Food Hydrocolloids 26 (2012) 108e117

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

Starchegelatin edible lms: 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
Article history: Received 20 May 2010 Accepted 14 April 2011 Keywords: Edible lm Sago starch Fish gelatin Glycerol Sorbitol DSC Glass transition

a b s t r a c t
Physical and mechanical properties of edible lms based on blends of sago starch and sh gelatin plasticized with glycerol or sorbitol (25%, w/w) were investigated. Film forming solutions of different ratios of sago starch to sh 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 sh gelatin was found to be 81.3%. The ndings of this study showed that the addition of sh gelatin in starch solutions has a signicant effect (p < 0.05), resulting in lms 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 lm samples plasticized with sorbitol showed signicantly 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 signicance differences was observed among the samples (p < 0.05) with glycerol with exception to lm with high protein content (21.6%) only and no trend was observed in % EAB among samples as well. Signicant difference (p < 0.05) was observed in TS and viscosity between different formulations with sorbitol. The morphology study of the sago starch/sh gelatin lms showed smoother surfaces with decreasing protein in the samples with either plasticizer. DSC scans showed that plasticizers and protein content incorporated with sago starch lms 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 sh gelatin polymers to form lms at the concentration levels used. 2011 Elsevier Ltd. All rights reserved.

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 lms 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, avor and aroma between food components and the surrounding atmosphere (Jongjareonrak, Benjakul, Visessanguan, Prodpran & Tananka, 2006). Such lms 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 lms can be prepared from protein, polysaccharides, lipids or the combination of these components

* Corresponding author. Tel.: 60 46535200; fax: 60 46573678. E-mail address: norziah@usm.my (M.H. Norziah). 0268-005X/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.04.015

(Cao, Fu, & He, 2007; Cuq, Gontard, Cuq, & Guilbert, 1997). Starch lms 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 lms and coatings can be formulated consisting of several biopolymers. As an example, the lipid component in the lm 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 lms 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 lms, several studies have investigated various additives, types of modication, sources of starch, and process parameters in order to improve these weaknesses in properties (Mali, Grossmann, Garcia, Martino, &

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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 lms (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 lm producing a exible and easy handling lm 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 lms 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 lms. The objective of this study was to investigate the physical and mechanical properties including tensile strength, elongation-atbreak, water vapor permeability, lm morphology and moisture isotherm and the compatibility of edible lms made from a combination of sago starch and sh 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 sh wastes provided by a local surimi processing plant. The extractions and preparation of sh 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 uorescence detector and a ow 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.

2.4. Preparation of starch/gelatin edible lms The lm forming solutions were prepared using blends of sago starch and sh gelatin with added plasticizers (glycerol or sorbitol). Different ratios of sago starch and sh 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 lms 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 lms were prepared in the same way without the addition of sh gelatin. Each lm formulation was prepared in triplicates. 2.5. Viscosity of lm forming solutions Viscosity of sago starch/gelatin solutions was measured using a digital viscometer (Model DV-E, Brookeld, 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 lm forming solution Measurement of pH value of sago starch and sago starch/gelatin lm forming solutions was taken using a pH meter (Mettler Toledo, FE20. China). 2.7. Film thickness and light absorption Thickness of the lms 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 lms were measured by exposing the lms to light absorption at wavelength 550 nm. Film transparency was measured according to the method of Bao, Xu, and Wang (2009) by placing rectangular lm samples into a spectrophotometer test cell directly. Absorbance was recorded using an UV-160A uvevis spectrophotometer (Shimadzu, Japan). The transparency (T) of lms was calculated according to the following equation: T A550/x where A550 is the absorbance at 550 nm and x is the lm thickness (mm). According to this equation, a higher value of T would indicate

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a lower degree of transparency. Tests were run in triplicates for each type of lm. 2.8. Tensile strength and elongation tests The mechanical properties of lms 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 lms 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 lm width of 2 cm. The results of tensile and elongation tests were expressed by MPa and percentage (%), respectively. Each test trial per lm consisted of ve replicate measurements. 2.9. Water vapor permeability Water vapor permeability (WVP) of the lms 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 lms 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 lms was determined as follows: WVP (WVTR L)/DP where WVTR is the water vapor transmission rate (g m2 h1) through a lm, calculated from the slope of the straight line divided by the exposed lm area (m2), L is the mean lm thickness (mm), and DP is the partial water vapor pressure difference (Pa) across the two sides of the lm. For each type of lm, WVP measurements were replicated three times for each batch of lms. 2.10. Moisture sorption isotherm The sorption isotherm of sago starch and sago starch/gelatin lms was determined at 30  C according to Chang, Cheah, and Seow (2005) with slight modications. Samples of sago starch and sago starch/sh 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 xed 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 xed 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 nally (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 lms Thermal properties of lms 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 156.6  C, and enthalpy DH 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 ow 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 Duncans test with condence 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, Dzulkiy, Saari, & Ghazali, 2007). 3.2. Protein content and amino acids prole Protein content of sh gelatin was found to be 81.3% soluble protein. Therefore sago starch/sh gelatin lms 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 lm ratios, respectively. Amino acids composition of the sh 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 (Gmez-Estaca, Montero,

A.A. Al-Hassan, M.H. Norziah / Food Hydrocolloids 26 (2012) 108e117 Table 1 Amino acids composition in gelatin sample. Amino acids Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Arginine Alanine Tyrosine Valine Methionine Phenylalanine Isoleucine Leucine Lysine Hydroxyproline Proline Residues/100 residues 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

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 lms Thickness of sago starch/sh gelatin lms ranged between 0.05 and 0.07 mm for all lms with plasticizers. Films of sago starch/sh gelatin prepared were transparent and homogeneous. All lms were found to be exible and easily removed from the acrylic plate with the exception of lms (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 lms. This observation was similarly reported by Thomazine, Carvalho, and Sobral (2005) in those lms with glycerol tend not to be as strong and are more stretchable than lms that contain sorbitol. The UVeVis absorption of sago starch/sh gelatin lms and sago starch lms plasticized with 25% glycerol or sorbitol is shown in Table 2. Higher transparency values indicate higher absorbance values. The results showed that no signicant differences in the level of light absorption at 550 nm were found in both control lms with either plasticizer. Glycerol plasticized lms exhibited higher absorbance values compared to sorbitol plasticized lms. In glycerol plasticized sago starch/sh gelatin lms, further increase of gelatin concentration did not have any signicant effects on the degree of transparency or light absorption, however all lms exhibited signicantly higher absorbance than control. In general, all sago starch/sh gelatin lms with glycerol showed signicantly higher light absorbance (p < 0.05) compared to lms with sorbitol. Higher light absorbance of lms 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 lms The tensile strength (TS), percentage of elongation-at-break (% EAB) and Youngs modulus (E) values of the starch (as control) and sago starch/sh gelatin edible lms prepared in this study are
Table 2 Mechanical properties, Youngs modulus and transparency of sago starch/sh gelatin lms plasticized with 25% glycerol or 25% sorbitol. Films (starch: gelatin) 1:0 (G) 1:0 (S) 2:1 3:1 4:1 5:1 2:1 3:1 4:1 5:1 (G) (G) (G) (G) (S) (S) (S) (S) TS (MPa) % EAB Youngs modulus, (E)(N/m2) 107 6.17 0.01a 59.35 0.01b 0.12 0.16 0.20 0.18 0.91 1.47 1.71 1.20 0.04c 0.03c 0.01c 0.02c 0.09d 0.14e 0.05f 0.10g Transparency

Fernndez-Martn, & Gmez-Guilln, 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 lm forming solutions The pH of the lm forming solutions ranged between pH 4.54 and 4.61. The viscosity of lm forming solutions of sago starch/sh gelatin added with either sorbitol or glycerol increased signicantly (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 lm ratio, however as the gelatin content decreased in lm ratios of 4:1 and 5:1, there was no signicant difference between lms added with sorbitol or glycerol (Fig. 1). Moreover, signicant difference was observed in comparing sago starch/sh gelatin lm forming solutions to sago starch lm forming solution plasticized as controls with either glycerol or sorbitol. Sago starch lm forming solutions showed higher viscosity from other sago starch/sh gelatin lm forming solution. Dickinson (1998) reported that proteinepolysaccharides interaction between two biopolymers is

9.87 0.64a 25.03 0.88b 1.28 1.57 1.70 1.67 10.27 17.59 18.06 13.44 0.25c 0.30cd 0.08d 0.12d 0.90ae 1.71f 0.55f 0.73g

17.11 6.11ad 4.33 0.78e 102.31 95.15 84.14 93.11 11.94 19.04 5.53 11.76 20.78c 10.99bc 2.77b 12.34bc 2.07a 1.00d 0.42e 2.98a

1.34 0.09a 1.24 0.10a 2.00 1.91 2.12 1.85 0.86 1.43 1.20 1.60 0.18b 0.02b 0.09b 0.17b 0.06c 0.04ad 0.09ae 0.18d

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

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

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shown in Table 2. Films with glycerol at all sago starch/sh gelatin ratios showed lower tensile strength (between 1.28 MPa and 1.67 MPa) than the sago starch control lm with glycerol (9.87 MPa) and lms 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 lms. Thus protein, polysaccharides and proteinepolysaccharides based lms with glycerol are more stretchable and exible due to the plasticization effect that increases the mobility of polymer chains. Plasticizers have been used to overcome the brittleness of lms resulting from high intermolecular forces by increasing the mobility of polymer chains which makes lms stretchable and exible. Films with sorbitol at all ratios had signicantly lower (p < 0.05) TS than lms containing sago starch (1:0) with sorbitol only (25.03 MPa). However, in all formulations, the presence of protein signicantly 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; lms plasticized with sorbitol presented higher intermolecular forces and showed a lower capacity to interact with water. Higher tensile strength leads to stronger lms. It was observed that reducing the gelatin content in lms with glycerol (ratio from 2:1 to 5:1) did not have much effect on the TS values. In general, for lms with sorbitol, tensile strength increased signicantly from lm ratios of 2:1 to of 4:1, however for a lm 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 lms 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 lms with glycerol with highest % EAB value (102.31%) for a starch to gelatin ratio of (2:1) which indicated that in these lms, gelatin seemed to act as a plasticizer which enhanced lm exibility 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 lms. Moreover, increasing the concentration of glycerol at constant polysaccharideseprotein composition reduces the mechanical properties. However, for sago starch/sh gelatin lms with sorbitol, no trend was observed in % EAB (varied from 5.53 to 19.04%). Varying gelatin content in the lms 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 lms 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 lm 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 lms 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 lms 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. Youngs modulus (E) signicantly decreased with incorporation of protein in all lms with either plasticizer (Table 2). Control lms (1:0) showed the highest in Youngs modulus (6.17 Pa) and (59.35 Pa) for glycerol and sorbitol lms, respectively. Higher TS and lower % EAB results in higher Youngs modulus. However, generally Youngs modulus (E) increased with decrease in protein content (from 21.6% to 13.0%) with highest E value for lm ratio of 4:1 in both types of lms. However, when the protein content was decreased further to 10.9% (in lm ratio of 4:1) E values did not increase further in both types of lms. The addition of more protein showed lower E values compared to lms with no protein added in both cases with glycerol or sorbitol. The ndings 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 Youngs 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 lm 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 lm 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 lm 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 exibility of the composite lms could be modied 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 Youngs Modulus values were obtained

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113
1:0 G 2:1 G

with lms plasticized with glycerol, indicating that glycerol exerted a more effective plasticization. 3.6. Water vapor permeability of lms As shown in Fig. 2, water vapor permeability (WVP) of different ratios of starch/gelatin lms plasticized with glycerol did not show any signicant differences. There was no clear trend observed in WVP for lms with sorbitol. However, higher WVP was observed with the incorporation of sorbitol into lms compared to glycerol for sh gelatinesago starch lms. Control starch lms exhibited higher WVP with glycerol added compared to sorbitol however, both types of lms with either sorbitol or glycerol added showed lower WVP. Adding gelatin to starch lms leads to interaction with starch chains breaking the intact network of starch inter chain hydrogen-bonding. Since gelatin is more hygroscopic than starch, the afnity for water molecules will be higher in these lms and therefore resulting in higher water diffusion giving lms with higher WVP. The results also showed that increasing protein content in the lms i.e. varying the starch to gelatin ratio from 5:1 to 2:1 did not affect the WVP of both lms 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 lms containing greater protein content. McHugh, Avena-Bustillos, and Krochta (1993) also reported that since gelatin contained a wide range of hydrophilic amino acids, lms with a higher amount of protein and thickness could absorb more water molecules from the environment. Thus, the lm 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 sh proteins lm 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 lms 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 lms. 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
35

3:1 G 4:1 G

30
Moisture content (dry basis) (%)

5:1 G

25

20

15

10

10

20

30

40

50

60

70

80

90

Relative humidity (%RH)

Fig. 3. Moisture sorption isotherm for sago starch/sh gelatin lms (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 lms 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 lms. Garcia et al., 2000a reported that WVP depends on many factors such as the ratio between crystalline and amorphous zone, polymeric chain mobility and specic 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

35

2:1 S 3:1 S

Moisture content (dry basis) (%)

30

4:1 S 5:1 S

25

20

15

10

0 0 10 20 30 40 50 60 70 80 90

Relative humidity (%RH)

Fig. 2. Water vapor permeability (WVP) of sago starch/sh gelatin lms with 25% plasticizers [glycerol or sorbitol]. Fig. 4. Moisture sorption isotherm for sago starch/sh gelatin lms (ratios 1:0, 2:1, 3:1, 4:1 & 5:1) with 25% sorbitol, conditioned at different relative humidity.

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the addition of gellan to gelatin lms signicantly 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 lm. 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/sh gelatin edible lms as a function of sago starch and sh gelatin ratio with added sorbitol or glycerol. The sorption isotherm curves of sago starch and sago starch/sh gelatin lms showed a typical behavior of water vapor sensitive hydrophilic

biopolymers when the relative humidity (% RH) increased. These lms behaved as in gluten, starch and cellulose lms (Gontard, Guilbert, & Cuq, 1993; Mali, Grossmann, Garca, 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 lms. Glycerol sago starch/sh gelatin lms presented higher equilibrium moisture contents than those of sago starch lms only (Fig. 3). In general, moisture content of these lms 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 lms plasticized with

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

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115

25% sorbitol show similar sorption isotherm proles (Fig. 4) i.e. low equilibrium moisture contents at low aw then increased at higher aw. However, sago starch lms at 84% RH showed slightly lower equilibrium moisture content than those of sago starch/sh gelatin lms. Generally, the equilibrium moisture content was higher in lms with added glycerol than those lms 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

resulting in less plasticizing effect compared to glycerol (Mali et al., 2005). Hu, Chen, and Gao (2009) also reported that oxidized potato starch lms 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 lms 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/sh gelatin lms plasticized with 25% sorbitol (a) 2:1, (b) 3:1, (c) 4:1, (d) 5:1 and (e) 1:0.

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3.8. Film morphology Figs. 5 and 6 show the scanning electron micrographs of sago starch and sago starch/sh gelatin lms plasticized with glycerol and sorbitol, respectively. It was observed that there were some differences in surface and internal structure morphology between glycerol and sorbitol lms at the various starch to gelatin ratios. Glycerol lms were observed to have rougher surfaces with presence of pores or cavities that could be related to the formation of channels whereas sorbitol lms showed a more compact and homogeneous surface. However, smoother lm surfaces were observed in both glycerol and sorbitol lms that has less protein content (Figs. 5 and 6aed). Nevertheless, surface roughness and formation of channels disappeared in samples of sago starch lms only (1:0) with either plasticizer. Liu, Liu, Fishman, and Hicks (2007) worked on pectin lms with addition of two different types of proteins (sh skin gelatin or soybean protein) and obtained pectin lms that showed relatively smoother surfaces than those lms 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 lms with sh gelatin and gellan or k-carrageenan mixtures produces lms that showed discontinuous zones with noticeable cracks randomly distributed along the network. However, they also concluded that addition of gellan to sh gelatin lms 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 lms 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/sh gelatin lms 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/sh gelatin lms unplasticized and plasticized with glycerol or sorbitol (25%). Films 1:0 NP 2:1 3:1 4:1 5:1 NP NP NP NP Tg 84.04 0.81a 52.82 55.32 55.71 53.81 2.28d 1.43ef 0.87f 1.41de Tm 167.41 3.23a 159.15 144.91 151.64 163.76 16.84hi 15.57fg 11.51gh 6.82ai

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 ndings of this study showed that unplasticized sago starch lms 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 lms signicantly decreased Tg, Tm and DH. However, greater decrease in the values was noticed when sorbitol was used compared to glycerol. Plasticizing sago starch lms with glycerol makes lms more hydrophilic and retain higher moisture compared to unplasticized lms when conditioned at the same humidity (RH %) and temperature. Mali et al. (2005) also reported that glycerol-containing lms of cassava starch affected lms properties due to the hygroscopic character of glycerol that tends to provide additional water into the lm matrix. In this study, sorbitol showed lower Tg, Tm and DH when added to sago starch lms compared to unplasticized ones. Addition of glycerol to sago starch/ sh gelatin lms showed an increase in Tg for all ratios (1:0, 2:1, 3:1, 4:1 & 5:1) but a signicant 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 lms 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/sh gelatin lms plasticized with sorbitol showed signicantly lower (p  0.05) Tg, Tm and DH for all ratios with the exception of ratio (3:1) for Tm values that showed no signicant change compared to unplasticized lms 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 lms which may reduce the interaction between the starch and gelatin. The presence of only one Tg could indicate that the sago starch and gelatin lms 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 lms from sago starch and sago starch/sh 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 exible lms with added glycerol but not at higher gelatin content (2:1). The results obtained show that different ratios of sago starch and sh gelatin blends plasticized with glycerol or sorbitol affected physical, mechanical and water vapor permeability of the lms produced. Changing the ratio of two polymers can modify the strength and extensibility of the composite lms. SEM micrographs show uneven surfaces in glycerol lms compared to sorbitol lm surfaces. However, less protein in the samples gave smoother lms 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 sh 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

DH
219.63 13.70f 240.73 239.63 227.00 217.73 25.31h 9.22h 2.96g 11.40f

1:0 G 1:0 S 2:1 3:1 4:1 5:1 2:1 3:1 4:1 5:1 G G G G S S S S

60.08 1.1gh 53.10 0.8d 62.28 61.36 58.50 61.95 49.85 40.88 48.15 49.60 0.8i 1.1hi 0.5g 1.4i 4.4c 6.7b 4.9c 7.7c

133.90 12.3bd 112.70 5.4c 157.61 133.15 143.12 139.04 129.01 151.36 135.20 143.72 10hi 10.5bdh 4.1efg 3.1def 8.4b 16.5g 15.2bde 16.9fg

171.83 8.3de 148.33 1.2b 177.43 172.50 171.65 161.08 162.50 168.08 140.13 151.97 11.1e 27.3de 2.2de 6.8c 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 signicantly different (p < 0.05). G: glycerol; S: sorbitol, NP: no plasticizer added.

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Arabia Government for the nancial support given for this research work. References
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