Al-Hassan y Norziah 2012 [PDF]

Citation preview

Food Hydrocolloids 26 (2012) 108e117



Contents lists available at ScienceDirect



Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd



Starchegelatin edible films: Water vapor permeability and mechanical properties as affected by plasticizers A.A. Al-Hassan, M.H. Norziah* Food Technology Department, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia



a r t i c l e i n f o



a b s t r a c t



Article history: Received 20 May 2010 Accepted 14 April 2011



Physical and mechanical properties of edible films based on blends of sago starch and fish gelatin plasticized with glycerol or sorbitol (25%, w/w) were investigated. Film forming solutions of different ratios of sago starch to fish gelatin (1:0, 2:1, 3:1, 4:1, and 5:1) were used and cast at room temperature. Amylose content of sago starch was between 32 and 34% and the protein content of the fish gelatin was found to be 81.3%. The findings of this study showed that the addition of fish gelatin in starch solutions has a significant effect (p < 0.05), resulting in films with lower tensile strength (TS) and higher water vapor permeability (WVP). On the other hand, increasing protein content (from 10.9% to 21.6%) in film samples plasticized with sorbitol showed significantly lower (p < 0.05) TS but no trend was observed in % elongation-at-break (EAB) and no differences in WVP. However, TS decreased with higher protein content in the samples when either plasticizers were used in general, but no significance differences was observed among the samples (p < 0.05) with glycerol with exception to film with high protein content (21.6%) only and no trend was observed in % EAB among samples as well. Significant difference (p < 0.05) was observed in TS and viscosity between different formulations with sorbitol. The morphology study of the sago starch/fish gelatin films showed smoother surfaces with decreasing protein in the samples with either plasticizer. DSC scans showed that plasticizers and protein content incorporated with sago starch films reduced the glass transition temperature (Tg) and melting temperature (Tm) and the melting enthalpy (DHm). In this study, observation of a single Tg is an indication of the compatibility of the sago starch and fish gelatin polymers to form films at the concentration levels used. Ó 2011 Elsevier Ltd. All rights reserved.



Keywords: Edible film Sago starch Fish gelatin Glycerol Sorbitol DSC Glass transition



1. Introduction Consumers demand higher quality and longer shelf life in foods, while reducing disposable packaging materials and increasing recyclability. Such demands have caused increased interest in edible and biodegradable films or materials that potentially are used to extend the shelf life and improve the quality of almost any food system by serving as mass transfer barriers to moisture, oxygen, carbon dioxide, lipid, flavor and aroma between food components and the surrounding atmosphere (Jongjareonrak, Benjakul, Visessanguan, Prodpran & Tananka, 2006). Such films may have the ability of decreasing the amounts of non-renewable conventional synthetic polymer packaging materials, and use ingredients of agricultural derived products (Soares, Lima, Oliveira, Pires, & Soldi, 2005). Edible films can be prepared from protein, polysaccharides, lipids or the combination of these components



* Corresponding author. Tel.: þ60 46535200; fax: þ60 46573678. E-mail address: [email protected] (M.H. Norziah). 0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.04.015



(Cao, Fu, & He, 2007; Cuq, Gontard, Cuq, & Guilbert, 1997). Starch films due to the hydrophilicity properties of the starch polymers, provide a minimal barrier to moisture. Films of protein or polysaccharides have overall suitable mechanical and optical properties but are highly sensitive to moisture and exhibit poor water vapor barriers (Guilbert, Gontard, & Gorris, 1996). Thus, composite films and coatings can be formulated consisting of several biopolymers. As an example, the lipid component in the film formulation can serve as a good barrier to water vapor permeability (Garcia, Martino, & Zaritzky, 2000a) and polysaccharides and proteins can be reasonably effective as gas barriers (O2 and CO2)(Arvanitoyannis, Psomiadou, & Nakayama, 1996; Baldwin, Nisperos-Carriedo, & Baker, 1995). Lipids films are more moisture resistance but vulnerable to oxidation. Therefore, the new trend is to combine different biopolymers for food packaging and coatings (GomezGuillen et al., 2008). As a result of poor mechanical strength and high moisture sensitivity exhibited by starch edible films, several studies have investigated various additives, types of modification, sources of starch, and process parameters in order to improve these weaknesses in properties (Mali, Grossmann, Garcia, Martino, &



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



Zaritzky, 2006; Viga-Santos, Oliveria, Cereda, & Scamparini, 2007). Several studies have investigated the use of sorbitol and glycerol as plasticizers in forming starch or gelatin based edible films (Arvanitoyannis, 2002; Arvanitoyannis et al., 1996; Sobral, Menegalli, Hubinger, & Roques, 2001). Vanin, Sobral, Menegalli, Carvalho, and Habitante (2005) concluded that glycerol was compatible with gelatin and showed the highest plasticizing effect on the mechanical properties of the film producing a flexible and easy handling film with no phase separation. Sobral et al. (2001) reported that the effects of sorbitol on the water vapor permeability, mechanical and thermal properties of edible films based on gelatin gave a reasonable plasticizing effect on the puncture force. They also reported that the water vapor permeability increased with the increase in sorbitol content and similarly the increase of sorbitol content did not reduce the formation of junction zones in the films. The objective of this study was to investigate the physical and mechanical properties including tensile strength, elongation-atbreak, water vapor permeability, film morphology and moisture isotherm and the compatibility of edible films made from a combination of sago starch and fish gelatin plasticized with glycerol and sorbitol. 2. Materials and methods 2.1. Materials and reagents Sago starch (Metroxylon sagu) was purchased from Nitsei Industrial Sdn. Bhd. (Malaysia). Fish gelatin was extracted from fish wastes provided by a local surimi processing plant. The extractions and preparation of fish gelatin was carried out as described by Norziah, Al-Hassan, Khairulnizam, Mordi, and Norita (2009). Sorbitol was kindly donated by Rhodia, Malaysia, glycerol from Sim Company Sdn. Bhd, Malaysia, sodium cacodylate trihydrate and osmium tetroxide (4%) from Sigma, glutaraldehyde from Ajax chemicals. All chemicals were of analytical grade. 2.2. Determination of amylose content Amylose content in sago starch was determined according to the method described by McGrance, Cornell, and Rix (1998) based on the colorimetric measurement of the iodine complexes formed with amylose and amylopectin. Absorbance was read at 600 nm wavelength in a 1 cm path length quartz cell using a uvevis spectrophotometer (UV-160A, Shimadzu). Amylose and amylopectin from potato starch (Fluka Biochemika) were used to prepare standards. All tests were conducted in triplicate. 2.3. Analysis of protein and amino acids content Protein content was measured by Kjeldahl method (AOAC, 1999). Fish gelatin (1 g) was dissolved in 100 mL distilled water at 60  C until clear solution was obtained and 0.3 mL gelatin solution was poured into digestion tubes. The protein content was determined using the nitrogen conversion factor for gelatin, which is 5.55 (AOAC, 1984). The test was carried out in triplicate. The amino acids composition of the gelatin was determined using High performance liquid chromatography (HPLC, Agilent 1200) with a fluorescence detector and a flow rate of 2 mL/min. The column used was ZORBAX Eclipse-AAA (4.6  75 mm, 5 mm). The mobile phase was A: 40 mM Na2HPO4 pH 7.8 and B: Acetone: methanol: water (45:45:10 (v/v/v)). Each sample was hydrolyzed in 6 N hydrochloric acid at 110  C for 24 h.



109



2.4. Preparation of starch/gelatin edible films The film forming solutions were prepared using blends of sago starch and fish gelatin with added plasticizers (glycerol or sorbitol). Different ratios of sago starch and fish gelatin solutions (1:0, 2:1, 3:1, 4:1 & 5:1) based on total weight basis (5 g) including 25% (w/w) plasticizers (glycerol or sorbitol) in 200 mL distilled water were prepared. Sago starch (db) was dissolved in distilled water and heated with magnetic stirring in a water bath at 85  C for 30 min until completely gelatinized. From previous DSC runs (data are not shown here) in which sago starch powder (w7 mg) added with distilled water (ratio 1:3 sago starch: water) was heated from 40 to 90  C, the gelatinization temperature was observed to be at 67.13  C and complete gelatinization was achieved at 76e77  C. Fish gelatin was dissolved in distilled water at 60  C for 30 min until a clear solution was obtained. Fish gelatin solution was added to gelatinized sago starch at 60  C and stirring was continued for 30 min, followed by addition of plasticizer with constant stirring for another 30 min. The mixture was then cooled to room temperature. The solution mixture (95 g) was cast onto polyacrylic plates (16 cm  16 cm  3 mm) and dried in a ventilated oven at 35  C for 24 h. The dry films obtained were peeled off and stored in a desiccator containing saturated sodium bromide (NaBr) solution with 56% (RH) at 30  C until for further analysis. Control films were prepared in the same way without the addition of fish gelatin. Each film formulation was prepared in triplicates. 2.5. Viscosity of film forming solutions Viscosity of sago starch/gelatin solutions was measured using a digital viscometer (Model DV-E, Brookfield, middle Boro) with speed 100 rpm and LV spindle no. 2 for all samples and values are expressed in (centipoises, cP). The measurement was conducted at room temperature (28  C). The spindle was immersed into the solution for about 3 min for thermal equilibrium between solution and spindle with continued shearing. Five readings of viscosity were recorded for each solution, and average values were taken. Tests were run in triplicate. 2.6. Determination of pH of film forming solution Measurement of pH value of sago starch and sago starch/gelatin film forming solutions was taken using a pH meter (Mettler Toledo, FE20. China). 2.7. Film thickness and light absorption Thickness of the films was measured using a manual micrometer (Mitutoyo, Japan) with an accuracy of 0.001 mm. Six different positions of the samples were measured and average thickness was calculated. Films prepared were observed visually for homogeneity without phase separation and with uniform colour. The light barrier properties of the films were measured by exposing the films to light absorption at wavelength 550 nm. Film transparency was measured according to the method of Bao, Xu, and Wang (2009) by placing rectangular film samples into a spectrophotometer test cell directly. Absorbance was recorded using an UV-160A uvevis spectrophotometer (Shimadzu, Japan). The transparency (T) of films was calculated according to the following equation: T ¼ A550/x where A550 is the absorbance at 550 nm and x is the film thickness (mm). According to this equation, a higher value of T would indicate



110



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



a lower degree of transparency. Tests were run in triplicates for each type of film. 2.8. Tensile strength and elongation tests The mechanical properties of films prepared were evaluated by conducting tensile strength and elongation-at-break (EAB) tests according to ASTM D882-00 method (ASTM, 2000a). Film specimen strips (14 cm  2 cm) were cut and conditioned in a desiccator containing saturated sodium bromide solution with 56% (RH) at 30  C for 48 h prior to testing. Films were tested for sorption isotherm in 2 h interval and equilibrium was achieved in 6 h in most ratios where water content in the films did not change up to 24 h. Tensile strength and EAB were performed as a tension test using a Texture analyzer TA XT2 (Stable Microsystems, Surrey, UK) with a load cell of 30 kg and crosshead speed of 60 mm/min. The samples were mounted between grips with initial grip gap of 100 mm and film width of 2 cm. The results of tensile and elongation tests were expressed by MPa and percentage (%), respectively. Each test trial per film consisted of five replicate measurements. 2.9. Water vapor permeability Water vapor permeability (WVP) of the films was determined according to the ASTM E96-00 method (ASTM, 2000b). Gas permeation cells with diameter 4.5 cm and height 2.8 cm were used. Glass permeation cell contained 25 g silica gel (0% RH) that was dried in oven at 120  C for 1 day initially and the headspace for the cell was 1.0 cm from the opening of the cell. The test films were sealed on top of the permeation cells. These cells were then placed in a desiccator containing distilled water (100% RH) and kept at 30  C. The cells were weighed at 24 h intervals over a 7-day period. The cells were recorded to the nearest 0.0001 g and plotted as a function of time. The slope of each line was calculated by linear regression (r2  0.99). The measured WVP of the films was determined as follows: WVP ¼ (WVTR L)/DP where WVTR is the water vapor transmission rate (g m2 h1) through a film, calculated from the slope of the straight line divided by the exposed film area (m2), L is the mean film thickness (mm), and DP is the partial water vapor pressure difference (Pa) across the two sides of the film. For each type of film, WVP measurements were replicated three times for each batch of films. 2.10. Moisture sorption isotherm The sorption isotherm of sago starch and sago starch/gelatin films was determined at 30  C according to Chang, Cheah, and Seow (2005) with slight modifications. Samples of sago starch and sago starch/fish gelatin were cut into small pieces (1  2 cm) and dried in a desiccator over Phosphorus pentoxide (P2O5) for 7 days. The dried samples were weighed to the nearest 0.0001 g into pre-weighed bottles and equilibrated versus saturated salt solutions with known relative humidity (RH) in air-tight containers in an incubator at 30  C in duplicate. Seven super saturated salt solutions, lithium chloride, potassium acetate, magnesium chloride, potassium carbonate, sodium bromide, sodium chloride, and potassium chloride with relative humidity (RH) of 11%, 22%, 32%, 43%, 56%, 75% and 84% at 30  C respectively, were used and the equilibrium was assumed to be achieved when changes in weight did not exceed 0.1% for 3 consecutive measurements.



2.11. Scanning electron microscopy (SEM) Films were observed by SEM following the procedures described by Denavi et al. (2009) using a Modal Leo Sipra So Vp Field Emission, CaH-Zeiss SMT, Oberkochen, Germany. Films were pretreated prior to SEM analysis. They were cut into small strips and fixed in a solution of 2% (v/v) glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h, washed with 0.1 M sodium cacodylate buffer (3 times in 10 min) and post fixed in 0.4% (w/v) osmium tetroxide (O5O4) and 0.2 M sodium cacodylate buffer (pH 7.2) for 2 h, then washed with 0.1 M sodium cacodylate buffer (3 times in 10 min). Samples then were dehydrated for 15 min through a graded ethanol series: 30, 50, 70 and 90%, (v/v) and finally (3 times; 15 min) at 99.5% (v/v) then were dried overnight. The dried samples were mounted on aluminum stubs and then coated with a layer of gold by “Baizer SCD SPUTTER coater”, allowing surface visualization using an accelerated voltage of 5 kV. 2.12. Analysis on thermal properties of films Thermal properties of films were determined by using differential scanning calorimetry. Measurements on a TA Instruments Calorimeter Q200 (TA Instruments Ltd., Leatherhead, UK) with a refrigerated cooling system (RCS) and a nitrogen DSC cell purge at 50 ml/min was used to achieve temperatures of 0  C. T-zero hermetic aluminum pans were used with a conventional MDSC with a modulation of (1/min). Samples (w15 mg  2 mg) were conditioned over P2O5 for 7 days, then at 56% RH and 30  C for 48 h. Samples were then hermetically sealed and heated from 0 to 200  C at 3  C/min. The reference was an empty pan and the equipment was calibrated with indium (Tm ¼ 156.6  C, and enthalpy DH ¼ 28.3 J/g). The transition temperature (Tm) was calculated as the temperature where the endothermic peak occurs, and the enthalpy (DH) of the transition was calculated as the area over the endothermic peak. The glass transition temperature (Tg) was calculated using a TA Instrument Universal analysis 2000 software as the shifting down of the reversible heat flow curve. 2.13. Statistical analyses SPSS 12.0 was used in this study to analyze the data. One-way variance analysis was carried out using Duncan’s test with confidence level as p  0.05. 3. Results and discussion 3.1. Amylose content The amylose content in the sago starch was found to be in the range of 32e34%. Most native starches like corn, tapioca, potato and sago have high percentage of amylopectin. Generally, most starches contain 15e30% amylose (Wong, Muhammad, Dzulkifly, Saari, & Ghazali, 2007). 3.2. Protein content and amino acids profile Protein content of fish gelatin was found to be 81.3% soluble protein. Therefore sago starch/fish gelatin films contained different protein mixtures which are 21.6%, 16.3%, 13.0%, and 10.9% (w/w) protein in 2:1, 3:1, 4:1 and 5:1 film ratios, respectively. Amino acids composition of the fish gelatin is shown in Table 1. The result shows that the gelatin has high contents of glutamic acid, glycine, alanine and arginine. Proline and hydroxyproline are present in low amounts (5.72 and 5.94 residues/100) compared to bovine-hide and tuna skin gelatins as reported by (Gómez-Estaca, Montero,



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117 Table 1 Amino acids composition in gelatin sample. Amino acids



Residues/100 residues



Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Arginine Alanine Tyrosine Valine Methionine Phenylalanine Isoleucine Leucine Lysine Hydroxyproline Proline



6.20 11.61 4.21 0.67 21.08 4.24 7.36 16.63 0.32 3.12 2.06 2.12 1.46 2.74 4.43 5.94 5.72



Fernández-Martín, & Gómez-Guillén, 2009). They reported proline content of 12.7 and 10.7 residues/100 and hydroxyproline of (8.3 and 7.8 residues/100 residues) for bovine-hide and tuna skin gelatin respectively. 3.3. Viscosity and pH of film forming solutions The pH of the film forming solutions ranged between pH 4.54 and 4.61. The viscosity of film forming solutions of sago starch/fish gelatin added with either sorbitol or glycerol increased significantly (p < 0.05) with decreasing gelatin protein content in the samples. Film samples 2:1 and 3:1 with glycerol added showed higher viscosity than those added with sorbitol of the same film ratio, however as the gelatin content decreased in film ratios of 4:1 and 5:1, there was no significant difference between films added with sorbitol or glycerol (Fig. 1). Moreover, significant difference was observed in comparing sago starch/fish gelatin film forming solutions to sago starch film forming solution plasticized as controls with either glycerol or sorbitol. Sago starch film forming solutions showed higher viscosity from other sago starch/fish gelatin film forming solution. Dickinson (1998) reported that proteinepolysaccharides interaction between two biopolymers is



111



made up from an average over the large number of different intermolecular forces arising between the various segments and side-chains on the two macromolecules. Depending on the aqueous environmental conditions and the distribution of the different kinds of groups (charged, hydrophobic, hydrogen bonding, etc.). The overall proteinepolysaccharide interaction maybe net attractive or net repulsive. 3.4. Thickness and light absorption of films Thickness of sago starch/fish gelatin films ranged between 0.05 and 0.07 mm for all films with plasticizers. Films of sago starch/fish gelatin prepared were transparent and homogeneous. All films were found to be flexible and easily removed from the acrylic plate with the exception of films (2:1) that contain glycerol which was found to be soft, sticky and easily shrink when removed from casting plates and left outside for short time at lab environment (RH% z 65) and this was due to being more hygroscopic with higher protein content than other films. This observation was similarly reported by Thomazine, Carvalho, and Sobral (2005) in those films with glycerol tend not to be as strong and are more stretchable than films that contain sorbitol. The UVeVis absorption of sago starch/fish gelatin films and sago starch films plasticized with 25% glycerol or sorbitol is shown in Table 2. Higher transparency values indicate higher absorbance values. The results showed that no significant differences in the level of light absorption at 550 nm were found in both control films with either plasticizer. Glycerol plasticized films exhibited higher absorbance values compared to sorbitol plasticized films. In glycerol plasticized sago starch/fish gelatin films, further increase of gelatin concentration did not have any significant effects on the degree of transparency or light absorption, however all films exhibited significantly higher absorbance than control. In general, all sago starch/fish gelatin films with glycerol showed significantly higher light absorbance (p < 0.05) compared to films with sorbitol. Higher light absorbance of films could be an excellent barrier to prevent light-induced lipid oxidation when applied in food system (Gomez-Guillen, Ihlb, Bifanib, Silvab, & Montero, 2007). 3.5. Mechanical properties of starchegelatin films The tensile strength (TS), percentage of elongation-at-break (% EAB) and Young’s modulus (E) values of the starch (as control) and sago starch/fish gelatin edible films prepared in this study are Table 2 Mechanical properties, Young’s modulus and transparency of sago starch/fish gelatin films plasticized with 25% glycerol or 25% sorbitol.



Fig. 1. Viscosity of film forming solutions of different ratios of sago starch and fish ] or 25% sorbitol [ ]. gelatin plasticized with 25% glycerol [



Films (starch: gelatin)



TS (MPa)



1:0 (G) 1:0 (S)



9.87  0.64a 25.03  0.88b



% EAB



17.11  6.11ad 4.33  0.78e



Young’s modulus, (E)(N/m2) 107



Transparency



6.17  0.01a 59.35  0.01b



1.34  0.09a 1.24  0.10a



2:1 3:1 4:1 5:1



(G) (G) (G) (G)



1.28 1.57 1.70 1.67



   



0.25c 0.30cd 0.08d 0.12d



102.31 95.15 84.14 93.11



   



20.78c 10.99bc 2.77b 12.34bc



0.12 0.16 0.20 0.18



   



0.04c 0.03c 0.01c 0.02c



2.00 1.91 2.12 1.85



   



0.18b 0.02b 0.09b 0.17b



2:1 3:1 4:1 5:1



(S) (S) (S) (S)



10.27 17.59 18.06 13.44



   



0.90ae 1.71f 0.55f 0.73g



11.94 19.04 5.53 11.76



   



2.07a 1.00d 0.42e 2.98a



0.91 1.47 1.71 1.20



   



0.09d 0.14e 0.05f 0.10g



0.86 1.43 1.20 1.60



   



0.06c 0.04ad 0.09ae 0.18d



Values were given as mean  standard deviation. Values with the same superscript letters within a column are not significantly different (p < 0.05). G: glycerol; S: sorbitol; TS: tensile strength; EAB: Elongation-at-break.



112



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



shown in Table 2. Films with glycerol at all sago starch/fish gelatin ratios showed lower tensile strength (between 1.28 MPa and 1.67 MPa) than the sago starch control film with glycerol (9.87 MPa) and films of the same ratios that contain sorbitol (varied from 10.27 MPa to 18.06 MPa). According to Sothornvit and Krochta (2001) glycerol is a smaller molecular weight and more hygroscopic (at constant aw from adsorption and desorption isotherms) compared to sorbitol and therefore, glycerol increase its effectiveness as plasticizer. Glycerol contributes more plasticization effect compared to sorbitol when used at the same mass content in protein, polysaccharides and proteinepolysaccharides based films. Thus protein, polysaccharides and proteinepolysaccharides based films with glycerol are more stretchable and flexible due to the plasticization effect that increases the mobility of polymer chains. Plasticizers have been used to overcome the brittleness of films resulting from high intermolecular forces by increasing the mobility of polymer chains which makes films stretchable and flexible. Films with sorbitol at all ratios had significantly lower (p < 0.05) TS than films containing sago starch (1:0) with sorbitol only (25.03 MPa). However, in all formulations, the presence of protein significantly reduced the TS as a result of the interaction between hydroxyl groups between starch and protein that may reduce the interaction between starch chains since polyols did not vary in all samples. Polyols such as sorbitol are often cited as good plasticizers due to their ability to reduce intermolecular hydrogenbonding while increasing intermolecular spacing (Vanin et al., 2005). Sorbitol interacts with water by hydrogen bonding due to the present of more hydroxyl groups in the molecule (Mali, Sakanaka, Yamashita, & Grossmann, 2005). Garcia, Martino, and Zaritzky (2000b) reported that the chances of sorbitol to interact with polymeric starch chains are higher than glycerol due to sorbitol being more similar to the molecular structure of glucose units therefore; films plasticized with sorbitol presented higher intermolecular forces and showed a lower capacity to interact with water. Higher tensile strength leads to stronger films. It was observed that reducing the gelatin content in films with glycerol (ratio from 2:1 to 5:1) did not have much effect on the TS values. In general, for films with sorbitol, tensile strength increased significantly from film ratios of 2:1 to of 4:1, however for a film ratio of 5:1, the tensile strength was much lower. This indicated that tensile strength increased with decreasing content of gelatin from 21.6% to 13.0%. However with further reduction in gelatin content, no further increase in TS was observed for films with sorbitol in the ratios used in this study. Arvanitoyannis, Psomiadou, Nakayama, Aiba, and Yamamoto (1997) reported that tensile strength and elongation-at-break are greatly affected by preparation temperature and relative humidity of conditioning. From the results (Table 2), in general % elongation-at-break (% EAB) increased with gelatin content in films with glycerol with highest % EAB value (102.31%) for a starch to gelatin ratio of (2:1) which indicated that in these films, gelatin seemed to act as a plasticizer which enhanced film flexibility and reduced brittleness. Su, Huang, Yuan, Wang, and Li (2010) concluded that glycerol is a small size molecule plasticizer that can penetrate between the polymer chains, and weaken the interaction between polysaccharides and proteins as in carboxymethyl cellulose and soy protein isolate films. Moreover, increasing the concentration of glycerol at constant polysaccharideseprotein composition reduces the mechanical properties. However, for sago starch/fish gelatin films with sorbitol, no trend was observed in % EAB (varied from 5.53 to 19.04%). Varying gelatin content in the films had both negative and positive effects on % EAB. Samples with sorbitol with ratios of 3:1 and 4:1 showed the highest (19.04%) and the lowest % EAB (5.53%), respectively. In general, the % EAB depends as TS on many factors in a mixed system between polysaccharides and protein including the hydroxyl



groups that are available for forming hydrogen bonding between gelatinestarchepolyolewater. Sorbitol has higher molecular weight with more hydroxyl groups than glycerol, which may react with starch and gelatin molecules and give less plasticizing affect compare to glycerol. Mali et al. (2005) reported that glycerol-containing films of cassava starch affected the mechanical properties and resulted in the lowest values for tensile stress due to its hygroscopic character that tends to provide additional water into the film matrix. Moreover, they reported that lower molecular weight of the plasticizer such glycerol has a plasticization affect than the higher molecule ones like sorbitol when used at the same mass basis where the number of moles of glycerol incorporated in the films would be higher than the sorbitol ones. Pranoto, Lee, and Park (2007) reported that the polysaccharide macromolecules with relatively long chains can cross-link with gelatin, leading to increased TS, and also possibly macromolecular relaxation leading to increase % EAB. Chambi and Grosso (2006) concluded that the mechanical properties of films are largely associated with distribution and density of intermolecular and intra-molecular interactions, which depend on the arrangements, and orientation of polymer chains in the network. Young’s modulus (E) significantly decreased with incorporation of protein in all films with either plasticizer (Table 2). Control films (1:0) showed the highest in Young’s modulus (6.17 Pa) and (59.35 Pa) for glycerol and sorbitol films, respectively. Higher TS and lower % EAB results in higher Young’s modulus. However, generally Young’s modulus (E) increased with decrease in protein content (from 21.6% to 13.0%) with highest E value for film ratio of 4:1 in both types of films. However, when the protein content was decreased further to 10.9% (in film ratio of 4:1) E values did not increase further in both types of films. The addition of more protein showed lower E values compared to films with no protein added in both cases with glycerol or sorbitol. The findings of this study suggested that among the different formulations plasticized with glycerol or sorbitol; there was a certain level where the interaction between the two biopolymers i.e. starch and protein that affected TS, % EAB and Young’s modulus (E). However, no linear trend was observed in TS, % EAB and E with different ratios when protein content was increased in all samples with either plasticizers. Values obtained in this study showed that there might be an optimum level of interactions between polysaccharides and protein that may affect TS and % EAB as in (4:1) and (5:1) with both glycerol or sorbitol. Pranoto et al. (2007) reported that there was an optimum level for interaction between polysaccharides and gelatin where gelatin was the major and dominant phase in the film system they used, and concluded that 2% was found to be beyond the optimum level of interaction between polysaccharides and gelatin. Fonkwe, Narsimhan, and Cha (2003) reported that the polysaccharides could form networks with gelatin molecules between anionic domains of the polysaccharides and cationic domains of the gelatin, which strengthen the film structures as a result. Lee, Shim, and Lee (2004) found that TS was improved by increasing the gellan (polysaccharides) to gelatin ratio, with highest TS at the film made from gellan indicating that there is a certain level of optimum interaction between polysaccharides and gelatin in general. Thus, concluded that the strength and flexibility of the composite films could be modified by changing the ratio of polysaccharides to protein. Tolstoguzov (1994) reported that interactions in gelatin/ water or gelatin/starch/water/polyol systems are between hydroxyl groups of starch chains, starchewater and starchepolyol molecules, as well as between polyolepolyol or waterepolyol molecules. The possibilities of gelatin or starch hydrogen-bonding within the blends are greatly enhanced by the introduction of comparatively small molecules such as water and polyols. Mali et al. (2005) reported lower stress and Young’s Modulus values were obtained



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



113



with films plasticized with glycerol, indicating that glycerol exerted a more effective plasticization.



1:0 G 2:1 G



4:1 G 5:1 G



30 Moisture content (dry basis) (%)



As shown in Fig. 2, water vapor permeability (WVP) of different ratios of starch/gelatin films plasticized with glycerol did not show any significant differences. There was no clear trend observed in WVP for films with sorbitol. However, higher WVP was observed with the incorporation of sorbitol into films compared to glycerol for fish gelatinesago starch films. Control starch films exhibited higher WVP with glycerol added compared to sorbitol however, both types of films with either sorbitol or glycerol added showed lower WVP. Adding gelatin to starch films leads to interaction with starch chains breaking the intact network of starch inter chain hydrogen-bonding. Since gelatin is more hygroscopic than starch, the affinity for water molecules will be higher in these films and therefore resulting in higher water diffusion giving films with higher WVP. The results also showed that increasing protein content in the films i.e. varying the starch to gelatin ratio from 5:1 to 2:1 did not affect the WVP of both films with added sorbitol or glycerol. This result is in contrast with results obtained by Jongjareonrak, Benjakul, Visessanguan, and Tanaka (2006) who reported that higher WVP was found with gelatin films containing greater protein content. McHugh, Avena-Bustillos, and Krochta (1993) also reported that since gelatin contained a wide range of hydrophilic amino acids, films with a higher amount of protein and thickness could absorb more water molecules from the environment. Thus, the film with higher protein content was most likely to be hygroscopic, compared with that containing the lower protein content. Bourtoom, Chinnan, Jantawat, and Sanguandeekul (2006) concluded that plasticizers modify the protein network structure and increase the water vapor permeability (WVP) of edible watersoluble fish proteins film when both plasticizers sorbitol or glycerol increased from 25% to 75%. Plasticizers modify the molecular organization of the protein network and increase the free volume resulting in less dense network that results in more films that are permeable to water. The hydrophilicity nature of plasticizer molecules could be the cause of permeability increase with an increase in plasticizer. Hydrophilic plasticizers such glycerol and sorbitol are known to enhance the water vapor permeability of hydrocolloidbased films. Arvanitoyannis, Nakayama, and Aiba (1998) reported that increasing the total plasticizer content (water and polyols) in the polymer matrix results in a proportional increase of water



3:1 G



35



3.6. Water vapor permeability of films



25



20



15



10



5



0



0



10



20



30



40



50



60



70



80



90



Relative humidity (%RH)



Fig. 3. Moisture sorption isotherm for sago starch/fish gelatin films (ratios 1:0, 2:1, 3:1, 4:1 & 5:1) with 25% glycerol, conditioned at different relative humidity.



vapor transfer rate. However, they reported that sorbitol showed higher WVP than glycerol in hydroxypropyl starch and gelatin films produced by both methods of high temperature casting and drying (60  C) with glycerol or sorbitol (15% and 25%) and low temperature casting and drying (20  C) with glycerol (5% and 25%); and sorbitol (3% and 24%). Similarly, Arvanitoyannis and Biliaderis (1998) found that sorbitol increased WVP compared to glycerol at all concentrations used (5, 15 and 25%) in sodium caseinate and soluble starch films. Garcia et al., 2000a reported that WVP depends on many factors such as the ratio between crystalline and amorphous zone, polymeric chain mobility and specific interaction between the functional groups of the polymers and the gases in the amorphous zone. Slight differences in WVP values maybe related to the difference in water molecule diffusion and hydrophilicehydrophobic ratio (Arvanitoyannis, Kalichevsky, Blanshard, & Psomiadou, 1994; Garcia et al., 2000a). However, Pranoto et al. (2007) reported that



1:0 S 2:1 S



35



Moisture content (dry basis) (%)



3:1 S 4:1 S



30



5:1 S



25



20



15



10



5



0 0



10



20



30



40



50



60



70



80



90



Relative humidity (%RH) Fig. 2. Water vapor permeability (WVP) of sago starch/fish gelatin films with 25% plasticizers [glycerol or sorbitol].



Fig. 4. Moisture sorption isotherm for sago starch/fish gelatin films (ratios 1:0, 2:1, 3:1, 4:1 & 5:1) with 25% sorbitol, conditioned at different relative humidity.



114



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



the addition of gellan to gelatin films significantly reduced the WVP that may due to the ionic interaction between gelatin and gellan that formed a denser polymeric matrix, thus hindering water molecule transfer through the film. K-carrageenan was also found to form ionic complexes with gelatin but weaker than that of gellanegelatin matrix. 3.7. Moisture sorption isotherm Figs. 3 and 4 show the water sorption isotherms of sago starch and sago starch/fish gelatin edible films as a function of sago starch and fish gelatin ratio with added sorbitol or glycerol. The sorption isotherm curves of sago starch and sago starch/fish gelatin films showed a typical behavior of water vapor sensitive hydrophilic



biopolymers when the relative humidity (% RH) increased. These films behaved as in gluten, starch and cellulose films (Gontard, Guilbert, & Cuq, 1993; Mali, Grossmann, García, Martino, & Zaritzky, 2002) which presented a relatively slight slope at low water activity (aw), and an exponential increase at high aw, (above 0.75). Such non-linear water sorption isotherms were possibly due to the swelling of hydrophilic matrix that resulted in different structural changes in the films. Glycerol sago starch/fish gelatin films presented higher equilibrium moisture contents than those of sago starch films only (Fig. 3). In general, moisture content of these films increased slowly at RHs lower than 40%, followed by a steep increase in moisture content when RH% changed from 56% to 84%. The equilibrium moisture content curves of the films plasticized with



Fig. 5. Scanning electron microscopy images (500) of sago starch/fish gelatin films plasticized with 25% glycerol (a) 2:1, (b) 3:1, (c) 4:1, (d) 5:1 and (e) 1:0.



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



25% sorbitol show similar sorption isotherm profiles (Fig. 4) i.e. low equilibrium moisture contents at low aw then increased at higher aw. However, sago starch films at 84% RH showed slightly lower equilibrium moisture content than those of sago starch/fish gelatin films. Generally, the equilibrium moisture content was higher in films with added glycerol than those films plasticized with sorbitol due to the fact that glycerol is more hydrophilic than that of sorbitol. Due to the presence of more hydroxyl groups in the molecule, sorbitol interacted with water by hydrogen bonding



115



resulting in less plasticizing effect compared to glycerol (Mali et al., 2005). Hu, Chen, and Gao (2009) also reported that oxidized potato starch films with higher glycerol level showed higher moisture absorbability with increase of % RH due to glycerol having excellent water-retaining ability. A lower water binding capacity of films with sorbitol has also been reported and this behavior was due to the molecular structure of sorbitol facilitating interactions with the polymeric chains (Garcia et al., 2000b).



Fig. 6. Scanning electron microscopy images (500) of sago starch/fish gelatin films plasticized with 25% sorbitol (a) 2:1, (b) 3:1, (c) 4:1, (d) 5:1 and (e) 1:0.



116



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



3.8. Film morphology Figs. 5 and 6 show the scanning electron micrographs of sago starch and sago starch/fish gelatin films plasticized with glycerol and sorbitol, respectively. It was observed that there were some differences in surface and internal structure morphology between glycerol and sorbitol films at the various starch to gelatin ratios. Glycerol films were observed to have rougher surfaces with presence of pores or cavities that could be related to the formation of channels whereas sorbitol films showed a more compact and homogeneous surface. However, smoother film surfaces were observed in both glycerol and sorbitol films that has less protein content (Figs. 5 and 6aed). Nevertheless, surface roughness and formation of channels disappeared in samples of sago starch films only (1:0) with either plasticizer. Liu, Liu, Fishman, and Hicks (2007) worked on pectin films with addition of two different types of proteins (fish skin gelatin or soybean protein) and obtained pectin films that showed relatively smoother surfaces than those films with the protein addition which produced rough, dense, and brittle in appearance surfaces with some irregular particles distributed uniformly within the pectin phase. Pranoto et al. (2007) also found in films with fish gelatin and gellan or k-carrageenan mixtures produces films that showed discontinuous zones with noticeable cracks randomly distributed along the network. However, they also concluded that addition of gellan to fish gelatin films contributed to eliminating those cracks thus giving a more compact surface appearance, while addition of k-carrageenan had no contribution in eliminating those cracks. Moreover, De Carvalho and Grosso (2004) mentioned that gelatin films plasticized with glycerol have the presence of discontinuous zones characterized by cracks distributed along the length of the network, however those discontinuous zones possibly present as a result of the preferential channels that occurred through drying. 3.9. Differential scanning calorimetry Differential scanning calorimetry (DSC) results obtained for the different ratios of sago starch/fish gelatin films unplasticized and plasticized with 25% (w/w) glycerol or sorbitol are given in Table 3. The variation of glass transition temperature (Tg) is another Table 3 DSC thermal characterization of sago starch/fish gelatin films unplasticized and plasticized with glycerol or sorbitol (25%). Films



Tg



Tm



DH



1:0 NP



84.04  0.81a



167.41  3.23a



219.63  13.70f



2:1 3:1 4:1 5:1



52.82 55.32 55.71 53.81



NP NP NP NP



   



2.28d 1.43ef 0.87f 1.41de



159.15 144.91 151.64 163.76



   



16.84hi 15.57fg 11.51gh 6.82ai



133.90  12.3bd 112.70  5.4c



240.73 239.63 227.00 217.73



   



25.31h 9.22h 2.96g 11.40f



1:0 G 1:0 S



60.08  1.1gh 53.10  0.8d



171.83  8.3de 148.33  1.2b



2:1 3:1 4:1 5:1



G G G G



62.28 61.36 58.50 61.95



   



0.8i 1.1hi 0.5g 1.4i



157.61 133.15 143.12 139.04



   



10hi 10.5bdh 4.1efg 3.1def



177.43 172.50 171.65 161.08



   



11.1e 27.3de 2.2de 6.8c



2:1 3:1 4:1 5:1



S S S S



49.85 40.88 48.15 49.60



   



4.4c 6.7b 4.9c 7.7c



129.01 151.36 135.20 143.72



   



8.4b 16.5g 15.2bde 16.9fg



162.50 168.08 140.13 151.97



   



4.5c 8.3cd 15.5a 2.3b



Values are given as mean  standard deviation. Values with the same superscript letters within a column are not significantly different (p < 0.05). G: glycerol; S: sorbitol, NP: no plasticizer added.



effective indicator of the compatibility of polymers. Although polysaccharides and protein materials have been widely studied, still there is poor understanding of the dynamic interaction between the molecules of such polymers. The findings of this study showed that unplasticized sago starch films had a Tg value of about (84.04  0.8  C), Tm (167.41  3.2) and DH of (219.63  13.7). Incorporating plasticizer (25% glycerol or sorbitol) into the starch films significantly decreased Tg, Tm and DH. However, greater decrease in the values was noticed when sorbitol was used compared to glycerol. Plasticizing sago starch films with glycerol makes films more hydrophilic and retain higher moisture compared to unplasticized films when conditioned at the same humidity (RH %) and temperature. Mali et al. (2005) also reported that glycerol-containing films of cassava starch affected films properties due to the hygroscopic character of glycerol that tends to provide additional water into the film matrix. In this study, sorbitol showed lower Tg, Tm and DH when added to sago starch films compared to unplasticized ones. Addition of glycerol to sago starch/ fish gelatin films showed an increase in Tg for all ratios (1:0, 2:1, 3:1, 4:1 & 5:1) but a significant decrease (p  0.05) in Tm for ratios (3:1 & 5:1) and DH for all ratios which indicated that plasticizers reduced the intermolecular forces and increased the mobility of polar polymer chains. Su et al. (2010) concluded that plasticizers like glycerol reduce Tg values when incorporated into carboxymethyl cellulose and soy protein isolate films that was probably due to the ability of glycerol to penetrate between the polymer chains and therefore weaken the interaction between polysaccharides and proteins. Sago starch/fish gelatin films plasticized with sorbitol showed significantly lower (p  0.05) Tg, Tm and DH for all ratios with the exception of ratio (3:1) for Tm values that showed no significant change compared to unplasticized films of the same ratio. The reduction in Tg, Tm and DH when sorbitol was used as plasticizer maybe due to the interaction between sorbitol hydroxyl groups with the polymers present in films which may reduce the interaction between the starch and gelatin. The presence of only one Tg could indicate that the sago starch and gelatin films are compatible. This phenomena with only one Tg observed in starchecaseinate blends plasticized with glycerol or sorbitol was also reported by Arvanitoyannis and Biliaderis (1998) in their DTA runs. Su et al. (2010) also reported that normally, a single Tg for blended polymers in a DSC scan indicates good compatibility of the component polymers. 4. Conclusion The physical and mechanical properties of edible films from sago starch and sago starch/fish gelatin plasticized by glycerol and sorbitol were evaluated. Starch/gelatin solution with ratios of 3:1, 4:1 and 5:1 appear to form good flexible films with added glycerol but not at higher gelatin content (2:1). The results obtained show that different ratios of sago starch and fish gelatin blends plasticized with glycerol or sorbitol affected physical, mechanical and water vapor permeability of the films produced. Changing the ratio of two polymers can modify the strength and extensibility of the composite films. SEM micrographs show uneven surfaces in glycerol films compared to sorbitol film surfaces. However, less protein in the samples gave smoother films with either sorbitol or glycerol. Based on the concentration levels used and the polymers sources, DSC runs revealed a single Tg which maybe an indication of the compatibility of sago starch and fish gelatin polymers in this study. Acknowledgments The authors wish to express their thanks to the Ministry of Science, Technology and Innovation, Malaysia and the Saudi



A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117



Arabia Government for the financial support given for this research work. References AOAC. (1984). Official methods of analysis (13th ed.). Washington, DC: Association of Official Analytical Chemists. AOAC. (1999). Official methods of analysis (14th ed.). Washington, DC: Association of Official Analytical Chemists. Arvanitoyannis, I. (2002). Formation and properties of collagen and gelatin films and coatings. In A. Gennadios (Ed.), Protein-based films and coatings. Boca Raton, FL, USA: CRC Press LCC. Arvanitoyannis, I., & Biliaderis, C. G. (1998). Physical properties of polyol-plasticized edible films made from sodium caseinate and soluble starch blends. Food Chemistry, 62(3), 333e342. Arvanitoyannis, I., Kalichevsky, M., Blanshard, J. M. V., & Psomiadou, E. (1994). Study of diffusion and permeation of gases in undrawn and uniaxially drawn films made from potato and rice starch conditioned at different relative humidities. Carbohydrate Polymers, 24(1), 1e15. Arvanitoyannis, I., Nakayama, A., & Aiba, S. (1998). Edible films made from hydroxypropyl starch and gelatin and plasticized by polyols and water. Carbohydrate Polymers, 36(2e3), 105e119. Arvanitoyannis, I., Psomiadou, E., & Nakayama, A. (1996). Edible films made from sodium caseinate, starches, sugars or glycerol: part 1. Carbohydrate Polymers, 31, 179e192. Arvanitoyannis, I., Psomiadou, E., Nakayama, A., Aiba, S., & Yamamoto, N. (1997). Edible films made from gelatin, soluble starch and polyols: part 3. Food Chemistry, 60(4), 593e604. ASTM. (2000a). Standard test methods for tensile properties of thin plastic sheeting, method D882-00. Philadelphia, PA: American Society for Testing and Materials. ASTM. (2000b). Standard test methods for water vapor transmission of materials, method E 96-00. Philadelphia, PA: American Society for Testing and Materials. Baldwin, E. A., Nisperos-Carriedo, M. O., & Baker, R. A. (1995). Use of edible coatings to preserve quality of lightly (and slightly) processed products. Critical Reviews in Food Science and Nutrition, 35, 509e524. Bao, S., Xu, S., & Wang, Z. (2009). Antioxidant activity and properties of gelatin films incorporated with tea polyphenol-loaded chitosan nanoparticles. Journal of the Science of Food and Agriculture, 89(15), 2692e2700. Bourtoom, T., Chinnan, M. S., Jantawat, P., & Sanguandeekul, R. (2006). Effect of plasticizer type and concentration on the properties of edible film from watersoluble fish proteins in surimi wash-water. Food Science and Technology International, 12(2), 119e126. Cao, N., Fu, Y., & He, J. (2007). Preparation and physical properties of soy protein isolate and gelatin composite films. Food Hydrocolloids, 21(7), 1153e1162. Chambi, H., & Grosso, C. (2006). Edible films produced with gelatin and casein cross-linked with transglutaminase. Food Research International, 39, 458e466. Chang, Y. P., Cheah, P. B., & Seow, C. C. (2005). Plasticizingeantiplasticizing effects of water on physical properties of tapioca starch films in the glassy state. Journal of Food Science, 65(3), 445e451. Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1997). Selected functional properties of fish myofibrillar protein-based films as affected by hydrophilic plasticizers. Journal of Agricultural and Food Chemistry, 45, 622e626. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18, 717e726. Denavi, G., Tapia-Blácido, D. R., Añón, M. C., Sobral, P. J. A., Mauri, A. N., & Menegalli, F. C. (2009). Effects of drying conditions on some physical properties of soy protein films. Journal of Food Engineering, 90(3), 341e349. Dickinson, E. (1998). Stability and rheological implications of electrostatic milk proteinepolysaccharide interaction. Trends in Food Science & Technology, 9, 347e354. Fonkwe, L. G., Narsimhan, G., & Cha, A. S. (2003). Characterization of gelation time and texture of gelatin and gelatinepolysaccharide mixed gels. Food Hydrocolloids, 17(6), 871e883, Food Hydrocolloids, 18, 717e726. Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2000a). Lipid addition to improve barrier properties of edible starch-based films and coatings. Journal of Food Science, 65, 941e947. Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2000b). Microstructural characterization of plasticized starch-based films. Starch/Stärke, 52(4), 118e124. Gómez-Estaca, J., Montero, P., Fernández-Martín, F., & Gómez-Guillén, M. C. (2009). Physico-chemical and film-forming properties of bovine-hide and tuna-skin gelatin: a comparative study. Journal of Food Engineering, 90(4), 480e486. Gomez-Guillen, M. C., Ihlb, M., Bifanib, V., Silvab, A., & Montero, P. (2007). Edible films made from tuna-fish gelatin with antioxidant extracts of two different



117



murta ecotypes leaves (Ugni molinae Turcz). Food Hydrocolloids, 21, 1133e1143. Gomez-Guillen, M. C., Perez-Mateos, M., Gomez-Estaca, E., Lopez-Caballero, E., Gimenez, B., & Montero, P. (2008). Fish gelatin: a renewable material for developing active biodegradable films. Trends in Food Science and Technology, 20, 3e16. Gontard, N., Guilbert, S., & Cuq, J. L. (1993). Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58(1993), 206e211. Guilbert, S., Gontard, N., & Gorris, L. G. M. (1996). Prolongation of the shelf life of perishable food products using biodegradable films and coatings. LebensmittelWissenschaft und-Technologie, 29, 10e17. Hu, G., Chen, j., & Gao, J. (2009). Preparation and characteristics of oxidized potato starch films. Carbohydrate Polymers, 76, 291e298. Jongjareonrak, A., Benjakul, S., Visessanguan, W., Prodpran, T., & Tanaka, M. (2006b). Characterization of edible films from skin gelatin of brown stripe red snapper and big eye snapper. Food Hydrocolloids, 20(4), 492e501. Jongjareonrak, A., Benjakul, S., Visessanguan, W., & Tanaka, M. (2006). Effects of plasticizers on the properties of edible films from skin gelatin of big eye snapper and brown stripe red snapper. European Food Research Technology, 222, 229e235. Lee, K. Y., Shim, J., & Lee, H. G. (2004). Mechanical properties of gellan and gelatin composite films. Carbohydrate Polymers, 56(2), 251e254. Liu, L., Liu, C. K., Fishman, M. L., & Hicks, K. B. (2007). Composite films from pectin and fish skin gelatin or soybean flour protein. Journal of Agricultural and Food Chemistry, 55(6), 2349e2355. Mali, S., Grossmann, M. V. E., García, M. A., Martino, M. N., & Zaritzky, N. E. (2002). Microstructural characterization of yam starch films. Carbohydrate Polymers, 50(2002), 379e386. Mali, S., Grossmann, M. V. E., Garcia, M. A., Martino, M. N., & Zaritzky, N. E. (2006). Effects of controlled storage on thermal, mechanical and barrier properties of plasticized films from different starch sources. Journal of Food Engineering, 75(4), 453e460. Mali, S., Sakanaka, L. S., Yamashita, F., & Grossmann, M. V. E. (2005). Water sorption and mechanical properties of cassava starch films and their relation to plasticizing effect. Carbohydrate Polymers, 60(3), 283e289. McGrance, S. J., Cornell, H. J., & Rix, C. J. (1998). A simple and rapid colorimetric method for the determination of amylose in starch products. Starch/Starke, 50, 158e163. McHugh, T. H., Avena-Bustillos, R., & Krochta, J. M. (1993). Hydrophilic edible films: modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science, 58(4), 899e903. Norziah, M. H., Al-Hassan, A., Khairulnizam, A. B., Mordi, M. M., & Norita, M. (2009). Characterization of fish gelatin from surimi processing wastes: thermal analysis and effect of transglutaminase on gel properties. Food Hydrocolloids, 23(6), 1610e1616. Pranoto, Y., Lee, C. M., & Park, H. J. (2007). Characterizations of fish gelatin films added with gellan and K-carrageenan. LWT e Food Science and Technology, 40(5), 766e774. Soares, R. M. D., Lima, A. M. F., Oliveira, R. V. B., Pires, A. T. N., & Soldi, V. (2005). Thermal degradation of biodegradable edible films based on xanthan and starches from different sources. Polymer Degradation and Stability, 90, 449e454. Sobral, P. J. A., Menegalli, F. C., Hubinger, M. D., & Roques, M. A. (2001). Mechanical, water vapor barrier and thermal properties of gelatin based edible films. Food Hydrocolloids, 15, 423e432. Sothornvit, R., & Krochta, J. M. (2001). Plasticizer effect on mechanical properties of b-lactoglobulin films. Journal of Food Engineering, 50(3), 149e155. Su, J.-F., Huang, Z., Yuan, X.-Y., Wang, X.-Y., & Li, M. (2010). Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions. Carbohydrate Polymers, 79(1), 145e153. Thomazine, M., Carvalho, R. A., & Sobral, P. J. A. (2005). Physical properties of gelatin films plasticized by blends of glycerol and sorbitol. Journal of Food Science, 70(3), E172eE176. Tolstoguzov, V. B. (1994). Some physicochemical aspects of protein processing in foods. In G. O. Philips, P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilizers for the food industry, Vol. 7 (pp. 115e154). Oxford: IRL Press. Vanin, F. M., Sobral, P. J. A., Menegalli, F. C., Carvalho, R. A., & Habitante, A. M. Q. B. (2005). Effects of plasticizers and their concentrations on thermal and functional properties of gelatin-based films. Food Hydrocolloids, 19(5), 899e907. Viga-Santos, P., Oliveria, L. M., Cereda, M. P., & Scamparini, A. R. P. (2007). Sucrose and inverted sugars as plasticizer. Effect on cassava starchegelatin film mechanical properties, hydrophilicity and water activity. Food Chemistry, 103, 255e262. Wong, C. W., Muhammad, S. K. S., Dzulkifly, M. H., Saari, N., & Ghazali, H. M. (2007). Enzymatic production of linear long-chain dextrin from sago (Metroxylon Sagu) starch. Food Chemistry, 100, 774e780.