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The effects of microbial transglutaminase on the properties of fish myofibrillar protein film
Food Packaging and Shelf Life 12 (2017) 91–99
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The effects of microbial transglutaminase on the properties of fish myofibrillar protein film
MARK
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Pimonpan Kaewprachua, Kazufumi Osakob, Wirongrong Tongdeesoontornc, , Saroat Rawdkuena a b c
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan Technology Management of Agricultural Produce and Packaging Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords: Biodegradable Fish myofibrillar protein Microbial transglutaminase Protein based films Low density polyethylene
In this study, the effects of microbial transglutaminase (MTGase) on the properties of fish myofibrillar protein (FMP) films were investigated. As MTGase content increased, the thickness and tensile strength of the FMP films increased. In contrast, their elongation at break (167.49–85.61%), film transparency, water vapor permeability (2.38–2.02 × 10−9 g m−1 s−1 Pa−1), moisture content, film solubility, and degree of swelling all decreased (P < 0.05). Lightness also decreased, and yellowness increased as MTGase content was increased (P < 0.05). MTGase action also significantly improved barrier properties and thermal stability of films. Electrophoretic and FT-IR studies revealed that cross-linking and conformational changes were pronounced in film treated with MTGase. Based on these results, the addition of MTGase produced a good alternative method for improving FMP film properties; however, the mechanical and water barrier properties of the resulting films need further development.
1. Introduction Pollution by non- or slowly-degradable packaging material waste is an increasing environmental concern. In this context, the packaging materials from natural resources have ability to reduce or replace the commercial synthetic packaging films. Biodegradable films can be made from natural resources including carbohydrates, proteins, and lipids. Among these materials, proteins are extensively used as the filmforming materials due to their film-forming ability, abundance, high nutritional value, and biodegradability (Kaewprachu, Osako, Benjakul, Tongdeesoontorn, & Rawdkuen, 2016b). Fish myofibrillar proteins have been used frequently as a polymer for film-forming (Kaewprachu, Osako, Benjakul, & Rawdkuen, 2016a; Kaewprachu et al., 2016b; Prodpran, Benjakul, & Phatcharat, 2012; Rostamzad, Paighambari, Shabanpour, Ojagh, & Mousavi, 2016). Myofibrillar proteins are generally dissolved in the solution that provides the pH away from the isoelectric point (pI ≅ 5) of the proteins (Iwata, Ishizaki, Handa, & Tanaka, 2000). The dissociation and solubilization of myofibrillar proteins are then sufficient for expecting film formation. Previous experiments (Kaewprachu et al., 2016a, 2016b) showed that fish myofibrillar protein based films (FMP) had UV light barrier properties (200–280 nm) better than the synthetic wrap film (polyvinyl chloride; PVC). However, the relatively poor mechanical properties and
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Corresponding author. E-mail address: [email protected] (W. Tongdeesoontorn).
http://dx.doi.org/10.1016/j.fpsl.2017.04.002 Received 2 January 2017; Received in revised form 30 March 2017; Accepted 4 April 2017 2214-2894/ © 2017 Published by Elsevier Ltd.
high water vapor permeability posed significant limitations for their applicability in food packaging. Therefore, protein film network modifications are required to enhance these apparent weaknesses. Various approaches have been used to enhance the protein based films’ properties such as the use of chemicals, irradiation, thermal, and enzymatic modification. There are many researches that studied the enhancement of protein based films via chemical modifications by glutaraldehyde or genipin (0.15, 0.30, and 0.67%, w/v) (Amadori et al., 2015), gamma-irradiation (0–50 kGy) (Xu et al., 2012), and enzymatic reticulation (0–8 units per gram of protein) (Wang, Liu, Ye, Wang, & Li, 2015). In recent years, enzymatic cross-linking treatment has been used extensively over chemical modifications because of the toxicity of chemical synthetic agents, which is clearly unsuitable for real food systems application. Microbial transglutaminase (MTGase; EC.2.3.2.13) is most commonly used for protein modification. MTGase is a protein-glutamine γglutamyltransferase, which can be catalyzed the formation of an isopeptide bond between the group of carboxyl amide (donor) of glutamine residues and the group of ε-amine (acyl acceptor) of lysine residues. These reactions can be induced the formation of the intra- and intermolecular ε-(γ-glutamyl) lysine covalent bonds via cross-linking process (Nielsen, 1995). The cross-linking are commonly induced by MTGase could improve the films’ mechanical and physical properties by
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2.4.2. Mechanical properties A film sample was cut into 2 cm wide and 5 cm long, and then conditioned at 50 ± 5% RH at 25 °C for 48 h prior to testing. Tensipresser (TTP-50BX II, Takemoto Electric Inc., Tokyo, Japan) was used to measure tensile strength and elongation at break according to the ASTM standard method D882–97 (American Society for Testing and Materials, 1999). The conditions of testing were 30 mm of initial grip separation with the cross-head speed at 1 mm/s. Measurement was performed until the films were broken.
increasing the tensile strength, but lowering the elongation at break and solubility of the protein film. In addition, the MTGase enzymatic activity and biochemical reactions are affected by the pre-treatment of the protein (denaturation), the pH of the film-forming solution, the reaction time, the temperature, as well as the concentration of MTGase (Schmid, Sängerlaub, Wege, & Stäbler, 2014). Particularly, the effect of concentrations of MTGase on the protein based films’ properties have been extensively studied on whey protein isolate (Schmid et al., 2014), gelatin-calcium carbonate (Wang et al., 2015), and jellyfish protein (Lee, Lee, Yang, Won, & Song, 2015). Nevertheless, information regarding the effect of MTGase on the protein based films properties, especially in FMP films, remains very scarce. Therefore, the aim of this study was to investigate the effect of MTGase on the mechanical, physical, chemical, barrier, and thermal properties of FMP based film against a commercial wrap control film (low density polyethylene; LDPE).
2.4.3. Film appearance and color After the films were dried and conditioned at 50 ± 5% RH at 25 °C for 48 h, the visual aspect of film samples were examined by using a digital camera (Fujifilm Finepix S4900; acquired from Fujifilm Thailand Co. Ltd., Bangkok, Thailand). Color Reader (CR-13, Konica Minolta, Inc., Japan) was used to determine color attributes of the film and expressed as L*, a*, and b*.
2. Materials and methods
2.4.4. Water vapor permeability (WVP) A modified ASTM standard method (American Society for Testing and Materials, 1989) was used to evaluate the WVP as described in Kaewprachu et al. (2016b). Measurements were conducted at 30 °C at 50 ± 5% RH, and recorded the weight gain of the cup at an hour interval to 8 h. Experiments were repeated three times and expressed as g m−1 s−1 Pa−1.
2.1. Materials Microbial transglutaminase (MTGase) (Activa TG-K: 100 activity units per gram) was supplied by Ajinomoto Co. Inc. (Tokyo, Japan). Coomassie Blue R-250 was purchased from Merck (Darmstadt, Germany). SDS was purchased from Wako (Osaka, Japan). Glycerol and other analytical grade reagents were obtained from Kokusan Chemical Co., Ltd. (Yokohama, Japan). Low density polyethylene wrap film (LDPE) of 10 μm thickness (Ube Film Company Ltd., Japan) was used as the commercial wrap film in this study.
2.4.5. Differential Scanning Calorimetry (DSC) A differential scanning calorimeter (DSC-50, Shimadzu Co., Kyoto, Japan) was used for examining the thermal properties of the films (Kaewprachu et al., 2016b). Each film sample (10–12 mg) contained in an aluminum DSC pan were heated in the temperature range of 25–180 °C at a heating rate of 10 °C/min in a nitrogen atmosphere (20 ml/min).
2.2. Preparation of fish myofibrillar protein (FMP) Firstly, minced fish (fresh tilapia; Orcochromis niloticus) was added with five volumes of 50 mM NaCl and then homogenized for 2 min at 11,000 rpm. The mixtures were centrifuged at 10,000 × g for 10 min at 4 °C. These processes were repeated two times (Kaewprachu et al., 2016a). Finally, FMP was subjected to freeze drying, packed in plastic bag with zipper and kept at −20 °C until use.
2.4.6. Moisture content, degree of swelling, and film solubility Moisture content of FMP films was analyzed following the methods of AOAC (Association of Official Analytical Chemists, 2000). The difference between the initial and final weighing was used to evaluate moisture content. The swelling of films was examined according to the method presented by Mayachiew, Devahastin, Mackey, and Niranjan (2010) with a slight modification. A film sample was cut into 2 cm wide and 2 cm long and then dried at 105 °C for 24 h in an oven (Advantec, Electric Drying Oven, model DRA430DA, Toyo Seisakusho Kaisha Ltd., Chuo-ku, Tokyo, Japan). After, the film was weighed, left in 30 ml of distilled water at 25 °C for 24 h, and then blotted with a filter paper. The mass of the swollen film was weighed and recorded. The degree of swelling was calculated using the following equation:
2.3. Preparation of FMP films with MTGase FMP was added with the distilled water to obtain the final protein concentrations of 1% (w/v). The mixtures were homogenized at 11,000 rpm for 1 min, and then the pH of the mixtures was adjusted to 11 by using 1 N NaOH. After, the solutions were centrifuged at 3000 × g for 10 min at room temperature. The obtained supernatants was mixed with glycerol (25% w/w, protein content) and referred to as the film-forming solution (FFS; Kaewprachu et al., 2016b). The FFS was subjected to an additional 30 min of stirring at room temperature. After stirring, the different concentrations (0, 1, 2, 3, and 4% w/w, based on protein content) of MTGase were added into the FFS. Then, the mixtures were stirred for 30 min at room temperature. The FFS was used for film casting by adding 4 g of FFS onto a rimmed silicone resin plate (50 × 50 mm), evaporated, and dried in a ventilated oven environmental chamber (EYELA, Environmental Chamber, model KCL-2000A, Tokyo Rikakikai Co., Ltd., Chuo-ku, Tokyo, Japan) at 25 ± 0.5 °C and 50 ± 5% relative humidity (RH) for 24 h. Finally, the dry film was peeled and its properties were determined.
Degree of swelling = [(mf − mi)/mi] × 100
(1)
where mf and mi are the swollen film and the mass of dried film, respectively. The films’ solubility was analyzed as described by Sai-Ut, Benjakul, and Rawdkuen (2014) and calculated by the weight of the dry matter of un-dissolved debris subtracted from the initial weight of the dry matter. The films’ solubility was expressed as the percentage of total weight. Experiments were repeated three times.
2.4. Determination of film properties
2.4.7. Light transmission and transparency of the film A UV spectrophotometer (UV-1800, Shimadzu Co., Kyoto, Japan) was used for measuring the light transmission of the films. A film sample was cut to a rectangular shape (40 × 40 mm). Measurement was performed at the wavelength between 200 and 800 nm. A film sample was cut to a rectangular shape (40 × 40 mm) and measured at wavelength of 600 nm using a spectrophotometer. The
2.4.1. Film thickness Film thickness was determined by using a hand-held micrometer (Bial Pipe Gauge, Peacock Co., Tokyo, Japan). The film samples were randomly measured at six locations around the film. Experiments were repeated ten times. 92
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3.2. Mechanical properties
transparency value was calculated (Han & Floros, 1997): Transparency = −log T600/x
(2)
FMP films treated with MTGase at various levels showed significant differences in both tensile strength (TS) and elongation at break (EAB) (P < 0.05) (Table 1). The TS and EAB of the developing films were clearly affected by the incorporation of MTGase. This is illustrated by the slightly increased TS value (7.16–13.10 MPa) and by the slightly decreased EAB value (167.49–85.61%) when MTGase content was increased from 0 to 4% (w/w). The FMP film treated with 4% MTGase exhibited the highest TS value (13.10 MPa) while the control film had the lowest (7.16 MPa). Tang, Jiang, Wen, and Yang (2005) reported that covalent cross-linking between protein chains could provide films’ strength by increasing the TS value of the film. MTGase can form strong inter-molecular cross-links with protein molecules, which increases TS. This result is consistent with Rostamzad et al. (2016) who reported that the use of MTGase improved the TS of FMP nanocomposite film. They reported a TS increase of about 62.3%. These results are also in accordance with jellyfish protein film added with MTGase (Lee et al., 2015), where the TS value increased with increasing concentrations of MTGase from 5 to 15 units per gram of protein. The EAB value of FMP films decreased as the MTGase level increased (P < 0.05). This is also consistent with Weng and Zheng (2015) who reported that the EAB value of the gelatin-soy protein isolate film reduced with increasing the concentration of MTGase. Tang et al. (2005) suggested that MTGase could improve the film’s stuctures of soy protein isolate by increasing the compactness, but lowering the elasticity of film structures, resulting in low EAB if compared to an untreated film. The EAB value was affected by the inter-molecular chain mobility of the film. The addition of MTGase could reduce chain mobility of films by lowering the EAB values (Weng & Zheng, 2015). In general, the highest TS values are accompanied by lower EAB values for protein based films treated by other modification techniques. This is true for gelatin film treated with glutaradehyde or genipin (Amadori et al., 2015), soy protein isolate/ starch film treated with γ-irradiation (Xu et al., 2012), and fish myofibrillar protein film cross-linked by phenolic compounds (Prodpran et al., 2012). Notably, the TS of FMP film treated with 3% (w/w) MTGase is comparable to LDPE film, while film treated with 4% (w/w) MTGase showed greater TS over the LDPE film by around 13%. However, the EAB value of FMP films were still about 4 to 7 times less flexibile than LDPE film. Khoramnejadian, Zavareh, and Khoramnejadian (2013) reported that LDPE film had the TS and EAB values at 2.83 MPa and 286.85%, respectively. In general, LDPE film has relatively low TS at a range of 4.1–15.9 MPa, and they had a wide range of EAB values (90–800%). The mechanical properties of film are an important factor because they have significant implications for food film application. The film requires different mechanical properties for different film applications and uses. For example, film wrap requires higher flexibility (EAB) than a generic plastic bag. However, the TS of film wrap also should also be considered because it has an essential role
where T600 is the fractional transmittance at 600 nm, and x is the film thickness (mm). 2.4.8. Protein pattern of the films The pattern of protein film was analyzed by SDS-PAGE according to the method described in Laemmli (1970) that used a 4% and 10% for stacking and running gels, respectively. A film sample was added with 5% SDS and blended for 1 min then stirred continuously at room temperature for 24 h, after it was centrifuged at 3000 × g for 10 min. The supernatant obtained was analyzed for SDS-PAGE. 2.4.9. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectroscopy analysis of films was performed as described in Kaewprachu et al. (2016a). Measurements were examined at room temperature (25 °C) in the range of 4000–650 cm−1 with 64 scans and 4 cm−1 resolution by using a FT-IR spectrometer (Nicolet 6700, Thermo Scientific Inc., Waltham, MA, USA). 2.5. Statistical analysis Analysis of variance (ANOVA) was used for statistical analysis and the differences between means were carried out by DMRT. SPSS package (SPSS Inc., Chicago, IL, USA) was used as a tool for statistical analysis. 3. Results and discussion 3.1. Film thickness The thickness of FMP films treated with MTGase compared to control LDPE films are shown in Table 1. The thickness of the FMP films showed to be in the range of 0.010–0.013 mm. Film treated with 4% (w/w) MTGase showed greater thickness compared to other concentrations of MTGase (P < 0.05). This is because MTGase could form a compact film network with the FMP molecule, which resulted in increased thickness. Rostamzad et al. (2016) reported that the thickness of FMP films treated with MTGase (0–3% based on protein) showed to be in the range of 0.02–0.06 mm. Porta, Di Pierro, Rossi-Marquez, Mariniello, Kadivar, and Arabestani (2015) also reported that the thickness of bitter vetch (Vicia ervilia) protein film treated with MTGase (0–0.2 units per milligram) increased from 79.50 to 105.20 μm. As compared with the control LDPE film, the FMP film showed a greater thickness value of 10 to 25%. In general, thickness had effects for the films’ mechanical, water barrier, and optical properties.
Table 1 Thickness, mechanical properties, water vapor permeability, and thermal properties of FMP films treated with different concentrations of MTGase in comparison with LDPE. MTGase (%, w/w)
0 1 2 3 4 LDPE
Thickness (mm)
0.010 0.011 0.012 0.012 0.013 0.010
± ± ± ± ± ±
Tensile strength (MPa)
0.0009bc 0.0014b 0.0005ab 0.0012ab 0.0028a 0.0005c
7.16 ± 0.39e 8.73 ± 0.46d 9.68 ± 0.32c 11.08 ± 0.47b 13.10 ± 0.45a 11.40 ± 0.18b
Elongation (%)
WVP (10−9 g m−1 s−1 Pa−1)
167.49 ± 7.31b 143.66 ± 3.60c 132.76 ± 4.87c 107.15 ± 3.88d 85.61 ± 4.21e 622.67 ± 22.03a
2.38 2.19 2.11 2.09 2.02 0.04
± ± ± ± ± ±
0.05a 0.10b 0.04bc 0.12bc 0.06c 0.02d
Thermal properties Tm (°C)
ΔH (J/g)
93.83 ± 0.15d 96.44 ± 0.66d 102.59 ± 3.13c 105.06 ± 0.72c 112.69 ± 3.69b 122.54 ± 0.02a
2.20 ± 2.53b 2.57 ± 0.71b 4.68 ± 0.89b 5.86 ± 1.46b 5.92 ± 0.28b 105.08 ± 4.47a
Different superscripts in each column are significantly difference (P < 0.05). Values are given as mean ± SD from n = 10 determination for thickness; n = 5 for determinations of tensile strength and elongation; n = 3 for determinations of WVP and thermal properties. MTGase: microbial transglutaminase; LDPE: low density polyethylene.
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Fig. 1. Surface morphology of FMP films treated with different concentrations of MTGase in comparison with LDPE. The numbers designate of MTGase concentrations (%, w/w based on protein content).
increased from 0 to 4% (w/w). This is related to the film transparency. Due to the resulting desirable appearance, FMP films have attractiveness for the packaging application, especially for food products. Consumers are more confident about the packaged product because they can see the packaged product through the clearly transparent film. The color attributes of FMP film treated with MTGase at various concentrations as compared to the control LDPE films are shown in Fig. 1. The color parameters (a* and b*) of the films were affected by the MTGase levels (P < 0.05). The reduction in L* value with the coincidental increases in b* value were found in the films treated with 1 to 4% (w/w) MTGase when compared to the control film. However, no significant differences were found in the L* value when compared with the control film (P > 0.05). Yi, Kim, Bae, Whiteside, and Park (2006) found that the L* value of gelatin film treated with MTGase decreased, and the b* value of films increased due to the MTGase reaction.
in protecting the packaged food products from mechanical damage during processing, transportation, as well as distribution of the packaged food to the consumer. 3.3. Film appearance and color The appearances of FMP films treated with MTGase comparison to the control LDPE film are shown in Fig. 1. All of the film specimens were homogeneous, slightly yellowish, and had smooth surfaces. From visual aspect, the film samples showed no significant difference between the films appearances when they were placed on the background containing the letters because of all the letters were clearly observed when observed through the films. However, the FMP film showed slightly more turbidity than the control film. Furthermore, the turbidity became more pronounced as the concentration of MTGase 94
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lysine bonds. The strong film network required higher energy for disrupting the inter-chain interactions. Higher thermal stability of the resulting films correlated with higher mechanical properties of the resulting films, as elucidated by the increased TS of the resulting films (Table 1). Wang et al. (2015) reported that the Tm of gelatin-calcium carbonate film, which was cross-linked by MTGase addition, increased from 69.8 to 75.0 °C when the concentration of MTGase increased from 2 to 8 units per gram. They suggested that the protein strands were catalyzed by MTGase, leading to an aggregation of high molecularweight proteins with formed compact structures, which required high energy to disrupt the inter-chain bonds. For the control LDPE film, Tm was observed at around 122.54 °C with an ΔH of 105.08 J/g. Khoramnejadian et al. (2013) and Sabetzadeh, Bagheri, and Masoomi (2015) reported that the Tm of LDPE film presented at around 111.17 and 110.90 °C, respectively.
Tongnuanchan, Benjakul, and Prodpran (2011) reported that red tilapia muscle film prepared in alkaline conditions had a higher b* value than the film prepared in acidic conditions. This might be related to Maillard reaction. The discoloration of this film condition is mostly occurred from lipid oxidation due to the carbonyl groups involved in a Maillard reaction, resulting in more yellowness (Tongnuanchan et al., 2011). Rostamzad et al. (2016) also reported that increases in a* and b* values of FMP nanocomposite film were pronounced as the MTGase level increased from 0 to 3% (based on protein). As compared with the control LDPE film, the experimental films had an L* value closer to the control, while b* value showed to be higher. Therefore, it can be said that FMP films modified with MTGase has a potential as application for food packaging due to their colorlessness when compared to other protein based films. 3.4. Water vapor permeability
3.6. Moisture content, degree of swelling, and film solubility The WVP of the MTGase-treated FMP films were in a range of 2.02–2.38 × 10−9 g m−1 s−1 Pa−1. The control film showed the highest WVP (2.38 × 10−9 g m−1 s−1 Pa−1), followed by the treated film with 1–4% (w/w) MTGase (2.02–2.19 × 10−9 g m−1 s−1 Pa−1), and then the LDPE film (0.04 × 10−9 g m−1 s−1 Pa−1), respectively (Table 1). Slight decreases were observed when MTGase content increased from 1 to 4% (w/w) (P < 0.05). This suggests that the reduction of free volume of the FMP film matrix induced by the addition of MTGase led to a higher degree of cross-linking in the protein structure; therefore, the permeability decreased in the resulting film (De Carvalho & Grosso, 2004). This is consistent with Weng and Zheng (2015) who reported that the WVP of tilapia scale gelatin films decreased from 1.80 to 1.53 × 10−10 g m−1 s−1 Pa−1 with increasing the concentration of MTGase (0–30%), also because of increased crosslinking in the protein structure. WVP of MTGase-induced gelatincalcium carbonate film was reduced when the concentrations of MTGase reached up to 4–8 units per gram (Wang et al., 2015). The result showed that the formation of film network with a high molecular weight induced by MTGase caused the reduction of WVP in the treated films (Wang et al., 2015). In addition, the permeability of films depended on the degree of cross-linking, molecular mass, the chemical nature of macromolecule, crystallinity, and orientation (De Carvalho & Grosso, 2004). The utilization of the film with low WVP could improve the moisture transfer by lowering the transfer of moisture between the food and the surrounding atmosphere. However, the selection of food packaging applications depends on the moisture content of packaged food and the relative humidity of surrounding atmosphere. From this study, it can be suggested that the FMP films would be suitable for fresh fruit and vegetable, meat based products, but it has limitation to apply on dry food.
The moisture content of the FMP film treated with MTGase at various levels is presented in Fig. 2a. The resulting films showed a moisture content ranging from 23.30–27.84%. Furthermore, gradually decreasing moisture content was found when MTGase level increased from 0 to 4% (w/w) (P < 0.05). This indicates that the cross-linking induced by MTGase could decrease moisture uptake by proteins, resulting in an increase of hydrophobic groups in FMP (Tang et al., 2005). Jiang and Tang (2013) reported that the reduction in moisture content of gelatin films from 21.2 to 19.3% was observed when 4 units per gram of MTGase were incorporated. In contrast, Masamba, Li, Hategekimana, Zehadi, Ma, and Zhong (2016) who reported that moisture content of a zein film decreased when 0.5% MTGase was added, but it gradually increased when raising the concentration of MTGase to 1.0%. The addition of MTGase at low concentrations may cause the film to form a more compact network due to the effective covalent cross-links. On the other hand, the incorporation of MTGase, especially at higher concentrations, may induce aggregation, which leads to the formation of less compact and inhomogeneous film networks (Masamba et al., 2016). As a result, more water would absorb in the film treated with MTGase at high concentrations. The swelling of FMP film treated with MTGase compared to the LDPE film is presented in Fig. 2b. The degree of swelling of film is an important property to determine the film’s water resistance. Polymer networks are generally un-dissolved in a liquid media, but they swell in contact with water. The interaction between the liquid and the polymer can be indicated as the degree of swelling. The structure and the properties of the solvent and the polymer typically affect the degree of swelling. In general, a higher degree of crosslinking can decrease the polymer’s capacity to swell of polymer (Schmid et al., 2014). In this study, the degree of swelling of the resulting films was in the range of 41.37–110.22%, while swelling could not be detected in the LDPE film. The MTGase-treated films had a low degree of swelling compared to the control film, regardless of the concentration of MTGase used. The degree of swelling of FMP films decreased gradually (110.22–41.37%) with increasing MTGase concentration (P < 0.05). The addition of MTGase could reduce the degree of swelling of FMP film by around 62% compared to the control. The degree of swelling of MTGaseinduced whey protein isolate film decreased slightly at higher MTGase concentration (Schmid et al., 2014). This was due to the activity of MTGase accompanied by a higher degree of cross-linking caused by inducing the reduction of swelling capacity. Other cross-linking techniques such as chemical treatment for reducing swelling in biodegradable film have been studied. Cao, Fu, and He (2007) observed that the swelling of cross-linked gelatin films decreased by around 30.91% and 42.15% when increasing the concentrations of ferulic acid and tannic acid, respectively. Li, Gao, Wang, Zhang, and Tong (2013) also reported that the addition of glutaraldehyde (3.0 × 10−6–1.2 × 10−5 mol/g) induced a significant decrease in the degree of swelling of chitosan-
3.5. Differential scanning calorimetry The melting temperature (Tm) and enthalpy (ΔH) of FMP films treated with MTGase at various levels and LDPE film are shown in Table 1. Generally, this parameter indicates the mechanical properties of the films (strength and stiffness) and the temperature range in which the film can be used. The Tm and ΔH of the resulting films were in the range of 93.83–112.69 °C and 2.20–5.92 J/g, respectively. The lowest and highest Tm and ΔH were observed in the control film (Tm: 93.83 °C; ΔH: 2.20 J/g) and LDPE film (Tm: 122.54 °C; ΔH: 105.08 J/g), respectively. A slightly increase in melting temperature and enthalpy was more pronounced when MTGase content increased from 1 to 4% (w/w) (P < 0.05). This suggests that the enzymatic modification enhances the thermal stability of FMP film. The Tm of the films indicated that the temperature caused a disruption of the polymer interaction that was generated during film formation. The higher Tm and ΔH found in film treated with MTGase 4% (w/w) might be due to the greater inter-chain interaction of the MTGase treated film, most likely via ε-(γ-glutamyl) 95
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The film solubility of the FMP films treated with various concentrations of MTGase compared to the control LDPE film is shown in Fig. 2c. From the visual observation, the resulting films maintained their integrity after 24 h of immersion in water. The film solubility of the resulting films was in the range of 14.41–22.22%, while the LDPE film showed to be un-dissolved in water. The control film (without MTGase) had the highest film solubility (22.22%) compared to the MTGasetreated films, regardless of concentration used. Furthermore, the solubility of the FMP films decreased gradually (22.22 -14.41%) with increasing concentrations of MTGase (P < 0.05). Decreasing solubility of the treated film might be due to the stronger structure of the film network through covalent cross-linked proteins as evidenced by the increasing strength of the film (Table 1). This would be consistent with Weng and Zheng (2015) who reported that the film solubility of tilapia scale gelatin film decreased from 89.36 to 35.83% when the concentration of MTGase increased. Kolodziejska and Piotrowska (2007) observed a reduction in film solubility from 65 to 26% for fish gelatinchitosan film treated with MTGase. Masamba et al. (2016) also found that the reduction in film solubility (∼19–4.4%) was found when 1% MTGase was added in zein film. The increase in the degree of crosslinking by enzyme treatment causes the reduction of the low molecular weight fractions, resulting in a decrease in the solubility of the films (De Carvalho & Grosso, 2004). Based on these results, it can be said that the cross-linking induced by MTGase could decrease solubility of FMPbased films. For food packaging applications, higher water solubility is needed for dissolving the packaging before product consumption. It might be useful for encapsulating food or food additives. Moreover, the higher water solubility of the film is an eco-friendly packaging material, and it can be used to reduce or replace the synthetic polymer based packaging in order to minimize the environmental impact caused by the packaging waste. 3.7. Light transmission and transparency Transmission of UV and visible light at selected wavelengths of 200–800 nm, as well as the transparency of the films, are shown in Table 2. The light transmission in the UV ranges was 0.00–87.37%, while the transmission in the visible range was 57.30–92.23%. The transmission of UV and visible light decreased with increased concentrations of MTGase. The transmission of UV light was very low at 200–280 nm for FMP films due to they contain high amount of aromatic amino acids, which have ability to absorb UV light (Hamaguchi, WuYin, & Tanaka, 2007). This result suggests that MTGase-treated film could prevent UV transmission and therefore could reduce the food deterioration, especially the lipid oxidation that is induced by UV light. Schmid et al. (2014) also found that the light transmission rate of MTGase-induced whey protein isolate film decreased when increasing the MTGase concentration. A lowered visibility of light transmission was observed when MTGase level increased. This suggests that the cross-linking, or aggregation induced by MTGase, especially at higher amounts, resulted in limiting the light transmission of the film at the visible ranges. According to these results, the light transmission of FMP film depended on the amount of MTGase in the film matrix, and the interaction between FMP molecules was mediated by the addition of MTGase. The transparency value of the resulting films was in the range of 3.76–3.90 while the transparency value of LDPE film was 3.97 (Table 2). All films showed significant differences in film transparency when different amounts of MTGase were added (P < 0.05). The transparency value of MTGase-treated FMP film decreased when increasing the MTGase concentration (P < 0.05). Tang et al. (2005) suggested that the addition of MTGase induced protein aggregation or the cross-linking and makes the FFS become more turbid, which could be affected the transparency by lowering the transparency value of the films. This result is in contrast with Rostamzad et al. (2016) who reported that MTGase concentration had no significant effect on the
Fig. 2. Moisture content (a), degree of swelling (b) and film solubility (c) of FMP films treated with different concentrations of MTGase in comparison with LDPE. The numbers designate of MTGase concentrations (%, w/w based on protein content). Bars represent the standard deviation (n = 3). Different letters indicate the significant difference (P < 0.05).
starch composite film from 66.31 to 1.48%. Swelling has critical implication for food packaging. The degree of swelling is an important property when the film directly comes into contact with food surfaces. The degree of swelling should be as low as possible in order to protect the product against damages and against more water absorption that favor microbial growth. 96
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Table 2 Light transmittance and transparency of FMP films treated with different concentrations of MTGase in comparison with LDPE. MTGase (%, w/w)
0 1 2 3 4 LDPE
Transmittance (%T) at wavelength (nm) 200 280 350
400
500
600
700
800
0.00 0.00 0.00 0.00 0.00 73.60
82.50 74.13 69.23 66.00 61.60 89.77
85.13 79.10 75.83 73.33 70.93 90.30
86.7 82.00 80.03 78.07 75.00 90.77
87.27 83.37 82.33 80.37 77.80 91.60
87.90 84.43 84.03 81.90 79.80 92.23
9.83 8.43 6.60 4.97 2.47 87.37
79.47 69.33 63.70 60.00 57.30 88.57
Transparency
3.90 3.87 3.86 3.81 3.76 3.97
± ± ± ± ± ±
0.0005b 0.0006c 0.0003d 0.0016e 0.0001f 0.0031a
Different superscripts in each column are significantly difference (P < 0.05). Values are given as mean ± SD from triplicate determinations. MTGase: microbial transglutaminase; LDPE: low density polyethylene.
transparency of FMP nanocomposite film. The most critical property for film applications, in term of visual aspect, is transparency, which is important in order to enhance product appearance. Therefore, MTGasetreated FMP film had an effect on the optical properties of the resulting films.
in the presence of the enzyme. The decrease in MHC band intensity coincided with the increase in TS of the films (Table 1). The protein aggregation via the covalent bond had a direct result on the development of a strong film network. 3.9. FT-IR
3.8. Protein patterns
The FT-IR spectrum of FMP film treated with MTGase at various concentrations compared to the control LDPE film is presented in Fig. 4. FT-IR spectroscopy can be used for monitoring the functional groups and interactions of FMP in the films. Slight changes in wavenumber are generally observed at each absorption peak in the resulting films when the film was treated with different MTGase levels. If the FT-IR spectra of the resulting film were similar to that of the control film, it suggests that there was no significant modification of the secondary structure of fish myofibrillar protein following MTGase action. FMP films displayed major bands at 3286 cm−1 (amide-A, representing NeH and/or OeH stretching), 2927 cm−1 (amide-B, representing CeH stretching), 1652 cm−1 (amide-I, represents C]O stretching), and 1544 cm−1 (amide-II, arises from angular deformation of NeH and CeN stretching) (Kaewprachu et al., 2016a). The resulting films showed the peak at wavenumber 1045 cm−1, which was associated with the interactions between the plasticizer (OH-group of glycerol) and the film structure. According to the spectra, all films treated with different MTGase levels showed no change in the vibrational wavenumber for amide-I and amide-II peaks, except for amide-A and amide-B. The wavenumber of amide-A peaks shifted from 3286.23 cm−1 for the FMP film (without MTGase) to 3278.88, 3286.13, 3278.50, and 3281.66 cm−1 for the films treated with different concentrations (1–4% w/w) of MTGase, respectively. The amide-A peak of MTGase-treated films shifted to a lower wavenumber, probably due to fewer formations of hydrogen bonding, which may suggest that the enzymatic cross-linking by MTGase might induce aggregation via the NH-domain of the peptides. Furthermore, the wavenumber of amide-B peaks shifted from 2927.78 cm−1 for the FMP film (without MTGase) to 2925.64, 2921.00, 2922.22 and 2919.77 cm−1 for the films treated with different concentrations (1–4% w/w) of MTGase, respectively. Eissa, Puhl, Kadla, and Khan (2006) reported that amide-B of β-lactoglobulin after crosslinking with MTGase shifted slightly due to changes in the CH2 asymmetrical stretching mode, and due to the reaction of the protein through the enzymatic catalysis. Lower amounts of the −NH2 or −NH3+ group obtained from enzymatic cross-linking more likely results in the lower wavenumber of the amide-A and amide-B peaks obtained (Hoque, Benjakul, & Prodpran, 2011). The addition of MTGase could induce the change in the functional group and also the conformation of proteins as indicated by the changes in the FT-IR spectra, especially for amide-A and amide-B. For the spectra of the control LDPE film, the major absorption bands were found at around 2918 cm−1 (CH2 asymmetric stretching), 2850 cm−1 (CH2 symmetric stretching), 1471 cm−1 (blending deformation) and 717 cm−1 (rocking deformation). Gulmine, Janissek, Heise, and Akcelrud (2002) also reported that
Protein patterns of FMP film in the presence of different concentrations of MTGase under reducing conditions (presence of β-mercaptoethanol) are shown in Fig. 3. The major proteins in FMP were included myosin heavy chain (MHC), actin, tropomyosin, and troponin. Decreases in the MHC band intensity were observed when MTGase was added, when compared to that observed in the control film. Furthermore, the MHC band intensity decreased gradually when increasing the MTGase level (lane 1 to 4). The decrease in the MHC band intensity of the FMP film treated with MTGase was most likely due to the formation of inter-molecular cross-linking via non-disulfide covalent bond induced by MTGase (Prodpran et al., 2012). These results indicate that FMP films underwent cross-linking induced by MTGase. Al-Saadi, Shaker, and Ustunol (2014) reported that the formation of new polymers was observed when a goat milk protein film was treated with MTGase. This was evidenced by the disappearance of lower molecular protein bands with the concomitant appearance of higher molecular weight bands resulting from cross-linked proteins. Porta et al. (2015) reported that bitter vetch (Vicia ervilia) protein film contained glutamine and lysine proteins that were susceptible to cross-linking polymers
Fig. 3. Protein pattern of FMP films treated with different concentrations of MTGase under reducing conditions (20 μg protein, 10% separating gel). M: protein marker; MHC: myosin heavy chain. The numbers designate of MTGase concentrations (%, w/w based on protein content).
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Fig. 4. FT-IR spectra of FMP films treated with different concentrations of MTGase. The numbers designate of MTGase concentrations (%, w/w based on protein content).
the main absorption peaks of LDPE film were found at the wavenumbers of 2919, 2851, 1473, 1463, and 731–720 cm−1.
Philadelphia, PA, USA: ASTM D882-97. AOAC-Association of Official Analytical Chemists (2000). Official methods of analysis of the association of analytical chemists international(17). Gaithersburg MD: AOAC. Cao, N., Fu, Y., & He, J. (2007). Mechanical properties of gelatin films cross-linked, respectively: By ferulic acid and tannin acid. Food Hydrocolloids, 21, 575–584. 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. Eissa, A. S., Puhl, C., Kadla, J. F., & Khan, S. A. (2006). Enzymatic cross-linking of βlactoglobulin: conformational properties using FTIR spectroscopy. Biomacromolecules, 7, 1707–1713. Gulmine, J. V., Janissek, P. R., Heise, H. M., & Akcelrud, L. (2002). Polyethylene characterization by FTIR. Polymer Testing, 21, 557–563. Hamaguchi, P. Y., WuYin, W., & Tanaka, M. (2007). Effect of pH on the formation of edible films made from the muscle proteins of Blue marlin (Makaira mazara). Food Chemistry, 100, 914–920. Han, J., & Floros, J. (1997). Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity. Journal of Plastic Film and Sheeting, 13, 287–298. Hoque, M. S., Benjakul, S., & Prodpran, T. (2011). Properties of film from cuttlefish (Sepia pharaonis) skin gelatin incorporated with cinnamon: clove and star anise extracts. Food Hydrocolloids, 25, 1085–1097. Iwata, K. I., Ishizaki, S. H., Handa, A. K., & Tanaka, M. U. (2000). Preparation and characterization of edible films from fish water-soluble proteins. Fisheries Science, 66, 372–378. Jiang, Y., & Tang, C. H. (2013). Effects of transglutaminase on sorption: mechanical and moisture-related properties of gelatin films. Food Science and Technology International, 19, 99–108. Kaewprachu, P., Osako, K., Benjakul, S., & Rawdkuen, S. (2016a). Effect of protein concentrations on the properties of fish myofibrillar protein based film compared with PVC film. Journal of Food Science and Technology, 53, 2083–2091. Kaewprachu, P., Osako, K., Benjakul, S., Tongdeesoontorn, W., & Rawdkuen, S. (2016b). Biodegradable protein-based films and their properties: a comparative study. Packaging Technology and Science, 29, 77–90. 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-dimethyl aminopropyl) carbodiimide (EDC) and plasticized with glycerol. Food Chemistry, 103, 295–300. Khoramnejadian, S., Zavareh, J. J., & Khoramnejadian, S. (2013). Effect of potato starch on thermal & mechanical properties of low density polyethylene. Current World Environment, 8, 215–220. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Lee, J.-H., Lee, J.-H., Yang, H.-J., Won, M., & Song, K. B. (2015). Characterisation of jellyfish protein films with added transglutaminase and wasabi extract. International Journal of Food Science & Technology, 50, 1683–1689. Li, H., Gao, X., Wang, Y., Zhang, X., & Tong, Z. (2013). Comparison of chitosan/starch composite film properties before and after cross-linking. International Journal of Biological Macromolecules, 52, 275–279.
4. Conclusion MTGase effectively induced protein cross-linking of FMP molecules as indicated by the properties of the FMP films obtained. The addition of MTGase induced the interaction of proteins to be in compact form, which includes significantly increased TS and decreased EAB, decreased film solubility, lower degree of swelling, and decreased transparency. Improvements in the water barrier properties, UV absorption, and thermal stability of FMP film due to protein cross-linking were clearly observed. Addition of MTGase was a good alternative method for improving the properties of FMP film as indicated in almost all parameters tested. However, the resulting films showed to be poor in EAB when compared to LDPE film, so in this regard, further development is still necessary. Acknowledgements Mae Fah Luang University and the Thailand Research Fund were appreciated for the financial support to Ms. Pimonpan Kaewprachu through the Royal Golden Jubilee Ph.D. Program (Grant NO. PHD/ 0029/2555). Tokyo University of Marine Science and Technology was also acknowledged for materials, reagents, and equipment during research work. Thank you to Matthew Robert Ferguson for language editing support. References Al-Saadi, J. S., Shaker, K. A., & Ustunol, Z. (2014). Effect of heat and transglutaminase on solubility of goat milk protein-based films. International Journal of Dairy Technology, 67, 420–426. Amadori, S., Torricelli, P., Rubini, K., Fini, M., Panzavolta, S., & Bigi, A. (2015). Effect of sterilization and crosslinking on gelatin films. Journal of Materials Science: Materials in Medicine, 26, 69. American Society for Testing and Materials (1989). Annual book of ASTM standards. Philadelphia, PA, USA: ASTM. American Society for Testing and Materials (1999). Annual book of ASTM standards.
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gelatin film incorporated with longan seed extract compared with BHT. Journal of Food Science and Technology, 52, 5842–5849. Schmid, M., Sängerlaub, S., Wege, L., & Stäbler, A. (2014). Properties of transglutaminase crosslinked whey protein isolate coatings and cast films. Packaging Technology and Science, 27, 799–817. Tang, C.-H., Jiang, Y., Wen, Q.-B., & Yang, X.-Q. (2005). Effect of transglutaminase treatment on the properties of cast films of soy protein isolates. Journal of Biotechnology, 120, 296–307. Tongnuanchan, P., Benjakul, S., & Prodpran, T. (2011). Roles of lipid oxidation and pH on properties and yellow discolouration during storage of film from red tilapia (Oreochromis niloticus) muscle protein. Food Hydrocolloids, 25, 426–433. Wang, Y., Liu, A., Ye, R., Wang, W., & Li, X. (2015). Transglutaminase-induced crosslinking of gelatin-calcium carbonate composite films. Food Chemistry, 166, 414–422. 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. Xu, W., Liu, B., Yang, H., Liu, K., Jia, S., & Chen, F. (2012). Effect of γ-irradiation on the physicochemical properties of mixed soy protein isolate/starch material. African Journal of Biotechnology, 11, 7238–7246. 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, E376–E383.
Masamba, K., Li, Y., Hategekimana, J., Zehadi, M., Ma, J., & Zhong, F. (2016). Evaluation of mechanical and water barrier properties of transglutaminase cross-linked zein films incorporated with oleic acid. International Journal of Food Science & Technology, 51, 1159–1167. Mayachiew, P., Devahastin, S., Mackey, B. M., & Niranjan, K. (2010). Effects of drying methods and conditions on antimicrobial activity of edible chitosan films enriched with galangal extract. Food Research International, 43, 125–132. Nielsen, P. M. (1995). Reactions and potential industrial applications of transglutaminase: Review of literature and patents. Food Biotechnology, 9, 119–156. Porta, R., Di Pierro, P., Rossi-Marquez, G., Mariniello, L., Kadivar, M., & Arabestani, A. (2015). Microstructure and properties of bitter vetch (Vicia ervilia) protein films reinforced by microbial transglutaminase. Food Hydrocolloids, 50, 102–107. Prodpran, T., Benjakul, S., & Phatcharat, S. (2012). Effect of phenolic compounds on protein cross-linking and properties of film from fish myofibrillar protein. International Journal of Biological Macromolecules, 51, 774–782. Rostamzad, H., Paighambari, S. Y., Shabanpour, B., Ojagh, S. M., & Mousavi, S. M. (2016). Improvement of fish protein film with nanoclay and transglutaminase for food packaging. Food Packaging and Shelf Life, 7, 1–7. Sabetzadeh, M., Bagheri, R., & Masoomi, M. (2015). Study on ternary low density polyethylene/linear low density polyethylene/thermoplastic starch blend films. Carbohydrate Polymers, 119, 126–133. Sai-Ut, S., Benjakul, S., & Rawdkuen, S. (2014). Retardation of lipid oxidation using
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The effects of microbial transglutaminase on the properties of fish myofibrillar protein film
MARK
⁎
Pimonpan Kaewprachua, Kazufumi Osakob, Wirongrong Tongdeesoontornc, , Saroat Rawdkuena a b c
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan Technology Management of Agricultural Produce and Packaging Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords: Biodegradable Fish myofibrillar protein Microbial transglutaminase Protein based films Low density polyethylene
In this study, the effects of microbial transglutaminase (MTGase) on the properties of fish myofibrillar protein (FMP) films were investigated. As MTGase content increased, the thickness and tensile strength of the FMP films increased. In contrast, their elongation at break (167.49–85.61%), film transparency, water vapor permeability (2.38–2.02 × 10−9 g m−1 s−1 Pa−1), moisture content, film solubility, and degree of swelling all decreased (P < 0.05). Lightness also decreased, and yellowness increased as MTGase content was increased (P < 0.05). MTGase action also significantly improved barrier properties and thermal stability of films. Electrophoretic and FT-IR studies revealed that cross-linking and conformational changes were pronounced in film treated with MTGase. Based on these results, the addition of MTGase produced a good alternative method for improving FMP film properties; however, the mechanical and water barrier properties of the resulting films need further development.
1. Introduction Pollution by non- or slowly-degradable packaging material waste is an increasing environmental concern. In this context, the packaging materials from natural resources have ability to reduce or replace the commercial synthetic packaging films. Biodegradable films can be made from natural resources including carbohydrates, proteins, and lipids. Among these materials, proteins are extensively used as the filmforming materials due to their film-forming ability, abundance, high nutritional value, and biodegradability (Kaewprachu, Osako, Benjakul, Tongdeesoontorn, & Rawdkuen, 2016b). Fish myofibrillar proteins have been used frequently as a polymer for film-forming (Kaewprachu, Osako, Benjakul, & Rawdkuen, 2016a; Kaewprachu et al., 2016b; Prodpran, Benjakul, & Phatcharat, 2012; Rostamzad, Paighambari, Shabanpour, Ojagh, & Mousavi, 2016). Myofibrillar proteins are generally dissolved in the solution that provides the pH away from the isoelectric point (pI ≅ 5) of the proteins (Iwata, Ishizaki, Handa, & Tanaka, 2000). The dissociation and solubilization of myofibrillar proteins are then sufficient for expecting film formation. Previous experiments (Kaewprachu et al., 2016a, 2016b) showed that fish myofibrillar protein based films (FMP) had UV light barrier properties (200–280 nm) better than the synthetic wrap film (polyvinyl chloride; PVC). However, the relatively poor mechanical properties and
⁎
Corresponding author. E-mail address: [email protected] (W. Tongdeesoontorn).
http://dx.doi.org/10.1016/j.fpsl.2017.04.002 Received 2 January 2017; Received in revised form 30 March 2017; Accepted 4 April 2017 2214-2894/ © 2017 Published by Elsevier Ltd.
high water vapor permeability posed significant limitations for their applicability in food packaging. Therefore, protein film network modifications are required to enhance these apparent weaknesses. Various approaches have been used to enhance the protein based films’ properties such as the use of chemicals, irradiation, thermal, and enzymatic modification. There are many researches that studied the enhancement of protein based films via chemical modifications by glutaraldehyde or genipin (0.15, 0.30, and 0.67%, w/v) (Amadori et al., 2015), gamma-irradiation (0–50 kGy) (Xu et al., 2012), and enzymatic reticulation (0–8 units per gram of protein) (Wang, Liu, Ye, Wang, & Li, 2015). In recent years, enzymatic cross-linking treatment has been used extensively over chemical modifications because of the toxicity of chemical synthetic agents, which is clearly unsuitable for real food systems application. Microbial transglutaminase (MTGase; EC.2.3.2.13) is most commonly used for protein modification. MTGase is a protein-glutamine γglutamyltransferase, which can be catalyzed the formation of an isopeptide bond between the group of carboxyl amide (donor) of glutamine residues and the group of ε-amine (acyl acceptor) of lysine residues. These reactions can be induced the formation of the intra- and intermolecular ε-(γ-glutamyl) lysine covalent bonds via cross-linking process (Nielsen, 1995). The cross-linking are commonly induced by MTGase could improve the films’ mechanical and physical properties by
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2.4.2. Mechanical properties A film sample was cut into 2 cm wide and 5 cm long, and then conditioned at 50 ± 5% RH at 25 °C for 48 h prior to testing. Tensipresser (TTP-50BX II, Takemoto Electric Inc., Tokyo, Japan) was used to measure tensile strength and elongation at break according to the ASTM standard method D882–97 (American Society for Testing and Materials, 1999). The conditions of testing were 30 mm of initial grip separation with the cross-head speed at 1 mm/s. Measurement was performed until the films were broken.
increasing the tensile strength, but lowering the elongation at break and solubility of the protein film. In addition, the MTGase enzymatic activity and biochemical reactions are affected by the pre-treatment of the protein (denaturation), the pH of the film-forming solution, the reaction time, the temperature, as well as the concentration of MTGase (Schmid, Sängerlaub, Wege, & Stäbler, 2014). Particularly, the effect of concentrations of MTGase on the protein based films’ properties have been extensively studied on whey protein isolate (Schmid et al., 2014), gelatin-calcium carbonate (Wang et al., 2015), and jellyfish protein (Lee, Lee, Yang, Won, & Song, 2015). Nevertheless, information regarding the effect of MTGase on the protein based films properties, especially in FMP films, remains very scarce. Therefore, the aim of this study was to investigate the effect of MTGase on the mechanical, physical, chemical, barrier, and thermal properties of FMP based film against a commercial wrap control film (low density polyethylene; LDPE).
2.4.3. Film appearance and color After the films were dried and conditioned at 50 ± 5% RH at 25 °C for 48 h, the visual aspect of film samples were examined by using a digital camera (Fujifilm Finepix S4900; acquired from Fujifilm Thailand Co. Ltd., Bangkok, Thailand). Color Reader (CR-13, Konica Minolta, Inc., Japan) was used to determine color attributes of the film and expressed as L*, a*, and b*.
2. Materials and methods
2.4.4. Water vapor permeability (WVP) A modified ASTM standard method (American Society for Testing and Materials, 1989) was used to evaluate the WVP as described in Kaewprachu et al. (2016b). Measurements were conducted at 30 °C at 50 ± 5% RH, and recorded the weight gain of the cup at an hour interval to 8 h. Experiments were repeated three times and expressed as g m−1 s−1 Pa−1.
2.1. Materials Microbial transglutaminase (MTGase) (Activa TG-K: 100 activity units per gram) was supplied by Ajinomoto Co. Inc. (Tokyo, Japan). Coomassie Blue R-250 was purchased from Merck (Darmstadt, Germany). SDS was purchased from Wako (Osaka, Japan). Glycerol and other analytical grade reagents were obtained from Kokusan Chemical Co., Ltd. (Yokohama, Japan). Low density polyethylene wrap film (LDPE) of 10 μm thickness (Ube Film Company Ltd., Japan) was used as the commercial wrap film in this study.
2.4.5. Differential Scanning Calorimetry (DSC) A differential scanning calorimeter (DSC-50, Shimadzu Co., Kyoto, Japan) was used for examining the thermal properties of the films (Kaewprachu et al., 2016b). Each film sample (10–12 mg) contained in an aluminum DSC pan were heated in the temperature range of 25–180 °C at a heating rate of 10 °C/min in a nitrogen atmosphere (20 ml/min).
2.2. Preparation of fish myofibrillar protein (FMP) Firstly, minced fish (fresh tilapia; Orcochromis niloticus) was added with five volumes of 50 mM NaCl and then homogenized for 2 min at 11,000 rpm. The mixtures were centrifuged at 10,000 × g for 10 min at 4 °C. These processes were repeated two times (Kaewprachu et al., 2016a). Finally, FMP was subjected to freeze drying, packed in plastic bag with zipper and kept at −20 °C until use.
2.4.6. Moisture content, degree of swelling, and film solubility Moisture content of FMP films was analyzed following the methods of AOAC (Association of Official Analytical Chemists, 2000). The difference between the initial and final weighing was used to evaluate moisture content. The swelling of films was examined according to the method presented by Mayachiew, Devahastin, Mackey, and Niranjan (2010) with a slight modification. A film sample was cut into 2 cm wide and 2 cm long and then dried at 105 °C for 24 h in an oven (Advantec, Electric Drying Oven, model DRA430DA, Toyo Seisakusho Kaisha Ltd., Chuo-ku, Tokyo, Japan). After, the film was weighed, left in 30 ml of distilled water at 25 °C for 24 h, and then blotted with a filter paper. The mass of the swollen film was weighed and recorded. The degree of swelling was calculated using the following equation:
2.3. Preparation of FMP films with MTGase FMP was added with the distilled water to obtain the final protein concentrations of 1% (w/v). The mixtures were homogenized at 11,000 rpm for 1 min, and then the pH of the mixtures was adjusted to 11 by using 1 N NaOH. After, the solutions were centrifuged at 3000 × g for 10 min at room temperature. The obtained supernatants was mixed with glycerol (25% w/w, protein content) and referred to as the film-forming solution (FFS; Kaewprachu et al., 2016b). The FFS was subjected to an additional 30 min of stirring at room temperature. After stirring, the different concentrations (0, 1, 2, 3, and 4% w/w, based on protein content) of MTGase were added into the FFS. Then, the mixtures were stirred for 30 min at room temperature. The FFS was used for film casting by adding 4 g of FFS onto a rimmed silicone resin plate (50 × 50 mm), evaporated, and dried in a ventilated oven environmental chamber (EYELA, Environmental Chamber, model KCL-2000A, Tokyo Rikakikai Co., Ltd., Chuo-ku, Tokyo, Japan) at 25 ± 0.5 °C and 50 ± 5% relative humidity (RH) for 24 h. Finally, the dry film was peeled and its properties were determined.
Degree of swelling = [(mf − mi)/mi] × 100
(1)
where mf and mi are the swollen film and the mass of dried film, respectively. The films’ solubility was analyzed as described by Sai-Ut, Benjakul, and Rawdkuen (2014) and calculated by the weight of the dry matter of un-dissolved debris subtracted from the initial weight of the dry matter. The films’ solubility was expressed as the percentage of total weight. Experiments were repeated three times.
2.4. Determination of film properties
2.4.7. Light transmission and transparency of the film A UV spectrophotometer (UV-1800, Shimadzu Co., Kyoto, Japan) was used for measuring the light transmission of the films. A film sample was cut to a rectangular shape (40 × 40 mm). Measurement was performed at the wavelength between 200 and 800 nm. A film sample was cut to a rectangular shape (40 × 40 mm) and measured at wavelength of 600 nm using a spectrophotometer. The
2.4.1. Film thickness Film thickness was determined by using a hand-held micrometer (Bial Pipe Gauge, Peacock Co., Tokyo, Japan). The film samples were randomly measured at six locations around the film. Experiments were repeated ten times. 92
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3.2. Mechanical properties
transparency value was calculated (Han & Floros, 1997): Transparency = −log T600/x
(2)
FMP films treated with MTGase at various levels showed significant differences in both tensile strength (TS) and elongation at break (EAB) (P < 0.05) (Table 1). The TS and EAB of the developing films were clearly affected by the incorporation of MTGase. This is illustrated by the slightly increased TS value (7.16–13.10 MPa) and by the slightly decreased EAB value (167.49–85.61%) when MTGase content was increased from 0 to 4% (w/w). The FMP film treated with 4% MTGase exhibited the highest TS value (13.10 MPa) while the control film had the lowest (7.16 MPa). Tang, Jiang, Wen, and Yang (2005) reported that covalent cross-linking between protein chains could provide films’ strength by increasing the TS value of the film. MTGase can form strong inter-molecular cross-links with protein molecules, which increases TS. This result is consistent with Rostamzad et al. (2016) who reported that the use of MTGase improved the TS of FMP nanocomposite film. They reported a TS increase of about 62.3%. These results are also in accordance with jellyfish protein film added with MTGase (Lee et al., 2015), where the TS value increased with increasing concentrations of MTGase from 5 to 15 units per gram of protein. The EAB value of FMP films decreased as the MTGase level increased (P < 0.05). This is also consistent with Weng and Zheng (2015) who reported that the EAB value of the gelatin-soy protein isolate film reduced with increasing the concentration of MTGase. Tang et al. (2005) suggested that MTGase could improve the film’s stuctures of soy protein isolate by increasing the compactness, but lowering the elasticity of film structures, resulting in low EAB if compared to an untreated film. The EAB value was affected by the inter-molecular chain mobility of the film. The addition of MTGase could reduce chain mobility of films by lowering the EAB values (Weng & Zheng, 2015). In general, the highest TS values are accompanied by lower EAB values for protein based films treated by other modification techniques. This is true for gelatin film treated with glutaradehyde or genipin (Amadori et al., 2015), soy protein isolate/ starch film treated with γ-irradiation (Xu et al., 2012), and fish myofibrillar protein film cross-linked by phenolic compounds (Prodpran et al., 2012). Notably, the TS of FMP film treated with 3% (w/w) MTGase is comparable to LDPE film, while film treated with 4% (w/w) MTGase showed greater TS over the LDPE film by around 13%. However, the EAB value of FMP films were still about 4 to 7 times less flexibile than LDPE film. Khoramnejadian, Zavareh, and Khoramnejadian (2013) reported that LDPE film had the TS and EAB values at 2.83 MPa and 286.85%, respectively. In general, LDPE film has relatively low TS at a range of 4.1–15.9 MPa, and they had a wide range of EAB values (90–800%). The mechanical properties of film are an important factor because they have significant implications for food film application. The film requires different mechanical properties for different film applications and uses. For example, film wrap requires higher flexibility (EAB) than a generic plastic bag. However, the TS of film wrap also should also be considered because it has an essential role
where T600 is the fractional transmittance at 600 nm, and x is the film thickness (mm). 2.4.8. Protein pattern of the films The pattern of protein film was analyzed by SDS-PAGE according to the method described in Laemmli (1970) that used a 4% and 10% for stacking and running gels, respectively. A film sample was added with 5% SDS and blended for 1 min then stirred continuously at room temperature for 24 h, after it was centrifuged at 3000 × g for 10 min. The supernatant obtained was analyzed for SDS-PAGE. 2.4.9. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectroscopy analysis of films was performed as described in Kaewprachu et al. (2016a). Measurements were examined at room temperature (25 °C) in the range of 4000–650 cm−1 with 64 scans and 4 cm−1 resolution by using a FT-IR spectrometer (Nicolet 6700, Thermo Scientific Inc., Waltham, MA, USA). 2.5. Statistical analysis Analysis of variance (ANOVA) was used for statistical analysis and the differences between means were carried out by DMRT. SPSS package (SPSS Inc., Chicago, IL, USA) was used as a tool for statistical analysis. 3. Results and discussion 3.1. Film thickness The thickness of FMP films treated with MTGase compared to control LDPE films are shown in Table 1. The thickness of the FMP films showed to be in the range of 0.010–0.013 mm. Film treated with 4% (w/w) MTGase showed greater thickness compared to other concentrations of MTGase (P < 0.05). This is because MTGase could form a compact film network with the FMP molecule, which resulted in increased thickness. Rostamzad et al. (2016) reported that the thickness of FMP films treated with MTGase (0–3% based on protein) showed to be in the range of 0.02–0.06 mm. Porta, Di Pierro, Rossi-Marquez, Mariniello, Kadivar, and Arabestani (2015) also reported that the thickness of bitter vetch (Vicia ervilia) protein film treated with MTGase (0–0.2 units per milligram) increased from 79.50 to 105.20 μm. As compared with the control LDPE film, the FMP film showed a greater thickness value of 10 to 25%. In general, thickness had effects for the films’ mechanical, water barrier, and optical properties.
Table 1 Thickness, mechanical properties, water vapor permeability, and thermal properties of FMP films treated with different concentrations of MTGase in comparison with LDPE. MTGase (%, w/w)
0 1 2 3 4 LDPE
Thickness (mm)
0.010 0.011 0.012 0.012 0.013 0.010
± ± ± ± ± ±
Tensile strength (MPa)
0.0009bc 0.0014b 0.0005ab 0.0012ab 0.0028a 0.0005c
7.16 ± 0.39e 8.73 ± 0.46d 9.68 ± 0.32c 11.08 ± 0.47b 13.10 ± 0.45a 11.40 ± 0.18b
Elongation (%)
WVP (10−9 g m−1 s−1 Pa−1)
167.49 ± 7.31b 143.66 ± 3.60c 132.76 ± 4.87c 107.15 ± 3.88d 85.61 ± 4.21e 622.67 ± 22.03a
2.38 2.19 2.11 2.09 2.02 0.04
± ± ± ± ± ±
0.05a 0.10b 0.04bc 0.12bc 0.06c 0.02d
Thermal properties Tm (°C)
ΔH (J/g)
93.83 ± 0.15d 96.44 ± 0.66d 102.59 ± 3.13c 105.06 ± 0.72c 112.69 ± 3.69b 122.54 ± 0.02a
2.20 ± 2.53b 2.57 ± 0.71b 4.68 ± 0.89b 5.86 ± 1.46b 5.92 ± 0.28b 105.08 ± 4.47a
Different superscripts in each column are significantly difference (P < 0.05). Values are given as mean ± SD from n = 10 determination for thickness; n = 5 for determinations of tensile strength and elongation; n = 3 for determinations of WVP and thermal properties. MTGase: microbial transglutaminase; LDPE: low density polyethylene.
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Fig. 1. Surface morphology of FMP films treated with different concentrations of MTGase in comparison with LDPE. The numbers designate of MTGase concentrations (%, w/w based on protein content).
increased from 0 to 4% (w/w). This is related to the film transparency. Due to the resulting desirable appearance, FMP films have attractiveness for the packaging application, especially for food products. Consumers are more confident about the packaged product because they can see the packaged product through the clearly transparent film. The color attributes of FMP film treated with MTGase at various concentrations as compared to the control LDPE films are shown in Fig. 1. The color parameters (a* and b*) of the films were affected by the MTGase levels (P < 0.05). The reduction in L* value with the coincidental increases in b* value were found in the films treated with 1 to 4% (w/w) MTGase when compared to the control film. However, no significant differences were found in the L* value when compared with the control film (P > 0.05). Yi, Kim, Bae, Whiteside, and Park (2006) found that the L* value of gelatin film treated with MTGase decreased, and the b* value of films increased due to the MTGase reaction.
in protecting the packaged food products from mechanical damage during processing, transportation, as well as distribution of the packaged food to the consumer. 3.3. Film appearance and color The appearances of FMP films treated with MTGase comparison to the control LDPE film are shown in Fig. 1. All of the film specimens were homogeneous, slightly yellowish, and had smooth surfaces. From visual aspect, the film samples showed no significant difference between the films appearances when they were placed on the background containing the letters because of all the letters were clearly observed when observed through the films. However, the FMP film showed slightly more turbidity than the control film. Furthermore, the turbidity became more pronounced as the concentration of MTGase 94
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lysine bonds. The strong film network required higher energy for disrupting the inter-chain interactions. Higher thermal stability of the resulting films correlated with higher mechanical properties of the resulting films, as elucidated by the increased TS of the resulting films (Table 1). Wang et al. (2015) reported that the Tm of gelatin-calcium carbonate film, which was cross-linked by MTGase addition, increased from 69.8 to 75.0 °C when the concentration of MTGase increased from 2 to 8 units per gram. They suggested that the protein strands were catalyzed by MTGase, leading to an aggregation of high molecularweight proteins with formed compact structures, which required high energy to disrupt the inter-chain bonds. For the control LDPE film, Tm was observed at around 122.54 °C with an ΔH of 105.08 J/g. Khoramnejadian et al. (2013) and Sabetzadeh, Bagheri, and Masoomi (2015) reported that the Tm of LDPE film presented at around 111.17 and 110.90 °C, respectively.
Tongnuanchan, Benjakul, and Prodpran (2011) reported that red tilapia muscle film prepared in alkaline conditions had a higher b* value than the film prepared in acidic conditions. This might be related to Maillard reaction. The discoloration of this film condition is mostly occurred from lipid oxidation due to the carbonyl groups involved in a Maillard reaction, resulting in more yellowness (Tongnuanchan et al., 2011). Rostamzad et al. (2016) also reported that increases in a* and b* values of FMP nanocomposite film were pronounced as the MTGase level increased from 0 to 3% (based on protein). As compared with the control LDPE film, the experimental films had an L* value closer to the control, while b* value showed to be higher. Therefore, it can be said that FMP films modified with MTGase has a potential as application for food packaging due to their colorlessness when compared to other protein based films. 3.4. Water vapor permeability
3.6. Moisture content, degree of swelling, and film solubility The WVP of the MTGase-treated FMP films were in a range of 2.02–2.38 × 10−9 g m−1 s−1 Pa−1. The control film showed the highest WVP (2.38 × 10−9 g m−1 s−1 Pa−1), followed by the treated film with 1–4% (w/w) MTGase (2.02–2.19 × 10−9 g m−1 s−1 Pa−1), and then the LDPE film (0.04 × 10−9 g m−1 s−1 Pa−1), respectively (Table 1). Slight decreases were observed when MTGase content increased from 1 to 4% (w/w) (P < 0.05). This suggests that the reduction of free volume of the FMP film matrix induced by the addition of MTGase led to a higher degree of cross-linking in the protein structure; therefore, the permeability decreased in the resulting film (De Carvalho & Grosso, 2004). This is consistent with Weng and Zheng (2015) who reported that the WVP of tilapia scale gelatin films decreased from 1.80 to 1.53 × 10−10 g m−1 s−1 Pa−1 with increasing the concentration of MTGase (0–30%), also because of increased crosslinking in the protein structure. WVP of MTGase-induced gelatincalcium carbonate film was reduced when the concentrations of MTGase reached up to 4–8 units per gram (Wang et al., 2015). The result showed that the formation of film network with a high molecular weight induced by MTGase caused the reduction of WVP in the treated films (Wang et al., 2015). In addition, the permeability of films depended on the degree of cross-linking, molecular mass, the chemical nature of macromolecule, crystallinity, and orientation (De Carvalho & Grosso, 2004). The utilization of the film with low WVP could improve the moisture transfer by lowering the transfer of moisture between the food and the surrounding atmosphere. However, the selection of food packaging applications depends on the moisture content of packaged food and the relative humidity of surrounding atmosphere. From this study, it can be suggested that the FMP films would be suitable for fresh fruit and vegetable, meat based products, but it has limitation to apply on dry food.
The moisture content of the FMP film treated with MTGase at various levels is presented in Fig. 2a. The resulting films showed a moisture content ranging from 23.30–27.84%. Furthermore, gradually decreasing moisture content was found when MTGase level increased from 0 to 4% (w/w) (P < 0.05). This indicates that the cross-linking induced by MTGase could decrease moisture uptake by proteins, resulting in an increase of hydrophobic groups in FMP (Tang et al., 2005). Jiang and Tang (2013) reported that the reduction in moisture content of gelatin films from 21.2 to 19.3% was observed when 4 units per gram of MTGase were incorporated. In contrast, Masamba, Li, Hategekimana, Zehadi, Ma, and Zhong (2016) who reported that moisture content of a zein film decreased when 0.5% MTGase was added, but it gradually increased when raising the concentration of MTGase to 1.0%. The addition of MTGase at low concentrations may cause the film to form a more compact network due to the effective covalent cross-links. On the other hand, the incorporation of MTGase, especially at higher concentrations, may induce aggregation, which leads to the formation of less compact and inhomogeneous film networks (Masamba et al., 2016). As a result, more water would absorb in the film treated with MTGase at high concentrations. The swelling of FMP film treated with MTGase compared to the LDPE film is presented in Fig. 2b. The degree of swelling of film is an important property to determine the film’s water resistance. Polymer networks are generally un-dissolved in a liquid media, but they swell in contact with water. The interaction between the liquid and the polymer can be indicated as the degree of swelling. The structure and the properties of the solvent and the polymer typically affect the degree of swelling. In general, a higher degree of crosslinking can decrease the polymer’s capacity to swell of polymer (Schmid et al., 2014). In this study, the degree of swelling of the resulting films was in the range of 41.37–110.22%, while swelling could not be detected in the LDPE film. The MTGase-treated films had a low degree of swelling compared to the control film, regardless of the concentration of MTGase used. The degree of swelling of FMP films decreased gradually (110.22–41.37%) with increasing MTGase concentration (P < 0.05). The addition of MTGase could reduce the degree of swelling of FMP film by around 62% compared to the control. The degree of swelling of MTGaseinduced whey protein isolate film decreased slightly at higher MTGase concentration (Schmid et al., 2014). This was due to the activity of MTGase accompanied by a higher degree of cross-linking caused by inducing the reduction of swelling capacity. Other cross-linking techniques such as chemical treatment for reducing swelling in biodegradable film have been studied. Cao, Fu, and He (2007) observed that the swelling of cross-linked gelatin films decreased by around 30.91% and 42.15% when increasing the concentrations of ferulic acid and tannic acid, respectively. Li, Gao, Wang, Zhang, and Tong (2013) also reported that the addition of glutaraldehyde (3.0 × 10−6–1.2 × 10−5 mol/g) induced a significant decrease in the degree of swelling of chitosan-
3.5. Differential scanning calorimetry The melting temperature (Tm) and enthalpy (ΔH) of FMP films treated with MTGase at various levels and LDPE film are shown in Table 1. Generally, this parameter indicates the mechanical properties of the films (strength and stiffness) and the temperature range in which the film can be used. The Tm and ΔH of the resulting films were in the range of 93.83–112.69 °C and 2.20–5.92 J/g, respectively. The lowest and highest Tm and ΔH were observed in the control film (Tm: 93.83 °C; ΔH: 2.20 J/g) and LDPE film (Tm: 122.54 °C; ΔH: 105.08 J/g), respectively. A slightly increase in melting temperature and enthalpy was more pronounced when MTGase content increased from 1 to 4% (w/w) (P < 0.05). This suggests that the enzymatic modification enhances the thermal stability of FMP film. The Tm of the films indicated that the temperature caused a disruption of the polymer interaction that was generated during film formation. The higher Tm and ΔH found in film treated with MTGase 4% (w/w) might be due to the greater inter-chain interaction of the MTGase treated film, most likely via ε-(γ-glutamyl) 95
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The film solubility of the FMP films treated with various concentrations of MTGase compared to the control LDPE film is shown in Fig. 2c. From the visual observation, the resulting films maintained their integrity after 24 h of immersion in water. The film solubility of the resulting films was in the range of 14.41–22.22%, while the LDPE film showed to be un-dissolved in water. The control film (without MTGase) had the highest film solubility (22.22%) compared to the MTGasetreated films, regardless of concentration used. Furthermore, the solubility of the FMP films decreased gradually (22.22 -14.41%) with increasing concentrations of MTGase (P < 0.05). Decreasing solubility of the treated film might be due to the stronger structure of the film network through covalent cross-linked proteins as evidenced by the increasing strength of the film (Table 1). This would be consistent with Weng and Zheng (2015) who reported that the film solubility of tilapia scale gelatin film decreased from 89.36 to 35.83% when the concentration of MTGase increased. Kolodziejska and Piotrowska (2007) observed a reduction in film solubility from 65 to 26% for fish gelatinchitosan film treated with MTGase. Masamba et al. (2016) also found that the reduction in film solubility (∼19–4.4%) was found when 1% MTGase was added in zein film. The increase in the degree of crosslinking by enzyme treatment causes the reduction of the low molecular weight fractions, resulting in a decrease in the solubility of the films (De Carvalho & Grosso, 2004). Based on these results, it can be said that the cross-linking induced by MTGase could decrease solubility of FMPbased films. For food packaging applications, higher water solubility is needed for dissolving the packaging before product consumption. It might be useful for encapsulating food or food additives. Moreover, the higher water solubility of the film is an eco-friendly packaging material, and it can be used to reduce or replace the synthetic polymer based packaging in order to minimize the environmental impact caused by the packaging waste. 3.7. Light transmission and transparency Transmission of UV and visible light at selected wavelengths of 200–800 nm, as well as the transparency of the films, are shown in Table 2. The light transmission in the UV ranges was 0.00–87.37%, while the transmission in the visible range was 57.30–92.23%. The transmission of UV and visible light decreased with increased concentrations of MTGase. The transmission of UV light was very low at 200–280 nm for FMP films due to they contain high amount of aromatic amino acids, which have ability to absorb UV light (Hamaguchi, WuYin, & Tanaka, 2007). This result suggests that MTGase-treated film could prevent UV transmission and therefore could reduce the food deterioration, especially the lipid oxidation that is induced by UV light. Schmid et al. (2014) also found that the light transmission rate of MTGase-induced whey protein isolate film decreased when increasing the MTGase concentration. A lowered visibility of light transmission was observed when MTGase level increased. This suggests that the cross-linking, or aggregation induced by MTGase, especially at higher amounts, resulted in limiting the light transmission of the film at the visible ranges. According to these results, the light transmission of FMP film depended on the amount of MTGase in the film matrix, and the interaction between FMP molecules was mediated by the addition of MTGase. The transparency value of the resulting films was in the range of 3.76–3.90 while the transparency value of LDPE film was 3.97 (Table 2). All films showed significant differences in film transparency when different amounts of MTGase were added (P < 0.05). The transparency value of MTGase-treated FMP film decreased when increasing the MTGase concentration (P < 0.05). Tang et al. (2005) suggested that the addition of MTGase induced protein aggregation or the cross-linking and makes the FFS become more turbid, which could be affected the transparency by lowering the transparency value of the films. This result is in contrast with Rostamzad et al. (2016) who reported that MTGase concentration had no significant effect on the
Fig. 2. Moisture content (a), degree of swelling (b) and film solubility (c) of FMP films treated with different concentrations of MTGase in comparison with LDPE. The numbers designate of MTGase concentrations (%, w/w based on protein content). Bars represent the standard deviation (n = 3). Different letters indicate the significant difference (P < 0.05).
starch composite film from 66.31 to 1.48%. Swelling has critical implication for food packaging. The degree of swelling is an important property when the film directly comes into contact with food surfaces. The degree of swelling should be as low as possible in order to protect the product against damages and against more water absorption that favor microbial growth. 96
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Table 2 Light transmittance and transparency of FMP films treated with different concentrations of MTGase in comparison with LDPE. MTGase (%, w/w)
0 1 2 3 4 LDPE
Transmittance (%T) at wavelength (nm) 200 280 350
400
500
600
700
800
0.00 0.00 0.00 0.00 0.00 73.60
82.50 74.13 69.23 66.00 61.60 89.77
85.13 79.10 75.83 73.33 70.93 90.30
86.7 82.00 80.03 78.07 75.00 90.77
87.27 83.37 82.33 80.37 77.80 91.60
87.90 84.43 84.03 81.90 79.80 92.23
9.83 8.43 6.60 4.97 2.47 87.37
79.47 69.33 63.70 60.00 57.30 88.57
Transparency
3.90 3.87 3.86 3.81 3.76 3.97
± ± ± ± ± ±
0.0005b 0.0006c 0.0003d 0.0016e 0.0001f 0.0031a
Different superscripts in each column are significantly difference (P < 0.05). Values are given as mean ± SD from triplicate determinations. MTGase: microbial transglutaminase; LDPE: low density polyethylene.
transparency of FMP nanocomposite film. The most critical property for film applications, in term of visual aspect, is transparency, which is important in order to enhance product appearance. Therefore, MTGasetreated FMP film had an effect on the optical properties of the resulting films.
in the presence of the enzyme. The decrease in MHC band intensity coincided with the increase in TS of the films (Table 1). The protein aggregation via the covalent bond had a direct result on the development of a strong film network. 3.9. FT-IR
3.8. Protein patterns
The FT-IR spectrum of FMP film treated with MTGase at various concentrations compared to the control LDPE film is presented in Fig. 4. FT-IR spectroscopy can be used for monitoring the functional groups and interactions of FMP in the films. Slight changes in wavenumber are generally observed at each absorption peak in the resulting films when the film was treated with different MTGase levels. If the FT-IR spectra of the resulting film were similar to that of the control film, it suggests that there was no significant modification of the secondary structure of fish myofibrillar protein following MTGase action. FMP films displayed major bands at 3286 cm−1 (amide-A, representing NeH and/or OeH stretching), 2927 cm−1 (amide-B, representing CeH stretching), 1652 cm−1 (amide-I, represents C]O stretching), and 1544 cm−1 (amide-II, arises from angular deformation of NeH and CeN stretching) (Kaewprachu et al., 2016a). The resulting films showed the peak at wavenumber 1045 cm−1, which was associated with the interactions between the plasticizer (OH-group of glycerol) and the film structure. According to the spectra, all films treated with different MTGase levels showed no change in the vibrational wavenumber for amide-I and amide-II peaks, except for amide-A and amide-B. The wavenumber of amide-A peaks shifted from 3286.23 cm−1 for the FMP film (without MTGase) to 3278.88, 3286.13, 3278.50, and 3281.66 cm−1 for the films treated with different concentrations (1–4% w/w) of MTGase, respectively. The amide-A peak of MTGase-treated films shifted to a lower wavenumber, probably due to fewer formations of hydrogen bonding, which may suggest that the enzymatic cross-linking by MTGase might induce aggregation via the NH-domain of the peptides. Furthermore, the wavenumber of amide-B peaks shifted from 2927.78 cm−1 for the FMP film (without MTGase) to 2925.64, 2921.00, 2922.22 and 2919.77 cm−1 for the films treated with different concentrations (1–4% w/w) of MTGase, respectively. Eissa, Puhl, Kadla, and Khan (2006) reported that amide-B of β-lactoglobulin after crosslinking with MTGase shifted slightly due to changes in the CH2 asymmetrical stretching mode, and due to the reaction of the protein through the enzymatic catalysis. Lower amounts of the −NH2 or −NH3+ group obtained from enzymatic cross-linking more likely results in the lower wavenumber of the amide-A and amide-B peaks obtained (Hoque, Benjakul, & Prodpran, 2011). The addition of MTGase could induce the change in the functional group and also the conformation of proteins as indicated by the changes in the FT-IR spectra, especially for amide-A and amide-B. For the spectra of the control LDPE film, the major absorption bands were found at around 2918 cm−1 (CH2 asymmetric stretching), 2850 cm−1 (CH2 symmetric stretching), 1471 cm−1 (blending deformation) and 717 cm−1 (rocking deformation). Gulmine, Janissek, Heise, and Akcelrud (2002) also reported that
Protein patterns of FMP film in the presence of different concentrations of MTGase under reducing conditions (presence of β-mercaptoethanol) are shown in Fig. 3. The major proteins in FMP were included myosin heavy chain (MHC), actin, tropomyosin, and troponin. Decreases in the MHC band intensity were observed when MTGase was added, when compared to that observed in the control film. Furthermore, the MHC band intensity decreased gradually when increasing the MTGase level (lane 1 to 4). The decrease in the MHC band intensity of the FMP film treated with MTGase was most likely due to the formation of inter-molecular cross-linking via non-disulfide covalent bond induced by MTGase (Prodpran et al., 2012). These results indicate that FMP films underwent cross-linking induced by MTGase. Al-Saadi, Shaker, and Ustunol (2014) reported that the formation of new polymers was observed when a goat milk protein film was treated with MTGase. This was evidenced by the disappearance of lower molecular protein bands with the concomitant appearance of higher molecular weight bands resulting from cross-linked proteins. Porta et al. (2015) reported that bitter vetch (Vicia ervilia) protein film contained glutamine and lysine proteins that were susceptible to cross-linking polymers
Fig. 3. Protein pattern of FMP films treated with different concentrations of MTGase under reducing conditions (20 μg protein, 10% separating gel). M: protein marker; MHC: myosin heavy chain. The numbers designate of MTGase concentrations (%, w/w based on protein content).
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Fig. 4. FT-IR spectra of FMP films treated with different concentrations of MTGase. The numbers designate of MTGase concentrations (%, w/w based on protein content).
the main absorption peaks of LDPE film were found at the wavenumbers of 2919, 2851, 1473, 1463, and 731–720 cm−1.
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4. Conclusion MTGase effectively induced protein cross-linking of FMP molecules as indicated by the properties of the FMP films obtained. The addition of MTGase induced the interaction of proteins to be in compact form, which includes significantly increased TS and decreased EAB, decreased film solubility, lower degree of swelling, and decreased transparency. Improvements in the water barrier properties, UV absorption, and thermal stability of FMP film due to protein cross-linking were clearly observed. Addition of MTGase was a good alternative method for improving the properties of FMP film as indicated in almost all parameters tested. However, the resulting films showed to be poor in EAB when compared to LDPE film, so in this regard, further development is still necessary. Acknowledgements Mae Fah Luang University and the Thailand Research Fund were appreciated for the financial support to Ms. Pimonpan Kaewprachu through the Royal Golden Jubilee Ph.D. Program (Grant NO. PHD/ 0029/2555). Tokyo University of Marine Science and Technology was also acknowledged for materials, reagents, and equipment during research work. Thank you to Matthew Robert Ferguson for language editing support. References Al-Saadi, J. S., Shaker, K. A., & Ustunol, Z. (2014). Effect of heat and transglutaminase on solubility of goat milk protein-based films. International Journal of Dairy Technology, 67, 420–426. Amadori, S., Torricelli, P., Rubini, K., Fini, M., Panzavolta, S., & Bigi, A. (2015). Effect of sterilization and crosslinking on gelatin films. Journal of Materials Science: Materials in Medicine, 26, 69. American Society for Testing and Materials (1989). Annual book of ASTM standards. Philadelphia, PA, USA: ASTM. American Society for Testing and Materials (1999). Annual book of ASTM standards.
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