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gelatin films
Food Packaging and Shelf Life 13 (2017) 15–19
Contents lists available at ScienceDirect
Food Packaging and Shelf Life journal homepage: www.elsevier.com/locate/fpsl
Effect of transglutaminase induced crosslinking on the properties of starch/ gelatin films
MARK
⁎
A.A. AL-Hassana, , M.H. Norziahb a b
Food Science and Human Nutrition Department, College of Agriculture & vet. Medicine, Qassim University, 51452, Burydah, Saudi Arabia School of Industrial Technology, Food Technology Department,UniversitiSains Malaysia, 11800, penang, Malaysia
A R T I C L E I N F O
A B S T R A C T
Keywords: Edible films Sago starch Fish gelatin Transglutaminase MDSC FTIR
This paper was to measure the effect of fish gelatin and Transglutaminase enzyme (TGs) on properties of sago starch and fish gelatin films. The concentration of glycerol was 30% of polymers (db) and the sago starch to fish gelatin ratios were (1:0 and 3:1) with TGs concentrations (1, 5 and 10 mg/g gelatin). The results were discussed in terms of ‘gelatin and TGs effect’. In a general manner, fish gelatin and TGs have an effect on both physicochemical and functional properties of the produced films. Addition of fish gelatin to sago starch films significantly reduced tensile strength (TS), water vapor permeability (WVP) but increased the percentage of elongation at break (%EAB). Positive effects of TGs addition on mechanical properties were observed. FTIR-ATR showed an evident of interaction between polysaccharides and protein. Furthermore, the transmittance percentage of amide I and amide II bands in treated films reduced with increasing enzyme concentration as an evident of enzyme crosslinking.
1. Introduction Quality and shelf life of foods can be improved by using edible films (starch and gelatin) that provide barriers to mass transfer. Edible films functional properties depend on the material characteristics and the method of their preparation (Flores, Famá, Rojas, Goyanes & Gerschenson, 2007). The main film-forming materials used and investigated to enhance the food qualities and shelf-life are polysaccharides, proteins and lipids; their derivatives and their mixtures. Polysaccharides materials including starch, carrageenan and alginate have been used as a result of their ability of form films. Hydrocolloid films are considered to be good oxygen and carbon dioxide barriers but less effective as moisture barriers (De Carvalho & Grosso, 2004). Polysaccharides-protein mixed systems have been extensively studies where understanding the interactions between these two biopolymers are of great importance in developing edible films to enhance film properties as well as for food processes and products. Several studies have examined different film properties including physical, mechanical and thermal such as mixed starch and gelatin films (Arvanitoyannis, Nakayama & Aiba, 1998), gelatin and chitosan films (Kolodziejska & Piotrowska, 2007), cassava and gelatin films (Veiga-Santos, Oliveira, Cereda & Scamparini, 2007) and soy protein and gelatin films (Cao, Fu & He, 2007). Modification of gelatin and gelatin based films could be achieved through several methods
⁎
Corresponding author. E-mail address: [email protected] (A.A. AL-Hassan).
http://dx.doi.org/10.1016/j.fpsl.2017.04.006 Received 5 October 2016; Received in revised form 11 April 2017; Accepted 13 April 2017 2214-2894/ © 2017 Elsevier Ltd. All rights reserved.
including electrostatic forces and establishment of salt bridges as conducted by Kaewruang, Benjakul, Prodpran, Encarnacion, and Nalinanon (2014) and (Sow & Yang, 2015) or through protein bonds cross-linking (De Jong & Koppelman, 2002). Transglutaminase enzyme (TGs) is a protein polymerizing agent that results in forming isopeptide bonds between proteins based foods which enhance their properties. It improves food products properties such as firmness, viscosity, elasticity and water binding capacity (Kieliszek & Misiewicz, 2014). Examining the affects of transglutaminase enzyme on the properties of films (mechanical, water vapor permeability (WVP) and solubility) have been conducted in some studies as in gelatin-casein films (Chambi & Grosso, 2006), gelatin films (De Carvalho & Grosso, 2004; Lim, Mine & Tung, 1999) and in ground beef to enhance its texture (Bilic et al., 2005). Addition of TGs to casein-protein blends resulted in formation of protein cross-linking and high molecular mass polymers, thus resulting in stronger gels between 22 and 37 °C (Mylla et al., 2007). In the this study, the objectives were to evaluate the effects of adding fish gelatin to sago starch based film and incorporating TGs enzyme on the rheological properties of the film forming solutions and the physical, thermal and mechanical properties of the developed sago starch/fish gelatin films.
Food Packaging and Shelf Life 13 (2017) 15–19
A.A. AL-Hassan, M.H. Norziah
2. Materials and methods
2.7. Measurements of mechanical properties
Biopolymers as sago starch (Metroxylon sagu) (Nitsei Industrial Sdn. Bhd, Malaysia) and gelatin was extracted from surimi fish wastes (Norziah et al., 2009). Transglutaminase enzyme was from Ajinomoto (Activa TG-S, Japan) with activity as 100 U/g powder. All reagents were of analytical grade.
Developed films were tested following the ASTM methods D882-00 (ASTM, 2000a) to measure the tensile strength and percentage of elongation at break (%EAB) plus Young’s modulus. The film specimen strips (14 × 2 cm) were conditions with saturated sodium bromide solution (56% RH, 30 ° C/48 h) prior to testing.
2.1. Starch/gelatin film forming solutions
2.8. Measurements of water vapor permeability
Biopolymers with glycerol were used to prepare the film forming solutions (FFS) following the procedures described by (AlHassan & Norziah, 2012). Sago starch/fish gelatin solutions were prepared with the ratios (1:0 and 3:1) giving a total weight of (5.2 g) in 200 mL including 30% (w/w) glycerol. Sago starch was dissolved and heated in distilled water (at 85 ° C/30 min) (solution A) followed by addition of plasticizer at 60 °C. Likewise, sago starch/fish gelatin film forming solutions (3:1) were prepared by adding fish gelatin to (solution A) at 60 °C and continue stirring (30 min), then adding glycerol (solution B). The mixture (3:1) was incubated with transglutaminase enzyme (TGs) at 50 °C/15 min as reported by (Kolodziejska & Piotrowska, 2007), then enzyme was deactivated (De Carvalho & Grosso, 2004). The concentrations of added TGs to starch:gelatin(3:1) were 1.0, 5.0 and 10.0 mg/g gelatin.
Developed films were tested for water vapor permeability (WVP) following ASTM E96-00 method (ASTM, 2000b). Glass permeation cells were used that contained silica gels (0% RH) and mounted films on top of the cell. Samples were placed in a desiccator (100% RH, 30 °C) and measurements were taken at 24 h intervals over a 7-day period.
2.2. Starch/gelatin edible film casting
The flow behaviour and viscosity profile of film forming solutions (FFS) with 30% glycerol are shown in Table 1. Film-forming solutions showed non-Newtonian behaviour with thixotropy shear thinning (pseudoplastic behaviour) i.e increase in shear stress decreased the viscosity indicative of structural breakdown due to shearing. All samples were fitted to Cross-model. Starch suspension was reported as non-Newtonian presented pseudoplastic or shear-thinning behaviour (Bertuzzi, Armada & Gottifredi, 2007). The highest viscosity was observed for sago starch samples only (1:0). Glycerol as the plasticizer, reduced the intra and inter-molecuar forces in sago starch due to formation of hydrogen bonds of starch molecule. Kurata and Tsunashima (1998) reported that bigger molecules resulted in higher solution viscosity. Addition of fish gelatin to sago starch FFS reduced the viscosity as well as the consistency due to a reduction in the molecular weight compared to sago starch solution. Sago starch/fish gelatin solution (3:1) presented a high shear stress-shear rate relationship compared to other film forming solutions with transglutaminase. Addition of transglutaminase enzyme to (3:1) film forming solutions reduced the shear stress-shear rate relationship for all different concentrations (1, 5 and 10 mg) compared to untreated samples (TGs 0 mg). The observation may be due to reduction in hydrogen-bonding formed between starch and gelatin due to the formation of the isopeptide in the gelatin, which maybe resulted in excessive crosslinking. Therefore, presence of gelatin in the sample may reduce inter and intra molecular interactions of the sago starch.
2.9. Statistical analyses The results of this study were analyzed using SPSS 18.0. ANOVA was applied using Duncan’s test with confidence level as p < 0.05. 3. Results and discussion 3.1. Flow analysis
Film forming solution was poured onto casting plates (16 × 16 cm & 3 mm height) and dried at 35 ° C/24 h using a ventilated oven. 2.3. Dynamic rheological measurements Starch/fish gelatin film forming solutions (FFS) were cooled to room temperature (∼28 °C) before dynamic rheological measurements (viscoelastic flow). An oscillatory shear test was applied to measure the rheological properties of the starch/gelatin mixtures using a controlled stress AR 1000 rheometer (TA Instruments, USA). 2.4. Measurement of crosslinking degree The crosslinking degree of transglutaminase treated films was preformed according to the method of Bubnis and Ofner (1992) as described by Prasertsung, Mongkolnavin, Kanokpanont, and Damrongsakkul (2010). Formation of a yellow soluble complex was used to determine un-crosslinking groups due to gelatin free amino groups react with 2,4,6-trinitrobenzene sulfonic acid (TNBS). 2.5. Solubility of films Films solubility was determined as described by (García, Pinotti, Martino, & Zaritzky, 2009). Sample films (2 × 3 cm) were placed in a desiccator containing P2O5 (0% RH/7days). Then samples were subject to constant agitation for (1 h/25 ± 2 °C) in test beakers with 80 mL deionized water and filtered (Whatman 4) and dried in an oven (60 °C) to a constant weight. Total soluble was expressed as percentage (% solubility) and calculated according to the follows equation: Percentage of Solubility = [(Initial dry weight-Final dry weight)/Initial dry weight] × 100.
Table 1 values of consistency, flow behaviour index and r2 obtained for film forming solution of sago starch (1:0), sago starch/fish gelatin (3:1) and (3:1) with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg).
2.6. FTIR spectroscopy
Sample
ɳ0a
ɳ∞ b
C (s)c
md
r2e
1:0 (G) 3:1 (G) TGs 1 mg TGs 5 mg TGs 10 mg
0.432 0.033 0.014 0.015 0.012
0.0060 0.0079 0.0071 0.0073 0.0058
1.627 0.113 0.016 0.018 0.009
0.516 0.536 0.936 0.806 0.976
0.9896 0.9990 0.9970 0.9918 0.9942
a b
Films spectra were recorded with Fourier transform infrared spectrometry-Attenuated total reflectance (Thermo Scientific Nicolet iS10 FT-IR Spectrometer, Massachusetts, USA) from 4000 to 400 cm−1.
c d e
16
infinite-rate viscosity. zero-rate viscosity. consistency. flow behaviour index. determination coefficient for the Cross model fit.
Food Packaging and Shelf Life 13 (2017) 15–19
A.A. AL-Hassan, M.H. Norziah
Fig. 1. Degree of enzyme crosslinking of films (3:1) treated with transglutaminase enzyme with incubation for 15 min at 50 °C at concentrations of 1, 5 and 10 mg/g (TGs/gelatin).
Fig. 3. FTIR spectra of sago starch/fish gelatin (3:1) and films (3:1) with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg).
linking degree to 47.2 ± 32.7%. Further increase in enzyme concentration to 10 mg/g, increased the crosslinking degree to 53.0 ± 31.6%. The reduction of available gelatin free amino groups with increase in enzyme concentration added showed evidence of crosslinking. The crosslinking-degree of the amino groups depends on the source of protein, in which amino groups number and reactivity and glutamine varied, therefore free amino groups for mTGase crosslinking depends on the steric and conformational constraints (Yang, Shi & Liang, 2015; Wang et al., 2013; Yang et al., 2014). Ardelean, Jaros and Rohm (2013) concluded that TGase showed different crosslink degree with proteins from cow milk (30%) compared to (9.6%) of the from goat source. De Carvalho and Grosso (2004) reported that TNBS method showed a decrease in ε-amino groups in gelatin films as a result of the enzyme treatment indicating an increase in molecular weight due to polymerization.
Table 2 Mechanical properties and Young’s modulus of sago starch (1:0), sago starch/fish gelatin films (3:1) and films (3:1) with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg). Films
TS (MPa)
1:0 (G) 3:1 (G) TGS 1 mg TGS 5 mg TGS10 mg
8.15 2.60 3.81 3.99 5.89
± ± ± ± ±
0.30a 0.16d 0.19b 0.59b 0.76c
% EAB
42.50 72.19 61.93 57.42 47.75
± ± ± ± ±
9.34c 6.73a 11.33b 12.81b 3.71c
Young’s modulus, (E) (N/m2) × 107
Solubility%
19.87 ± 4.78a 3.76 ± 1.10d 6.35 ± 1.18c 7.46 ± 2.44c 12.29 ± 0.69b
46.56 30.19 38.04 20.63 16.33
± ± ± ± ±
1.75a 2.74c 0.82b 0.89d 0.92e
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; TGS: transglutaminase enzyme; TS: tensile strength and%EAB: percentage of elongation at break.
3.3. Ftir-atr FTIR-ATR spectrum of blend films is shown in Fig. 3. The OeH stretching broad band of sago starch film (1:0) presented at 3309.38 cm−1. The band at 2941. 08 cm−1 related to the CeH stretching, while the band of 1339.32 cm−1 presented the OeH of water. Likewise, in sago starch/fish gelatin (TGs 0 mg) and films with transglutaminase (TGs 1 mg, TGs 5 mg and TGs 10 mg) band at 3309. 38 cm−1 (OeH stretching as in 1:0), while band at 1543.13 cm−1 was the eNH twisting (amide II). The band at 1647.98 cm−1 was due to the C]O stretching as amide I which also present in sago starch films (1:0) as a results of protein presence (Amide I). Transmittance percentage of amide I and II bands in treated films reduced with higher enzyme concentration added. Pshenitsyna, Molotkova, Noskova and Aksel'rod (1979) mentioned that both Amide I and Amide II (1650 cm−1 & 1550 cm−I) associated with C]O and CeN groups stretching vibrations and the deformation vibrations of NH groups, respectively. In addition, they have stated that absorption band of Amide II is often used for structural characterization. An ether group can be observed in the big band at 997.37 cm−1 as stated by Bourtoom and Chinnan (2008) where they observed 993 band in starch-chitosan films. Hydrogen interactions between both biopolymers can be considers by moving in band location of the amide-1 and amide-3 groups. (Benbettaïeb, Kurek, Bornaz & Debeaufort, 2014).
Fig. 2. Water vapor permeability of sago starch (1:0), sago starch/fish gelatin (3:1) films and films (3:1) treated with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg).
3.2. Crosslinking degree of films Crosslinking degree of sago starch/fish gelatin films (3:1) treated with TGs with the concentrations of (1, 5 and 10 mg/g) is shown in Fig. 1. Incubation of the films with transglutaminase enzyme with the concentration 1 mg/g showed a reduction in amino acid groups resulting in a crosslinking percentage of 30.9 ± 3.1% when incubated for 15 min. Increasing the enzyme concentration to 5 mg/g showed further reduction of available amino groups with increasing cross-
3.4. Mechanical properties All thickness of biopolymers films with plasticizers ranged between 0.05–0.07 mm. Developed biopolymer films were transparent, homogeneous, flexible and easily removed from the acrylic plates. Table 2 shows tensile strengths (TS), percentage of elongation at break (% EAB) 17
Food Packaging and Shelf Life 13 (2017) 15–19
A.A. AL-Hassan, M.H. Norziah
and Young’s modulus (E) of the films plasticized with 30% glycerol. The findings showed there is a reduction in TS (from 8.15 MPa to 2.60 MPa) associated with increase in%EAB (from 42.50% to 72.19%) when fish gelatin incorporated to sago starch film. Since the plasticizer concentration was constant, hydrogen bonding formed between protein and starch may result in TS reduction which might reduce the interaction between starch chains. Transglutaminase significantly increase TS with all con concentration (TGs 1 mg, TGs 5 mg and TGs 10 mg) compared to control (3:1 G) with no enzyme added. The possibility of increasing in TS associated with a reduction in%EAB as well as an increase in Young’s modulus resulted from the transglutaminase induced crosslinking between amino groups. Mariniello et al. (2003) reported that pectinsoy flour films treated with transglutaminase was less extensible than untreated films resulted in two times increase in TS associated with two times reduction in%EAB.
creases the WVP. Kolodziejska and Piotrowska (2007) reported that crosslinking of fish gelatin-chitosan films with chemical (EDC) or TGase had different WVP in the presence of different glycerol concentrations (0 to 30%). 4. Conclusion The effect of fish gelatin and transglutaminase incorporation into sago starch based film plasticized with 30% glycerol were tested. The findings indicated that addition of fish gelatin to sago starch FFS significantly reduced WVP of the developed films and has an effect on TS and%EAB. Adding TGs with concentrations beyond 1 mg/g to the sago starch/fish gelatin FFS reduced flow consequently and has an effect on viscosity. The results also showed that TS and% EAB changed with enzyme incorporation. Moreover, FTIR-ATR showed a reduction in the% transmittance of the peaks involving ε-amino groups due to the reduction of amino acids as a results of gelatin molecules polymerization.
3.5. Solubility of films Table 2 shows solubility of films. Addition of fish gelatin to sago starch significantly reduced solubility from 46.56% to 30.19%, this may be due to stronger hydrogen bonding formation between sago starch and fish gelatin than starch chains intra and intermolecular or hydrogen bonding with water molecules that makes films more intact and less soluble. Addition of transglutaminase enzyme by 1 mg to films (3:1) significantly increased solubility from 30.19% to 38.04%. Formation of small molecular fractions as crosslinked occurred reduced hydrogen bonding between biopolymers that makes films more soluble. More glycerol became exposed to water molecular resulting in more solubility of films. Increase in enzyme concentration significantly reduced solubility of treated films compared to films with no enzyme added. Reduction in solubility was observed in treated films from 30.19% to 20.63% and 16.33% for 5 mg and 10 mg respectively; due to isopeptide formation between amino acids of the gelatin however, starch protein may involve in the crosslinking formation. The findings of this study was in agreement with Kolodziejska and Piotrowska (2007) who concluded that modified fish gelatin–chitosan films with the enzyme transglutaminase decreased the solubility even in acidic pH and high temperature compare to uncross linked films however, plasticization with glycerol had no effect on the mixture solubility in aqueous medium. Fish gelatin films treatment with TGs enzyme reduced the solubility by 50% at pH 3.0 and to 40% at pH 6 (Piotrowska, Sztuka, Kolodziejska & Dobrosielska, 2008). De Carvalho and Grosso (2004) mentioned that increase in crosslinking degree could result in decreasing low molecular fractions weight that led to the reduction in solubility.
References 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 9600. Philadelphia, PA: American Society for Testing and Materials. Al-Hassan, A. A., & Norziah, M. H. (2012). Starch-gelatin edible films: water vapor permeability and mechanical properties as affected by plasticizers. Food Hydrocolloids, 26(1), 108–117. Ardelean, A. I., Jaros, D., & Rohm, H. (2013). Influence of microbial transglutaminase cross-linking on gelationkinetics and texture of acid gels made from whole goats and cows milk. Dairy Science & Technology, 93(1), 63–71. Arvanitoyannis, I., Nakayama, A., & Aiba, S.-I. (1998). Edible films made from hydroxypropyl starch and gelatin and plasticized by polyols and water. Carbohydrate Polymers, 36(2–3), 105–119. Benbettaïeb, N., Kurek, M., Bornaz, S., & Debeaufort, F. (2014). Barrier, structural and mechanical properties of bovine gelatin-chitosan blend films related to biopolymer interactions. Journal of the Science of Food and Agriculture, 94(12), 2409–2419. Bertuzzi, M. A., Armada, M., & Gottifredi, J. C. (2007). Physicochemical characterization of starch based films. Journal of Food Engineering, 82(1), 17–25. Bourtoom, T., & Chinnan, M. S. (2008). Preparation and properties of rice starch-chitosan blend biodegradable film. LWT – Food Science and Technology, 41(9), 1633–1641. Bubnis, W. A., & Ofner, C. M. (1992). The determination of ε-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid. Analytical Biochemistry, 207(1), 129–133. Cao, N., Fu, Y., & He, J. (2007). Preparation and physical properties of soy protein isolate and gelatin composite films. Food Hydrocolloids, 21(7), 1153–1162. Chambi, H., & Grosso, C. (2006). Edible films produced with gelatin and caseincrosslinked with transglutaminase. Food Research International, 39(0), 458–466. 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(3), 622–626. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food, 2016 Food Hydrocolloids, 2016. Food Hydrocolloids, 2016(18), 717–726. Flores, S., Famá, L., Rojas, A. M., Goyanes, S., & Gerschenson, L. (2007). Physical properties of tapioca-starch edible films: Influence of filmmaking and potassium sorbate. Food Research International, 40(2), 257–265. García, M. A., Pinotti, A., Martino, M., & Zaritzky, N. (2009). Electrically treated composite FILMS based on chitosan and methylcellulose blends. Food Hydrocolloids, 23(0), 722–728. Kaewruang, P., Benjakul, S., Prodpran, T., Encarnacion, A. B., & Nalinanon, S. (2014). Impact of divalent salts and bovine gelatin on gel properties of phosphorylated gelatin from the skin of unicorn leatherjacket. LWT e Food Science and Technology, 55(0), 477–482. Kieliszek, M., & Misiewicz, A. (2014). Microbial transglutaminase and its application in the food industry. A review. Folia Microbiologica, 59, 241–250. Kolodziejska, I., & Piotrowska, B. (2007). The water vapour permeability, mechanical properties and solubility of fish gelatin-chitosan films modified with transglutaminase or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and plasticized with glycerol. Food Chemistry, 103(2), 295–300. Kurata, M., & Tsunashima, Y. (1998). Viscosity–molecular weight relationships and unperturbed dimensions of linear chain molecules. In J. Brandrup, & E. H. Immergut (Eds.), Polymer handbook(3rd ed.). New York: John Wiley & sons. Lim, L. T., Mine, Y., & Tung, M. A. (1999). Barrier and tensile properties of transglutaminase cross-linked gelatin films as affected by relative humidity, temperature, and glycerol content. Journal of Food Science, 64(4), 616–622. Mariniello, L., Di Pierro, P., Esposito, C., Sorrentino, A., Masi, P., & Porta, R. (2003). Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase. Journal of Biotechnology, 102(2), 191–198.
3.6. Water vapor permeability Water vapor permeability of developed films is shown in Fig. 2. Film thickness ranged between 0.05–0.07 mm. Sago starch film (1:0) has a WVP value of (4.3 × 10−4 g mm/m2 h Pa). Incorporation of fish gelatin to sago starch films significantly reduced WVP to (2.4 × 10−4 g mm/m2 h Pa). The possibility of the reduction may due to glycerol plasticizer effect as a small molecule that penetrates between the molecular of the two biopolymers resulting in a reduction of the intra and inter hydrogen bonding within a single polymer, which makes more mobility of the biopolymers to form hydrogen-bonding between them resulted in intact molecules with less WVP. Addition of transglutaminase enzyme to films (3:1) had no significant effect on WVP films which may be due to hydrophilicity nature of material used. Even though the transglutaminase-induced crosslinking implicated covalent iso-peptide bonds, the sorption sites responsible for making films hydrophilic in the two biopolymers had not changed. The result may be due to the effect of the present of the hydrophilic plasticizer. Cuq, Gontard, Cuq and Guilbert (1997) reported that plasticization of protein and polysaccharide films with a hydrophilic plasticizer in18
Food Packaging and Shelf Life 13 (2017) 15–19
A.A. AL-Hassan, M.H. Norziah
Sow, L. C., & Yang, H. (2015). Effects of salt and sugar addition on the physicochemical properties and nanostructure of fish gelatin. Food Hydrocolloids, 45(0), 72–82. Veiga-Santos, P., Oliveira, L. M., Cereda, M. P., & Scamparini, A. R. P. (2007). Sucrose and inverted sugar as plasticizer. Effect on cassava starch-gelatin film mechanical properties, hydrophilicity and water activity. Food Chemistry, 103(2), 255–262. Wang, P., Liu, H., Wen, P., Zhang, H., Guo, H., & Ren, F. (2013). The composition size and hydration of yak casein micelles. International Dairy Journal, 31(2), 107–110. Yang, M., Shi, Y., Wang, P., Liu, H., Wen, P., & Ren, F. (2014). Effect of succinylation on the functional properties of yak caseins a comparison with cow caseins. Dairy Sci Technol, 94(4), 359–372. Yang, M., Shi, Y., & Liang, Q. (2015). Effect of microbial transglutaminase crosslinking on the functional properties of yak caseins: A comparison with cow caseins. Dairy Sci. Technol. 96(0), 39–51.
Norziah, M. H., Al-Hassan, A., Khairulnizam, A. B., Mordi, M. N., & Norita, M. (2009). Characterization of fish gelatin from surimi processing wastes: Thermal analysis and effect of transglutaminase on gel properties. Food Hydrocolloids, 23(6), 1610–1616. Piotrowska, B., Sztuka, K., Kolodziejska, I., & Dobrosielska, E. (2008). Influence of transglutaminase or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) on the properties of fish-skin gelatin films. Food Hydrocolloids, 22(7), 1362–1371. Prasertsung, I., Mongkolnavin, R., Kanokpanont, S., & Damrongsakkul, S. (2010). The effects of pulsed inductively coupled plasma (PICP) on physical properties and biocompatibility of crosslinked gelatin films. International Journal of Biological Macromolecules, 46(1), 72–78. Pshenitsyna, V. P., Molotkova, N. N., Noskova, M. P., & Aksel'rod, B. Y. (1979). Display of the amide II absorption band in the spectra of reaction products of urea with formaldehyde. Journal of Applied Spectroscopy, 29(3).
19
Contents lists available at ScienceDirect
Food Packaging and Shelf Life journal homepage: www.elsevier.com/locate/fpsl
Effect of transglutaminase induced crosslinking on the properties of starch/ gelatin films
MARK
⁎
A.A. AL-Hassana, , M.H. Norziahb a b
Food Science and Human Nutrition Department, College of Agriculture & vet. Medicine, Qassim University, 51452, Burydah, Saudi Arabia School of Industrial Technology, Food Technology Department,UniversitiSains Malaysia, 11800, penang, Malaysia
A R T I C L E I N F O
A B S T R A C T
Keywords: Edible films Sago starch Fish gelatin Transglutaminase MDSC FTIR
This paper was to measure the effect of fish gelatin and Transglutaminase enzyme (TGs) on properties of sago starch and fish gelatin films. The concentration of glycerol was 30% of polymers (db) and the sago starch to fish gelatin ratios were (1:0 and 3:1) with TGs concentrations (1, 5 and 10 mg/g gelatin). The results were discussed in terms of ‘gelatin and TGs effect’. In a general manner, fish gelatin and TGs have an effect on both physicochemical and functional properties of the produced films. Addition of fish gelatin to sago starch films significantly reduced tensile strength (TS), water vapor permeability (WVP) but increased the percentage of elongation at break (%EAB). Positive effects of TGs addition on mechanical properties were observed. FTIR-ATR showed an evident of interaction between polysaccharides and protein. Furthermore, the transmittance percentage of amide I and amide II bands in treated films reduced with increasing enzyme concentration as an evident of enzyme crosslinking.
1. Introduction Quality and shelf life of foods can be improved by using edible films (starch and gelatin) that provide barriers to mass transfer. Edible films functional properties depend on the material characteristics and the method of their preparation (Flores, Famá, Rojas, Goyanes & Gerschenson, 2007). The main film-forming materials used and investigated to enhance the food qualities and shelf-life are polysaccharides, proteins and lipids; their derivatives and their mixtures. Polysaccharides materials including starch, carrageenan and alginate have been used as a result of their ability of form films. Hydrocolloid films are considered to be good oxygen and carbon dioxide barriers but less effective as moisture barriers (De Carvalho & Grosso, 2004). Polysaccharides-protein mixed systems have been extensively studies where understanding the interactions between these two biopolymers are of great importance in developing edible films to enhance film properties as well as for food processes and products. Several studies have examined different film properties including physical, mechanical and thermal such as mixed starch and gelatin films (Arvanitoyannis, Nakayama & Aiba, 1998), gelatin and chitosan films (Kolodziejska & Piotrowska, 2007), cassava and gelatin films (Veiga-Santos, Oliveira, Cereda & Scamparini, 2007) and soy protein and gelatin films (Cao, Fu & He, 2007). Modification of gelatin and gelatin based films could be achieved through several methods
⁎
Corresponding author. E-mail address: [email protected] (A.A. AL-Hassan).
http://dx.doi.org/10.1016/j.fpsl.2017.04.006 Received 5 October 2016; Received in revised form 11 April 2017; Accepted 13 April 2017 2214-2894/ © 2017 Elsevier Ltd. All rights reserved.
including electrostatic forces and establishment of salt bridges as conducted by Kaewruang, Benjakul, Prodpran, Encarnacion, and Nalinanon (2014) and (Sow & Yang, 2015) or through protein bonds cross-linking (De Jong & Koppelman, 2002). Transglutaminase enzyme (TGs) is a protein polymerizing agent that results in forming isopeptide bonds between proteins based foods which enhance their properties. It improves food products properties such as firmness, viscosity, elasticity and water binding capacity (Kieliszek & Misiewicz, 2014). Examining the affects of transglutaminase enzyme on the properties of films (mechanical, water vapor permeability (WVP) and solubility) have been conducted in some studies as in gelatin-casein films (Chambi & Grosso, 2006), gelatin films (De Carvalho & Grosso, 2004; Lim, Mine & Tung, 1999) and in ground beef to enhance its texture (Bilic et al., 2005). Addition of TGs to casein-protein blends resulted in formation of protein cross-linking and high molecular mass polymers, thus resulting in stronger gels between 22 and 37 °C (Mylla et al., 2007). In the this study, the objectives were to evaluate the effects of adding fish gelatin to sago starch based film and incorporating TGs enzyme on the rheological properties of the film forming solutions and the physical, thermal and mechanical properties of the developed sago starch/fish gelatin films.
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2. Materials and methods
2.7. Measurements of mechanical properties
Biopolymers as sago starch (Metroxylon sagu) (Nitsei Industrial Sdn. Bhd, Malaysia) and gelatin was extracted from surimi fish wastes (Norziah et al., 2009). Transglutaminase enzyme was from Ajinomoto (Activa TG-S, Japan) with activity as 100 U/g powder. All reagents were of analytical grade.
Developed films were tested following the ASTM methods D882-00 (ASTM, 2000a) to measure the tensile strength and percentage of elongation at break (%EAB) plus Young’s modulus. The film specimen strips (14 × 2 cm) were conditions with saturated sodium bromide solution (56% RH, 30 ° C/48 h) prior to testing.
2.1. Starch/gelatin film forming solutions
2.8. Measurements of water vapor permeability
Biopolymers with glycerol were used to prepare the film forming solutions (FFS) following the procedures described by (AlHassan & Norziah, 2012). Sago starch/fish gelatin solutions were prepared with the ratios (1:0 and 3:1) giving a total weight of (5.2 g) in 200 mL including 30% (w/w) glycerol. Sago starch was dissolved and heated in distilled water (at 85 ° C/30 min) (solution A) followed by addition of plasticizer at 60 °C. Likewise, sago starch/fish gelatin film forming solutions (3:1) were prepared by adding fish gelatin to (solution A) at 60 °C and continue stirring (30 min), then adding glycerol (solution B). The mixture (3:1) was incubated with transglutaminase enzyme (TGs) at 50 °C/15 min as reported by (Kolodziejska & Piotrowska, 2007), then enzyme was deactivated (De Carvalho & Grosso, 2004). The concentrations of added TGs to starch:gelatin(3:1) were 1.0, 5.0 and 10.0 mg/g gelatin.
Developed films were tested for water vapor permeability (WVP) following ASTM E96-00 method (ASTM, 2000b). Glass permeation cells were used that contained silica gels (0% RH) and mounted films on top of the cell. Samples were placed in a desiccator (100% RH, 30 °C) and measurements were taken at 24 h intervals over a 7-day period.
2.2. Starch/gelatin edible film casting
The flow behaviour and viscosity profile of film forming solutions (FFS) with 30% glycerol are shown in Table 1. Film-forming solutions showed non-Newtonian behaviour with thixotropy shear thinning (pseudoplastic behaviour) i.e increase in shear stress decreased the viscosity indicative of structural breakdown due to shearing. All samples were fitted to Cross-model. Starch suspension was reported as non-Newtonian presented pseudoplastic or shear-thinning behaviour (Bertuzzi, Armada & Gottifredi, 2007). The highest viscosity was observed for sago starch samples only (1:0). Glycerol as the plasticizer, reduced the intra and inter-molecuar forces in sago starch due to formation of hydrogen bonds of starch molecule. Kurata and Tsunashima (1998) reported that bigger molecules resulted in higher solution viscosity. Addition of fish gelatin to sago starch FFS reduced the viscosity as well as the consistency due to a reduction in the molecular weight compared to sago starch solution. Sago starch/fish gelatin solution (3:1) presented a high shear stress-shear rate relationship compared to other film forming solutions with transglutaminase. Addition of transglutaminase enzyme to (3:1) film forming solutions reduced the shear stress-shear rate relationship for all different concentrations (1, 5 and 10 mg) compared to untreated samples (TGs 0 mg). The observation may be due to reduction in hydrogen-bonding formed between starch and gelatin due to the formation of the isopeptide in the gelatin, which maybe resulted in excessive crosslinking. Therefore, presence of gelatin in the sample may reduce inter and intra molecular interactions of the sago starch.
2.9. Statistical analyses The results of this study were analyzed using SPSS 18.0. ANOVA was applied using Duncan’s test with confidence level as p < 0.05. 3. Results and discussion 3.1. Flow analysis
Film forming solution was poured onto casting plates (16 × 16 cm & 3 mm height) and dried at 35 ° C/24 h using a ventilated oven. 2.3. Dynamic rheological measurements Starch/fish gelatin film forming solutions (FFS) were cooled to room temperature (∼28 °C) before dynamic rheological measurements (viscoelastic flow). An oscillatory shear test was applied to measure the rheological properties of the starch/gelatin mixtures using a controlled stress AR 1000 rheometer (TA Instruments, USA). 2.4. Measurement of crosslinking degree The crosslinking degree of transglutaminase treated films was preformed according to the method of Bubnis and Ofner (1992) as described by Prasertsung, Mongkolnavin, Kanokpanont, and Damrongsakkul (2010). Formation of a yellow soluble complex was used to determine un-crosslinking groups due to gelatin free amino groups react with 2,4,6-trinitrobenzene sulfonic acid (TNBS). 2.5. Solubility of films Films solubility was determined as described by (García, Pinotti, Martino, & Zaritzky, 2009). Sample films (2 × 3 cm) were placed in a desiccator containing P2O5 (0% RH/7days). Then samples were subject to constant agitation for (1 h/25 ± 2 °C) in test beakers with 80 mL deionized water and filtered (Whatman 4) and dried in an oven (60 °C) to a constant weight. Total soluble was expressed as percentage (% solubility) and calculated according to the follows equation: Percentage of Solubility = [(Initial dry weight-Final dry weight)/Initial dry weight] × 100.
Table 1 values of consistency, flow behaviour index and r2 obtained for film forming solution of sago starch (1:0), sago starch/fish gelatin (3:1) and (3:1) with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg).
2.6. FTIR spectroscopy
Sample
ɳ0a
ɳ∞ b
C (s)c
md
r2e
1:0 (G) 3:1 (G) TGs 1 mg TGs 5 mg TGs 10 mg
0.432 0.033 0.014 0.015 0.012
0.0060 0.0079 0.0071 0.0073 0.0058
1.627 0.113 0.016 0.018 0.009
0.516 0.536 0.936 0.806 0.976
0.9896 0.9990 0.9970 0.9918 0.9942
a b
Films spectra were recorded with Fourier transform infrared spectrometry-Attenuated total reflectance (Thermo Scientific Nicolet iS10 FT-IR Spectrometer, Massachusetts, USA) from 4000 to 400 cm−1.
c d e
16
infinite-rate viscosity. zero-rate viscosity. consistency. flow behaviour index. determination coefficient for the Cross model fit.
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Fig. 1. Degree of enzyme crosslinking of films (3:1) treated with transglutaminase enzyme with incubation for 15 min at 50 °C at concentrations of 1, 5 and 10 mg/g (TGs/gelatin).
Fig. 3. FTIR spectra of sago starch/fish gelatin (3:1) and films (3:1) with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg).
linking degree to 47.2 ± 32.7%. Further increase in enzyme concentration to 10 mg/g, increased the crosslinking degree to 53.0 ± 31.6%. The reduction of available gelatin free amino groups with increase in enzyme concentration added showed evidence of crosslinking. The crosslinking-degree of the amino groups depends on the source of protein, in which amino groups number and reactivity and glutamine varied, therefore free amino groups for mTGase crosslinking depends on the steric and conformational constraints (Yang, Shi & Liang, 2015; Wang et al., 2013; Yang et al., 2014). Ardelean, Jaros and Rohm (2013) concluded that TGase showed different crosslink degree with proteins from cow milk (30%) compared to (9.6%) of the from goat source. De Carvalho and Grosso (2004) reported that TNBS method showed a decrease in ε-amino groups in gelatin films as a result of the enzyme treatment indicating an increase in molecular weight due to polymerization.
Table 2 Mechanical properties and Young’s modulus of sago starch (1:0), sago starch/fish gelatin films (3:1) and films (3:1) with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg). Films
TS (MPa)
1:0 (G) 3:1 (G) TGS 1 mg TGS 5 mg TGS10 mg
8.15 2.60 3.81 3.99 5.89
± ± ± ± ±
0.30a 0.16d 0.19b 0.59b 0.76c
% EAB
42.50 72.19 61.93 57.42 47.75
± ± ± ± ±
9.34c 6.73a 11.33b 12.81b 3.71c
Young’s modulus, (E) (N/m2) × 107
Solubility%
19.87 ± 4.78a 3.76 ± 1.10d 6.35 ± 1.18c 7.46 ± 2.44c 12.29 ± 0.69b
46.56 30.19 38.04 20.63 16.33
± ± ± ± ±
1.75a 2.74c 0.82b 0.89d 0.92e
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; TGS: transglutaminase enzyme; TS: tensile strength and%EAB: percentage of elongation at break.
3.3. Ftir-atr FTIR-ATR spectrum of blend films is shown in Fig. 3. The OeH stretching broad band of sago starch film (1:0) presented at 3309.38 cm−1. The band at 2941. 08 cm−1 related to the CeH stretching, while the band of 1339.32 cm−1 presented the OeH of water. Likewise, in sago starch/fish gelatin (TGs 0 mg) and films with transglutaminase (TGs 1 mg, TGs 5 mg and TGs 10 mg) band at 3309. 38 cm−1 (OeH stretching as in 1:0), while band at 1543.13 cm−1 was the eNH twisting (amide II). The band at 1647.98 cm−1 was due to the C]O stretching as amide I which also present in sago starch films (1:0) as a results of protein presence (Amide I). Transmittance percentage of amide I and II bands in treated films reduced with higher enzyme concentration added. Pshenitsyna, Molotkova, Noskova and Aksel'rod (1979) mentioned that both Amide I and Amide II (1650 cm−1 & 1550 cm−I) associated with C]O and CeN groups stretching vibrations and the deformation vibrations of NH groups, respectively. In addition, they have stated that absorption band of Amide II is often used for structural characterization. An ether group can be observed in the big band at 997.37 cm−1 as stated by Bourtoom and Chinnan (2008) where they observed 993 band in starch-chitosan films. Hydrogen interactions between both biopolymers can be considers by moving in band location of the amide-1 and amide-3 groups. (Benbettaïeb, Kurek, Bornaz & Debeaufort, 2014).
Fig. 2. Water vapor permeability of sago starch (1:0), sago starch/fish gelatin (3:1) films and films (3:1) treated with transglutaminase enzyme (TGs 1 mg, TGs 5 mg and TGs 10 mg).
3.2. Crosslinking degree of films Crosslinking degree of sago starch/fish gelatin films (3:1) treated with TGs with the concentrations of (1, 5 and 10 mg/g) is shown in Fig. 1. Incubation of the films with transglutaminase enzyme with the concentration 1 mg/g showed a reduction in amino acid groups resulting in a crosslinking percentage of 30.9 ± 3.1% when incubated for 15 min. Increasing the enzyme concentration to 5 mg/g showed further reduction of available amino groups with increasing cross-
3.4. Mechanical properties All thickness of biopolymers films with plasticizers ranged between 0.05–0.07 mm. Developed biopolymer films were transparent, homogeneous, flexible and easily removed from the acrylic plates. Table 2 shows tensile strengths (TS), percentage of elongation at break (% EAB) 17
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and Young’s modulus (E) of the films plasticized with 30% glycerol. The findings showed there is a reduction in TS (from 8.15 MPa to 2.60 MPa) associated with increase in%EAB (from 42.50% to 72.19%) when fish gelatin incorporated to sago starch film. Since the plasticizer concentration was constant, hydrogen bonding formed between protein and starch may result in TS reduction which might reduce the interaction between starch chains. Transglutaminase significantly increase TS with all con concentration (TGs 1 mg, TGs 5 mg and TGs 10 mg) compared to control (3:1 G) with no enzyme added. The possibility of increasing in TS associated with a reduction in%EAB as well as an increase in Young’s modulus resulted from the transglutaminase induced crosslinking between amino groups. Mariniello et al. (2003) reported that pectinsoy flour films treated with transglutaminase was less extensible than untreated films resulted in two times increase in TS associated with two times reduction in%EAB.
creases the WVP. Kolodziejska and Piotrowska (2007) reported that crosslinking of fish gelatin-chitosan films with chemical (EDC) or TGase had different WVP in the presence of different glycerol concentrations (0 to 30%). 4. Conclusion The effect of fish gelatin and transglutaminase incorporation into sago starch based film plasticized with 30% glycerol were tested. The findings indicated that addition of fish gelatin to sago starch FFS significantly reduced WVP of the developed films and has an effect on TS and%EAB. Adding TGs with concentrations beyond 1 mg/g to the sago starch/fish gelatin FFS reduced flow consequently and has an effect on viscosity. The results also showed that TS and% EAB changed with enzyme incorporation. Moreover, FTIR-ATR showed a reduction in the% transmittance of the peaks involving ε-amino groups due to the reduction of amino acids as a results of gelatin molecules polymerization.
3.5. Solubility of films Table 2 shows solubility of films. Addition of fish gelatin to sago starch significantly reduced solubility from 46.56% to 30.19%, this may be due to stronger hydrogen bonding formation between sago starch and fish gelatin than starch chains intra and intermolecular or hydrogen bonding with water molecules that makes films more intact and less soluble. Addition of transglutaminase enzyme by 1 mg to films (3:1) significantly increased solubility from 30.19% to 38.04%. Formation of small molecular fractions as crosslinked occurred reduced hydrogen bonding between biopolymers that makes films more soluble. More glycerol became exposed to water molecular resulting in more solubility of films. Increase in enzyme concentration significantly reduced solubility of treated films compared to films with no enzyme added. Reduction in solubility was observed in treated films from 30.19% to 20.63% and 16.33% for 5 mg and 10 mg respectively; due to isopeptide formation between amino acids of the gelatin however, starch protein may involve in the crosslinking formation. The findings of this study was in agreement with Kolodziejska and Piotrowska (2007) who concluded that modified fish gelatin–chitosan films with the enzyme transglutaminase decreased the solubility even in acidic pH and high temperature compare to uncross linked films however, plasticization with glycerol had no effect on the mixture solubility in aqueous medium. Fish gelatin films treatment with TGs enzyme reduced the solubility by 50% at pH 3.0 and to 40% at pH 6 (Piotrowska, Sztuka, Kolodziejska & Dobrosielska, 2008). De Carvalho and Grosso (2004) mentioned that increase in crosslinking degree could result in decreasing low molecular fractions weight that led to the reduction in solubility.
References 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 9600. Philadelphia, PA: American Society for Testing and Materials. Al-Hassan, A. A., & Norziah, M. H. (2012). Starch-gelatin edible films: water vapor permeability and mechanical properties as affected by plasticizers. Food Hydrocolloids, 26(1), 108–117. Ardelean, A. I., Jaros, D., & Rohm, H. (2013). Influence of microbial transglutaminase cross-linking on gelationkinetics and texture of acid gels made from whole goats and cows milk. Dairy Science & Technology, 93(1), 63–71. Arvanitoyannis, I., Nakayama, A., & Aiba, S.-I. (1998). Edible films made from hydroxypropyl starch and gelatin and plasticized by polyols and water. Carbohydrate Polymers, 36(2–3), 105–119. Benbettaïeb, N., Kurek, M., Bornaz, S., & Debeaufort, F. (2014). Barrier, structural and mechanical properties of bovine gelatin-chitosan blend films related to biopolymer interactions. Journal of the Science of Food and Agriculture, 94(12), 2409–2419. Bertuzzi, M. A., Armada, M., & Gottifredi, J. C. (2007). Physicochemical characterization of starch based films. Journal of Food Engineering, 82(1), 17–25. Bourtoom, T., & Chinnan, M. S. (2008). Preparation and properties of rice starch-chitosan blend biodegradable film. LWT – Food Science and Technology, 41(9), 1633–1641. Bubnis, W. A., & Ofner, C. M. (1992). The determination of ε-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid. Analytical Biochemistry, 207(1), 129–133. Cao, N., Fu, Y., & He, J. (2007). Preparation and physical properties of soy protein isolate and gelatin composite films. Food Hydrocolloids, 21(7), 1153–1162. Chambi, H., & Grosso, C. (2006). Edible films produced with gelatin and caseincrosslinked with transglutaminase. Food Research International, 39(0), 458–466. 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(3), 622–626. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food, 2016 Food Hydrocolloids, 2016. Food Hydrocolloids, 2016(18), 717–726. Flores, S., Famá, L., Rojas, A. M., Goyanes, S., & Gerschenson, L. (2007). Physical properties of tapioca-starch edible films: Influence of filmmaking and potassium sorbate. Food Research International, 40(2), 257–265. García, M. A., Pinotti, A., Martino, M., & Zaritzky, N. (2009). Electrically treated composite FILMS based on chitosan and methylcellulose blends. Food Hydrocolloids, 23(0), 722–728. Kaewruang, P., Benjakul, S., Prodpran, T., Encarnacion, A. B., & Nalinanon, S. (2014). Impact of divalent salts and bovine gelatin on gel properties of phosphorylated gelatin from the skin of unicorn leatherjacket. LWT e Food Science and Technology, 55(0), 477–482. Kieliszek, M., & Misiewicz, A. (2014). Microbial transglutaminase and its application in the food industry. A review. Folia Microbiologica, 59, 241–250. Kolodziejska, I., & Piotrowska, B. (2007). The water vapour permeability, mechanical properties and solubility of fish gelatin-chitosan films modified with transglutaminase or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and plasticized with glycerol. Food Chemistry, 103(2), 295–300. Kurata, M., & Tsunashima, Y. (1998). Viscosity–molecular weight relationships and unperturbed dimensions of linear chain molecules. In J. Brandrup, & E. H. Immergut (Eds.), Polymer handbook(3rd ed.). New York: John Wiley & sons. Lim, L. T., Mine, Y., & Tung, M. A. (1999). Barrier and tensile properties of transglutaminase cross-linked gelatin films as affected by relative humidity, temperature, and glycerol content. Journal of Food Science, 64(4), 616–622. Mariniello, L., Di Pierro, P., Esposito, C., Sorrentino, A., Masi, P., & Porta, R. (2003). Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase. Journal of Biotechnology, 102(2), 191–198.
3.6. Water vapor permeability Water vapor permeability of developed films is shown in Fig. 2. Film thickness ranged between 0.05–0.07 mm. Sago starch film (1:0) has a WVP value of (4.3 × 10−4 g mm/m2 h Pa). Incorporation of fish gelatin to sago starch films significantly reduced WVP to (2.4 × 10−4 g mm/m2 h Pa). The possibility of the reduction may due to glycerol plasticizer effect as a small molecule that penetrates between the molecular of the two biopolymers resulting in a reduction of the intra and inter hydrogen bonding within a single polymer, which makes more mobility of the biopolymers to form hydrogen-bonding between them resulted in intact molecules with less WVP. Addition of transglutaminase enzyme to films (3:1) had no significant effect on WVP films which may be due to hydrophilicity nature of material used. Even though the transglutaminase-induced crosslinking implicated covalent iso-peptide bonds, the sorption sites responsible for making films hydrophilic in the two biopolymers had not changed. The result may be due to the effect of the present of the hydrophilic plasticizer. Cuq, Gontard, Cuq and Guilbert (1997) reported that plasticization of protein and polysaccharide films with a hydrophilic plasticizer in18
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Sow, L. C., & Yang, H. (2015). Effects of salt and sugar addition on the physicochemical properties and nanostructure of fish gelatin. Food Hydrocolloids, 45(0), 72–82. Veiga-Santos, P., Oliveira, L. M., Cereda, M. P., & Scamparini, A. R. P. (2007). Sucrose and inverted sugar as plasticizer. Effect on cassava starch-gelatin film mechanical properties, hydrophilicity and water activity. Food Chemistry, 103(2), 255–262. Wang, P., Liu, H., Wen, P., Zhang, H., Guo, H., & Ren, F. (2013). The composition size and hydration of yak casein micelles. International Dairy Journal, 31(2), 107–110. Yang, M., Shi, Y., Wang, P., Liu, H., Wen, P., & Ren, F. (2014). Effect of succinylation on the functional properties of yak caseins a comparison with cow caseins. Dairy Sci Technol, 94(4), 359–372. Yang, M., Shi, Y., & Liang, Q. (2015). Effect of microbial transglutaminase crosslinking on the functional properties of yak caseins: A comparison with cow caseins. Dairy Sci. Technol. 96(0), 39–51.
Norziah, M. H., Al-Hassan, A., Khairulnizam, A. B., Mordi, M. N., & Norita, M. (2009). Characterization of fish gelatin from surimi processing wastes: Thermal analysis and effect of transglutaminase on gel properties. Food Hydrocolloids, 23(6), 1610–1616. Piotrowska, B., Sztuka, K., Kolodziejska, I., & Dobrosielska, E. (2008). Influence of transglutaminase or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) on the properties of fish-skin gelatin films. Food Hydrocolloids, 22(7), 1362–1371. Prasertsung, I., Mongkolnavin, R., Kanokpanont, S., & Damrongsakkul, S. (2010). The effects of pulsed inductively coupled plasma (PICP) on physical properties and biocompatibility of crosslinked gelatin films. International Journal of Biological Macromolecules, 46(1), 72–78. Pshenitsyna, V. P., Molotkova, N. N., Noskova, M. P., & Aksel'rod, B. Y. (1979). Display of the amide II absorption band in the spectra of reaction products of urea with formaldehyde. Journal of Applied Spectroscopy, 29(3).
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