Effect of transglutaminase treatment on properties of coconut protein-guar gum composite film

LWT - Food Science and Technology 115 (2019) 108422

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Effect of transglutaminase treatment on properties of coconut protein-guar gum composite film

T

Karuna L. Sorde, Laxmi Ananthanarayan∗ Food Engineering and Technology Department, Institute of Chemical Technology, N. M. Parekh Marg, Mumbai-19, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Coconut protein Edible packaging Transglutaminase Mechanical and barrier properties Scanning electron microscopy

Polysaccharides, proteins and lipids have been utilized for synthesis of polymer matrices to minimize environmental pollution caused by plastic food packaging. Edible coatings are one of the promising alternative in this regard. The coconut protein-guar gum composite films were prepared and studied for their mechanical and barrier properties. Addition of transglutaminase to the composite film forming solution significantly increased the mechanical strength from 1.76 MPa to 3.79 MPa and elongation from 56 mm to 184.87 mm. Improved barrier properties such as water vapour permeability and oxygen transfer rates were observed after treatment of composite films with microbial transglutaminase (MTGase). Differential scanning calorimetric analysis demonstrated an increase in melting temperature with increasing concentration of MTGase. FTIR spectroscopy showed changes in the secondary structure of the protein (amide I and amide II band) and its interaction with the polysaccharide. Major coconut protein having a molecular weight of 53 kDa was not visible in MTGase treated films due to MTGase induced polymerization of protein as analyzed using SDS-PAGE electrophoresis. Scanning electron microscopy exhibited a fibrous microstructure of the surface of the MTGase treated coconut protein guar gum composite films.

1. Introduction Environmental concerns in regard to problems caused by non-biodegradable plastic packaging materials is ever increasing and this has compelled researchers to look for alternate approaches. Edible films and coatings are in great demand as substitutes for plastic materials in food packaging. Edible films are thin films wrapped around the food material to improve the shelf-life and quality attributes of food products. The major advantages of using natural biopolymer is that they are bio-degradable, edible and can be consumed with the food. Natural biopolymers can bring about an improvement in the nutritional value, reduced packaging volume, protection from deterioration, and preservation of organoleptic properties such as texture, colour, and flavor of food (Marquez, Di Pierro, Esposito, Mariniello, & Porta, 2013). They can be incorporated with anti-oxidants, flavors, colours, and nutraceuticals (Bifani et al., 2007; Guilbert, Cuq, & Gontard, 1997; Kester & Fennema, 1986). Carbohydrates are in great demand in preparation of films. Carbohydrates are abundantly available biopolymers and some of them can readily form a gel after heating. Proteins, on the other hand, have multiple film forming mechanisms involving intermolecular bondings, structural conformations, denaturation properties, electrostatic charges that gives them distinctive characteristics (Wittaya, ∗

2012). Coconut (Coconut nucifera L.) is an important oilseed indigenous to the Indian subcontinent, distributed mainly in coastal regions playing a significant role in the agrarian economy of India. Desiccated coconut is considered as a waste in the coconut oil industry, having a protein content of around 15–23% with appreciable nutritional value (Kwon, Bae, Park, & Rhee, 1996). However, most of the coconut proteins are insoluble in water. Besides being highly nutritive and possessing excellent functional properties, coconut protein is still relatively unexplored (Naik, Prakash, & Raghavrao, 2013). Based on the scientific literature, film-forming properties of coconut proteins have not been studied. Studies on coconut proteins and its gelling properties is still fragmented and very little information is available. Introduction of cross-linking agent is one of the options to improve the polymeric network to give structural strength and peeling ability to the gels and films formed thereof (Babin & Dickinson, 2001). The cross-linking agents are generally chemical or enzymatic in nature. Many chemical cross-linkers such as formaldehyde, glyoxal, glutaraldehyde, and genipin were used in this regards. However due to their toxic nature, use of these cross-linkers is not advisable (De Carvalho & Grosso, 2004). Transglutaminases (TGases) are a class of enzymes known to crosslink proteins and polypeptides by catalyzing acyl transfer reaction

Corresponding author. E-mail address: [email protected] (L. Ananthanarayan).

https://doi.org/10.1016/j.lwt.2019.108422 Received 6 February 2019; Received in revised form 18 July 2019; Accepted 19 July 2019 Available online 20 July 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

LWT - Food Science and Technology 115 (2019) 108422

K.L. Sorde and L. Ananthanarayan

between γ-carboxamide group of protein-bound glutamine and lysine (Kieliszek & Misiewicz, 2014). In the absence of lysine, primary amine acts as acyl acceptor. Apart from that variety of protein glutamyl derivatives are formed by substituting lysine with other compounds containing primary amino acids. These bonds are stable to proteolysis and enhance enzymatic, chemical and mechanical stability (Greenberg, Birckbichler, & Rice, 1991). Microbial transglutaminase (MTGase) has been applied in the formation of films and edible coatings to alter their mechanical and barriers properties (Wu, Wang, Liu, Liu, & Ye, 2017). There is a vast literature available on transglutaminase modified protein films showing improvements in mechanical strength, water barrier property and thermal stability of soy protein isolate (SPI) (Weng & Zheng, 2015), whey protein isolate (WPI) (Yildirim & Hettiarachchy, 1998), gelatin (Liu et al., 2017) egg white (Lim, Mine, & Tung, 1998) and casein (Motoki, Aso, Seguo, & Nio, 1987). High resistance to water vapour permeability was observed in MTGase modified gelatin films (De Carvalho & Grosso, 2004). Proteins and polysaccharides being different in their molecular structure and nature exhibit various electrostatic, hydrophilic and hydrophobic interactions. These interactions between protein and polysaccharides are of importance in changing viscosity and gelling properties of the solution (Amaral et al., 2018). Interaction between positively charged proteins and anionic polysaccharides are involved in gelation, co-acervation or multilayer formation (Di Pierro et al., 2013). The present study deals with effect of transglutaminase on coconut protein-guar gum composite films. Furthermore, the film properties based on mechanical strength, water vapour permeability, gas barrier properties and thermal properties were studied.

incubated at 80 °C in water bath for 15 min to deactivate the enzyme and then allowing it to cool by standing for 30 min at 30 °C. One gram of MTGase (Activa TG-S) commercial enzyme, dissolved in 10 mL of phosphate buffer (0.2 mol/L, pH 6) was added to the film forming solution. The MTGase activity was determined by the hydroxamate method described by Folk and Cole (1965) and modified by Macedo, Sette, and Sato (2007). For hydroxamate assay, a substrate solution containing 0.03 mol/L Z-Gln- Gly, 0.02 mol/L glutathione and 0.2 mol/L hydroxylamine was prepared in 0.2 mol/L citrate buffer (pH 6). Fifty microliters of the commercial enzyme preparation was pre-equilibrated by incubating it at 37°C for 10 min one hundred and 50 μL of the substrate solution was added to it and the reaction mixture was incubated at 37 °C for 60 min. The reaction was stopped by addition of 150 μL of stop solution [5 g ferric chloride in 15 g tri-carboxylic acid prepared in 100 mL of distilled water]. One unit of MTGase activity is calculated as the amount of enzyme required to produce 1 μmol of hydroxamate per minute at 37°C. Calibration curve was prepared using glutamyl γ mono hydroxamate. The films obtained were peeled from the plates and conditioned in desiccators at 25 ± 2 °C by maintaining the relative humidity (RH) of 50 ± 2% with saturated solution of Mg (NO3)2 6H2O. For further analysis, the films were conditioned for at least 48 h prior to testing. Thickness of the films formed varied from 0.10 to 0.13 mm. 2.2.3. Measurement of tensile strength and extensibility Tensile strength and extensibility of the developed films was measured using texture analyzer (Stable Micro System, Surrey, UK). Sample of dimensions 30 × 70 mm was placed on a base grip positioned on a heavy-duty platform (HDP/90). A load of 50 kg was applied to the film with a speed of 1 mm/s to measure the force until the film breaks. The force measured was then divided by the cross-section area to calculate tensile strength. Extensibility of the films was calculated as the mean distance till breaking of the film.

2. Materials and methods 2.1. Materials Desiccated coconut meal (Good life R, Marketed by Reliance Ltd. India) was purchased from local market, Transglutaminase (Activa TGS) a commercial MTGase was obtained from Ajinomoto, Japan. Carbobenzoxy glutamine glycine (CBZ-Gln-Gly), glutathione (reduced form), and glutamyl-γ monohydroxamate were purchased from SigmaAldrich (St.Louis, MO, USA). Hydroxylamine, trichloroacetic acid (TCA), ferric chloride were purchased from S.D. Fine Chemicals Ltd., India. All the other chemicals and reagents were of analytical grade and procured from reliable sources.

2.2.4. Transparency and colour Transparency of the CG films was determined using UV visible spectrophotometer (UV-1800, Shimadzu Scientific Instruments, Kyoto, Japan) by recording absorbance at 600 nm. Air was considered as reference and the values were determined as 100% transmittance. Colour of the films was measured using colorimeter (LabScan XE, Hunterlab US). The films were placed on the surface of a white standard plate. The values of colour parameter L*, a*, b* were determined. Whiteness index and yellowness index were determined by using the following formulae-

2.2. Methodology 2.2.1. Extraction of proteins from coconut meal The desiccated coconut meal was defatted using petroleum ether (60–80 °C) and its protein content was determined by Kjeldahl method (Kjeldahl, 1883). Twenty gram of defatted coconut meal was dispersed in 200 mL distilled water. Extraction of coconut protein was carried out by continuous heating at 250 rpm on a magnetic stirrer at 30 °C for 4 h. The pH during extraction was maintained at pH 8 by addition of 0.05 mol/L NaOH. After extraction, the solution was filtered thorough 2 layers of cheese cloth and centrifuged at 5018×g, at 4 °C for 5 min. Protein content was estimated by Bradford method (Bradford, 1976). The extracted proteins were dried in vacuum oven at 40 °C for 24 h.

Wi = 100 −

Yi =

142.86 b L

[(100 − L2) + a2 + b2]

(1)

(2)

2.2.5. Measurement of barrier properties Water vapour permeability (WVP) was measured gravimetrically by ASTM method (ASTM E96-95) with certain modifications (Shah, Vishwasrao, Singhal, & Ananthanarayan, 2016). The preconditioned films CG-0, CG-5, and CG-10 were cut in disc form of diameter 7 cm and sealed over a cup containing completely dried silica gel. An air gap of 1 cm was maintained inside the cup and 18 cm2 area was exposed for permeability. The cups sealed with films were placed in a desiccator with a humidity level of 50 ± 2% RH maintained by using a saturated solution of Mg(NO3)2.6H2O. The cups were pre-weighed and increase in weight was checked at an interval of 2 h. The thickness of the films was determined by taking seven to eight readings across the diameter by using micrometer screw gauge. Water vapour transmission rate (WVTR) and water vapour permeability were determined by the following equations-

2.2.2. Preparation and casting of films The film-forming mixture used in film preparation and casting (100 mL) contained the following composition: Reaction mixtures containing 2 g of coconut protein dissolved in distilled water, different concentrations of MTGase, 5 U/mL (CG-5), 10 U/mL (CG-10) and no enzyme (CG-0) and 0.75 g of guar gum, to a final volume of 100 mL were incubated at 45 °C for 3 h with shaking. After incubation 0.75 g of glycerol/100 g solution was added and pH was adjusted to pH 7.0 with 0.05 mol/L NaOH solution. The mixtures were 2

LWT - Food Science and Technology 115 (2019) 108422

K.L. Sorde and L. Ananthanarayan

WVTR = WVP =

Slope of weight gain vs time plot A

(3)

WVTR × h Δp

(4)

where A is the area of the film exposed (cm ), h is the thickness of the film (mm). WVP is measured as WVTR across the film where Δp is partial vapour pressure difference in kPa on both sides of the film measured as saturation pressure at a specific temperature. It is calculated as the difference in the relative humidity R1 = 51.4% at 30 °C inside the desiccator on the outer side of the cup and R2 = 0 inside the cup. The oxygen transfer rate (OTR) was measured using BTY-B1P (Labthink, Jianin, China) gas permeability tester. The difference in the pressure was calculated and it was maintained at 0.1 MPa. Change in pressure from the lower pressure side and OTR was calculated on a software-based data acquisition by changing pressure from the lower pressure side (Mendoza-Mendoza et al., 2013). The area exposed for permeation was 75.39 cm2 and the test was carried out at 30 ± 2 °C. 2

2.2.6. Differential scanning calorimetry Thermal properties are one of the important characteristics of the films to be used in food, pharmaceutical or textile applications. Thermal properties of the coconut protein-guar gum composite films were determined using DSC-60 (Shimadzu Scientific Instruments, Kyoto, Japan). A sample of 3.5 mg was placed in DSC pan. The pan was hermetically sealed and scanned over a temperature range from 30 to 300 °C with a heating rate of 5 K/min. An empty pan was used for reference. The values of enthalpy (ΔH) and melting temperature (Tm) were obtained by the instrumental assembly mentioned above.

Fig. 1. Effect of transglutaminase on (a) tensile strength and (b) extensibility of the coconut protein-guar gum composite films The results are mean of values of experiments performed in triplicate. Different letters denoted as superscript indicates the significant differences (p < 0.05).

2.2.7. FTIR FTIR analysis was performed to evaluate changes in the structure due to MTGase induced cross-linking. This was examined by blending 5 mg of the film sample with 100 mg KBr in a mortar pestle till a fine powder was obtained. The sample was placed in a sample holder of Diffused Reflectance Spectrometer using IR Prestige 21 (Shimadzu India Pvt.Ltd.) and scanned over a range of 400–4500/cm.

3. Results and discussion 3.1. Tensile strength and extensibility

2.2.8. Scanning electron microscopy The microstructure of the upper surface of the films was analyzed using scanning electron microscope (JSM-6380LA, Jeol Ltd., Japan) following the method described by Prashanth et al. (2006) with slight modifications. Films were conditioned at 50% RH for 48 h and then transferred to a desiccator containing dried silica gel with vacuum to dehydrate. The dehydrated films were mounted on the aluminum platform, sputter coated with platinum under vacuum at 25 °C and examined at 15 kV.

The desiccated coconut meal was first defatted and protein concentration determined by Kjeldahl method was found to be 21.45 g/ 100 g of the defatted meal. It has been studied that molecular size, the configuration of molecules, number of hydroxyl groups and other linkages, plasticizer and its compatibility with polymer influences mechanical properties of the film (Yang et al., 2000). The tensile strength was found to increase with the addition of MTGase as compared to that of CG-0 film (Fig. 1a). The reason can be attributed to cross-linking induced by MTGase which is a result of the reaction between glutamine residue of the enzyme with the lysine and primary amines in coconut protein to form a complex matrix which gives strength to the films formed. As demonstrated by other researchers, the tensile strength of gelatin films increased significantly with the addition of 1% MTGase (Weng & Zheng, 2015). Furthermore, the mechanical properties of polymer blends depend upon intermolecular forces, molecular symmetry and chain stiffness of the individual polymer (Joseph & Thomas, 2002). MTGase modified films were found to have better ductile and peel-ability than that of the unmodified CG films. Similar findings were obtained by Weng and Zheng (2015) where the tensile strength of gelatin-based film increased significantly after treatment with TGase. As shown in Fig. 1b, the extensibility of the films was observed to increase with the addition of MTGase. Similar findings were obtained by Mariniello et al. (2003); where the authors found almost five times increase in elongation at break in the pectin-protein films modified in the presence of MTGase than that of unmodified pectin-based films.

2.2.9. SDS-PAGE electrophoresis For determination of cross-linking of coconut proteins by MTGase, gel electrophoresis method using SDS PAGE was performed as per the method reported by Laemmli (1970). The samples were prepared by dissolving 10 mg of the films with MTGase at concentrations CG-5 and CG-10 (Concentrations mentioned in section 2.2) in 1 mL of water. Fifty microliters of the sample was added to 100 μL of sample buffer (0.5 mol/L Tris–HCl, pH 6.8). The sample buffer contained following components per 100 mL buffer: SDS, 10 g/100 mL buffer; glycerol, 10 mL; β-mercaptoethanol 5 mL; and 0.1 g bromophenol blue. The samples were heated at 95 °C for 10 min and loaded in gel comprised of 4% stacking gel (pH 6.8) and 12% resolving gel (pH 8.8). Electrophoresis was performed at a constant voltage of 70 V using Bio-Rad mini PROTEAN® 3-cell gel apparatus, Bio-Rad, USA. The gel was stained with 2 g silver nitrate in 100 mL water for exactly 25 min and developed in a developing solution (100 mL) containing 3 g sodium carbonate and 0.5 g formaldehyde. 3

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Table 1 Effect of addition of transglutaminase on transparency and colour of coconut protein-guar gum composite films. Samples CG-0 CG-5 CG-10

%T

L* a

a* a

33.69 ± 0.31 36.36 ± 0.25b 39.58 ± 0.28c

80.96 ± 1.11 77.34 ± 0.67b 75.53 ± 1.05c

b*

−0.54 ± 0.22 −0.77 ± 0.23b −1.13 ± 0.18c a

WI a

0.14 ± 0.11 0.69 ± 0.18b 0.27 ± 0.15c

33.69 ± 0.105 36.35 ± 0.03b 39.57 ± 0.04c

YI a

0.62 ± 0.49a 2.27 ± 0.5b 1.31 ± 0.69c

Table 2 Effect of transglutaminase on melting temperature and ΔH of coconut proteinguar gum composite films. Samples

Tm (°C)

ΔH (J/g)

CG-0 CG-5 CG-10

118 121.4 134.08

−228.7 −246.8 −308.8

Glycerol as a plasticizer plays an important role in the film forming properties as it has the ability to form a hydrogen bond network with the polymer (Sanyang, Sapuan, Jawaid, Ishak, & Sahari, 2016). Fig. 2. Effect of addition of transglutaminase on water vapour permeability of coconut protein-guar gum composite films The results are mean of values of experiments performed in triplicate. Different letters denoted as superscript indicates the significant differences (p < 0.05).

3.2. Transparency and colour The transparency of the control CG films was higher than that of MTGase treated films (Table 1). It was observed that the transparency of the films decreased with the addition of MTGase. For food packaging material, transparency of the films is important. Similar results were observed by Tang and Jiang (2007) in films prepared from soy protein isolate (SPI). The reduction in the transparency is probably due to the formation of cross-links altering the refractive index and preventing the passage of light through the film matrix (Ortega -Toro, Jimenez, Talens, & Chiralt, 2014). The films prepared showed a whiteness index (WI) ranging from 33 to 39% as shown in Table 1. A slight decline was observed in a* values of TGase added films. This indicates redness/ greenness of the films. The films were observed to be slightly yellow as indicated by positive b* value suggesting an increase in yellowness index with the addition of TGase. It was studied that fish skin gelatin films slightly changed to yellowish and greenish after treatment with TGase (Yi, Kim, Bae, Whiteside, & Park, 2006) (see Table 2). 3.3. Measurement of barrier properties

Fig. 3. Effect of addition of transglutaminase on oxygen transfer rate of coconut protein-guar gum composite films The results are mean of values of experiments performed in triplicate. Different letters denoted as superscript indicates the significant differences (p < 0.05).

3.3.1. Water vapour permeability Water vapour permeability (WVP) is an important characteristic of food packaging material as it is directly related to the deterioration of food material. At molecular level, there are many factors involved in the permeability of film such as crystallinity, density, molecular orientation, weight and cross-linking of materials (Jasse, Seuvre, & Mathloutht, 1994; Miller & Krochta, 1997). WVP of the films decreased with increasing concentration of MTGase as shown in Fig. 2. Similar results were obtained in SPI incorporated gelatin films modified with transglutaminase and TGaseinduced lizardfish scale gelatin films proposing the decrease in the free volume of protein films due to MTGase induced cross-linking (Wangtueai, Noomhorm, & Regenstein, 2010; Weng & Zheng, 2015). The results are in agreement with Weng and Zheng (2015), where the authors have reported a decrease in water vapour permeability of SPI films with increasing concentration of transglutaminase. WVP is associated with the availability of polar groups such as –OH, –COOH, –NH2. The probable reason for this might be cross-linking induced by MTGase reducing the availability of free polar groups.

reduce with increasing concentration of MTGase [CG-0 (8.543 × 10 cm3/m2 d. Pa, CG-5 (6.7 × 10 −4 cm3/m2 d. Pa, CG-10 (5.33 × 10 −4 cm3/m2 d. Pa)]. The results obtained in this investigation are in agreement with results reported on chitosan films and pectin-soy protein films modified by MTGase (Di Pierro et al., 2013). However, the oxygen permeability of these CG films was remarkably low as compared to other protein films. Oxygen transfer rate of casein films varied between 829.5 and 856.3 cc/m2.atm.day whereas it is 1548.4–1964.5 cc/ m2.atm.day for WPC film (Wagh, Pushpadass, Emerald, & Nath, 2014). This can be due to mixture of protein and gum interacting with each other forming a complex structure (Oliveira et al., 2011). −4

3.4. Differential scanning calorimetry Thermal properties of the films were investigated to determine the thermal stability of the coconut protein after addition of MTGase. Melting temperature increased with an increase in the concentration of MTGase. It was hypothesized that with the addition of transglutaminase, the proteins would cross-link and polysaccharide and the cross-

3.3.2. Oxygen transfer rate Reduced oxygen permeability was observed in the films prepared as shown in Fig. 3 using MTGase. Oxygen transfer rate was found to 4

LWT - Food Science and Technology 115 (2019) 108422

K.L. Sorde and L. Ananthanarayan

Fig. 4. Effect of transglutaminase on FTIR spectra of coconut protein-guar gum composite films. The spectra represents effect of treatment of transglutaminase at different concentrations.

Fig. 5. Scanning electron micrographs of transglutaminase treated and untreated films. a. (CG-0): untreated sample b. (CG-5) and c. (CG-10) samples with MTGase.

level. The FTIR spectra of the MTGase treated films are depicted in Fig. 4. With the increase in MTGase concentration, increase in the intensity of amide I and amide II band was observed, whereas, least intensity of these regions was observed in CG-0 sample. Stretching in the amide I band (∼1651) is associated with C=O while amide II band (1556) is associated with N–H twisting and bending vibration of C–H group. These bands are associated with the secondary structure of proteins. Covalent linkages formed by the action of MTGase lead to the formation of iso-peptide bonds thus increasing the number of bound N–H groups (Staroszczyk, Pielichowska, Sztuka, Stangret, & Kołodziejska, 2012). This increase was reflected by an increase in the intensity of amide I and amide II band.

linked molecules will be intertwined together to give a complex structure. Further, there are many interactions of the protein backbone and the polymeric network strongly held by transglutaminase. Therefore, more energy is required to break the structure. A gradual upshift (65–87 °C) in the melting temperature (Tm) was observed with increase in the degree of crosslinking in the gelatin films (De Carvalho & Grosso, 2004). Identical findings were reported by Kopp, Bonnet, and Renou (1989) for collagen with an increase in the degree of crosslinking. Furthermore, glass transition temperature (Tg) is associated with molecular structure, intermolecular interactions and chain stiffness (Ahmad, Benjakul, Prodpran, & Agustini, 2012).

3.5. FTIR The FTIR studies reveal the structural changes at the molecular 5

LWT - Food Science and Technology 115 (2019) 108422

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MTGase treatment. The results presented in the study suggest that the coconut protein-guar gum composite films modified by MTGase can be used as an edible packaging for food products, thereby providing an alternative to the non-biodegradable plastic coatings. Acknowledgment The authors are grateful to University Grants Commission, Government of India (Grant no. F.4-1/2006(BSR)/5-62/2007(BSR)) for financial support. References Ahmad, M., Benjakul, S., Prodpran, T., & Agustini, T. W. (2012). Physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils. Food Hydrocolloids, 28, 189–199. Amaral, T. N., Junqueira, L. A., Prado, M. E. T., Cirillo, M. A., Abreu, L.,R., Costa, F.,F., et al. (2018). Blends of Pereskia aculeata Miller mucilage, guar gum, and gum Arabic added to fermented milk beverages. Food Hydrocolloids, 79, 331–342. Babin, H., & Dickinson, E. (2001). Influence of transglutaminase treatment on the thermoreversible gelation of gelatin. Food Hydrocolloids, 15 271-176. Bifani, V., Ramirez, C., Ihl, M., Rubilar, M., Garcia, A., & Zaritzky, N. (2007). Effects of murta (Ugni molinae Turcz) extract on gas and water vapour permeability of carboxymethylcellulose-based edible films. LWT-Food Science and Technology, 40, 1473–1481. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72, 248–254. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18, 717–726. Di Pierro, P., Rossi Marquez, G., Mariniello, L., Sorrentino, A., Villalonga, R., & Porta, R. (2013). Effect of transglutaminase on the mechanical and barrier properties of whey protein/pectin films prepared at complexation pH. Journal of Agricultural and Food Chemistry, 61, 4593–4598. Folk, J. E., & Cole, P. W. (1965). Structural requirements of specific substrates for Guinea pig liver transglutaminase. Journal of Bological Chemistry, 240, 2951–2960. Greenberg, C. S., Birckbichler, P. J., & Rice, R. H. (1991). Transglutaminases: Multifunctional cross-linking enzymes that stabilize tissues. The FASEB Journal, 5, 3071–3077. Guilbert, S., Cuq, B., & Gontard, N. (1997). Recent innovations in edible and biodegradable packaging biodegradable packaging materials. Food Additives & Contaminants, 14, 741–751. Jasse, B., Seuvre, A. M., & Mathloutht, M. (1994). Permeability and structure in polymeric packaging materials. In M. Mathlouthi (Ed.). Food packaging and preservation. Boston, MA: Springer. Joseph, S., & Thomas, S. (2002). Modeling of tensile moduli in polystyrene/polybutadiene blends. Journal of Polymer Physics, 40, 755–764. Kester, J. J., & Fennema, O. R. (1986). Edible films and coatings: A review. Food Technology, 12, 47–59. Kieliszek, M., & Misiewicz, A. (2014). Microbial transglutaminase and its application in the food industry. A review. Folia Microbiologica, 59, 241–250. Kjeldahl, J. (1883). A new method for the determination of nitrogen in organic matter. Fresenius' Journal of Analytical Chemistry, 22, 366–382. Kopp, J., Bonnet, M., & Renou, J. P. (1989). Effect of collagen crosslinking on collagen–water interaction. Matrix, 9, 443–450. Kwon, K. S., Bae, D., Park, K. H., & Rhee, K. C. (1996). Aqueous extraction and membrane techniques improve coconut protein concentrate functionality. Journal of Food Science, 61, 753–756. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Lim, L., Mine, Y., & Tung, M. A. (1998). Transglutaminase cross- linked egg white protein films: Tensile properties and oxygen permeability. Journal of Agricultural and Food Chemistry, 46, 4022–4029. Liu, F., Chiou, B., Avena-bustillos, R. J., Zhang, Y., Li, Y., Mchugh, T. H., et al. (2017). Study of combined effects of glycerol and transglutaminase on properties of gelatin film. Food Hydrocolloids, 65, 1–9. Macedo, J. A., Sette, L. D., & Sato, H. H. (2007). Optimization of medium composition for transglutaminase production by a Brazilian soil Streptomyces sp. Electronic Journal Of Biotechnology, 10, 618–626. Mariniello, L., Di Pierro, P., Esposito, C., Sorrentino, A., Masi, P., & Porta, L. (2003). Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase. Journal of Biotechnology, 102, 191–198. Marquez, G. R., Di Pierro, P., Esposito, M., Mariniello, L., & Porta, R. (2013). Application of transglutaminase-crosslinked whey protein/pectin films as water barrier coatings in fried and baked foods. Food and Bioprocess Technology, 7, 447–455. Mendoza-Mendoza, B., Rodriiguez-Hernandez, A. I., Vargas-Torres, A., Diiaz-Ruiz, G., Montiel, R., & Ramos-Aboites, H. E. (2013). Characterization of the effects on the growth kinetics of Listeria monocytogenes in solid culture in contact with caseinate base edible films added with antilisterial activity from Streptococcus sp. ABMX isolated from Pozol, an indigenous Mexican beverage. International Food Research Journal, 20, 2917–2925.

Fig. 6. SDS PAGE analysis of the samples treated with different concentrations of transglutaminase.

3.6. Scanning electron microscopy Micro-structure of the films was determined by the SEM. Fig. 5 presents micrographs of the surface of MTGase treated and untreated films. In the CG-0 films, smooth and homogeneous surface and distribution was observed. Aggregation and fibrous structure was observed in the films treated with MTGase. However, these became more obvious as the concentration of MTGase increased. The film matrix exhibited more densely packed and heterogeneous structural morphology suggesting the formation of compact structure caused by cross-linking. Moreover, the fibrous structure formed might have the ability to enhance the strength of the films and other properties. 3.7. SDS PAGE electrophoresis Fig. 6 shows the molecular mass profile of the films produced with and without MTGase. Lane 2 is CG-0 untreated sample which shows three major bands with molecular masses, below 25 kDa, at around 33 kDa and 53 kDa identified as major protein bands in coconut protein fractions. The band near 53 kDa is cococin, a major 11S globulin present in coconut. Partial disappearance of the bands observed in lane 3 and lane 4 is attributed to polymerization of proteins. 4. Conclusions MTGase was successfully utilized for the preparation of coconut protein-guar gum composite film. Higher concentration of MTGase substantially affected the tensile strength, water vapour permeability, and oxygen transfer rate. Further, the thermal properties were affected by the addition of MTGase. A change in melting temperature was observed which in turn may affect the stability of the composite film. FTIR analysis revealed the changes in the secondary structure of protein probably due to MTGase mediated cross-linking. SDS PAGE analysis revealed the polymerization of proteins. Microstructure analysis suggested the formation of aggregates and fibrous structure as a result of 6

LWT - Food Science and Technology 115 (2019) 108422

K.L. Sorde and L. Ananthanarayan

Toro, R., Jimenez, A., Talens, P., & Chiralt, A. (2014). Properties of starch–hydroxypropyl methylcellulose based films obtained by compression molding. Carbohydrate Polymers, 109, 155–165. Wagh, Y. R., Pushpadass, H. A., Emerald, F. M. E., & Nath, B. S. (2014). Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese. Journal of Food Science and Technology, 51, 3767–3775. Wangtueai, S., Noomhorm, A., & Regenstein, J. M. (2010). Effect of microbial transglutaminase on gel properties and film characteristics of gelatin from lizardfish (Saurida spp.) scales. Journal of Food Science, 75, C731–C739. Weng, W., & Zheng, H. (2015). Effect of transglutaminase on properties of tilapia scale gelatin films incorporated with soy protein isolate. Food Chemistry, 169, 255–260. Wittaya, T. (2012). Protein-based edible films: Characteristics and improvement of properties. In A. A. Eissa (Ed.). Structure and function of food engineering. London, United Kingdom: Intech Open. Wu, X., Wang, K., Liu, Y., Liu, A., & Ye, R. (2017). Microstructure of transglutaminaseinduced gelatin-natamycin fungistatic composite films. International Journal of Food Properties, 20, 3191–3203. Yang, L., & Paulson, A. T. (2000). Mechanical and water vapour barrier properties of edible gellan films. Food Research International, 33, 563–570. Yi, J. B., Kim, Y. T., Bae, H. J., Whiteside, W. S., & Park, H. J. (2006). Influence of transglutaminase-induced cross-linking on properties of fish gelatin films. Journal of Food Science, 71, 376–383. Yildirim, M., & Hettiarachchy, N. S. (1998). Properties of films produced by cross-linking whey protein and 11S globulin using transglutaminase. Journal of Food Science, 63, 248–252.

Miller, K. S., & Krochta, J. M. (1997). Oxygen and aroma barrier properties of edible films: A review. Trends in Food Science & Technology, 8, 228–237. Motoki, M., Aso, H., Seguo, K., & Nio, N. (1987). as1-Casein film prepared using transglutaminase. Agricultural & Biological Chemistry, 51, 993–996. Naik, A., Prakash, M., R, R., & Raghavrao, K. (2013). Storage study and quality evaluation of coconut protein powder. Journal of Food Science, 78, 1784–1792. Oliveira, N. M., Dourado, F. Q., Peres, A. M., Silva, M. V., Maia, J. M., & Teixeira, J. A. (2011). Effect of guar gum on the physicochemical, thermal, rheological and textural properties of green edam cheese. Food and Bioprocess Technology, 4, 1414–1421. Prashanth, M. S., Parvathy, K. S., Susheelamma, N. S., Prashanth, K. H., Tharanathan, R. N., Cha, A., et al. (2006). Galactomannan esters—a simple, cost-effective method of preparation and characterization. Food Hydrocolloids, 20, 1198–1205. Sanyang, M. L., Sapuan, S. M., Jawaid, M., Ishak, M. R., & Sahari, J. (2016). Effect of plasticizer type and concentration on physical properties of biodegradable films based on sugar palm (Arenga pinnata) starch for food packaging. Journal of Food Science & Technology, 53, 326–336. Shah, N. N., Vishwasrao, C., Singhal, R. S., & Ananthanarayan, L. (2016). N-Octenyl succinylation of pullulan: Effect on its physico-mechanical and thermal properties and application as an edible coating on fruits. Food Hydrocolloids, 55, 179–188. Staroszczyk, H., Pielichowska, J., Sztuka, K., Stangret, J., & Kołodziejska, I. (2012). Molecular and structural characteristics of cod gelatin films modified with EDC and TGase. Food Chemistry, 130, 335–343. Tang, C. H., & Jiang, Y. (2007). Modulation of mechanical and surface hydrophobic properties of food protein films by transglutaminase treatment. Food Research International, 40, 504–509.

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