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

Control

217.5 ± 2.1d

3.43 ± 0.18d

NaCl

214.5 ± 2.3e

MgSO4 Gallic acid

241.7 ± 5.2b

Citric acid

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25.65 ± 0.33c

231.8 ± 4.0c

3.74 ± 0.09c

26.33 ± 0.35b

4.53 ± 0.34b

27.34 ± 0.42a

4.06 ± 0.21bc

26.85 ± 0.63ab

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3.26 ± 0.14e

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Common carp

25.64 ± 0.30c

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Nile tilapia

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227.2 ± 2.1c

Bovine

221.7 ± 3.3d

A

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(p ≤ 0.05).

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*Means values ± standard deviation (n=3). Different letters in the same line for each crosslinked chemical agent show significant differences

29

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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 Elongation break (%)*

(g s-1 m-1 Pa-1) ×10-11* (mm)*

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

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

M

ED

Common carp

Thickness

(MPa)*

A

A

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Nile tilapia

at WVP

*Means values ± standard deviation (n=3). Different letters in the same line for each crosslinked chemical agent shw significant differences (p ≤ 0.05).

30