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Effect of transglutaminase treatment on the properties of cast films of soy protein isolates
Journal of Biotechnology 120 (2005) 296–307
Effect of transglutaminase treatment on the properties of cast films of soy protein isolates Chuan-He Tang a,b,∗,1 , Yan Jiang a,1 , Qi-Biao Wen a , Xiao-Quan Yang a,b b
a Department of Food Science and Technology, South China University of Technology, GuangZhou 510640, PR China Research and Development Center of Food Proteins, South China University of Technology, GuangZhou 510640, PR China
Received 27 December 2004; received in revised form 6 June 2005; accepted 16 June 2005
Abstract The objective of this work was to investigate the effect of microbial transglutaminase (MTGase) treatment on the properties and microstructures of soy protein isolate (SPI) films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectively. Tensile strength (TS), elongation at break (EB), water vapor transmission rate (WVTR) or water vapor permeability (WVP), moisture content (MC), total soluble matter (TSM), lipid barrier property and surface hydrophobicity of control and MTGase-treated films were evaluated after conditioning film specimens at 25 ◦ C and 50% relative humidity (RH) for 48 h. The treatment by 4 units per SPI (U g−1 ) of MTGase increased the TS and surface hydrophobicity by 10–20% and 17–56%, respectively, and simultaneously significantly (P ≤ 0.05) decreased the E, MC and transparency. The WVTR or TSM of SPI films seemed to be not significantly affected by enzymatic treatment (P > 0.05). The MTGase treatment also slowed down the moisture loss rate of film-forming solutions with various plasticizers during the drying process, which was consistent with the increase of surface hydrophobicity of SPI films. Microstructural analyses indicated that the MTGase-treated films of SPI had a rougher surface and more homogeneous or compact cross-section compared to the controls. These results suggested that the MTGase treatment of film-forming solutions of SPI prior to casting could greatly modify the properties and microstructures of SPI films. © 2005 Elsevier B.V. All rights reserved. Keywords: Edible films; Microbial tranglutaminase (MTGase); Soy protein isolates (SPI); Film property
1. Introduction
∗ Corresponding author. Tel.: +86 20 87114262; fax: +86 20 87114263. E-mail address: [email protected] (C.-H. Tang). 1 Contributed equally to this work.
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.06.020
Biodegradable films have received considerable research interest as selective barriers of gases, vapors and solutes as well as for mechanical protection. A lot of biopolymers, including polysaccharide, protein and other components have been applied to the fabri-
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cation of biodegradable films. The film-forming ability of several plant proteins have been widely investigated, including soy proteins (Gennadios and Weller, 1991; Gennadios et al., 1994; Stuchell and Krochta, 1994; Kunte et al., 1997; Rhim et al., 2000), corn zein (Yamada et al., 1995; Parris and Coffin, 1997), wheat proteins (Gennadios and Weller, 1990; Gennadios et al., 1993; Gontard et al., 1992, 1993; Sanchez et al., 1998), cotton seed proteins (Marqui´e et al., 1995), pea proteins (Gu´eguen et al., 1998), peanut protein (Jangchud and Chinnan, 1999), and sunflower proteins (Orliac et al., 2002, 2003). A number of studies have concentrated on the mechanical and barrier properties of soy protein isolates (SPI) films in particular. Numerous physical, chemical or enzymatic treatments have been used to modify the properties of soy protein films, especially those mechanical and surface properties. Such treatments include heat curing (Gennadios et al., 1996; Rangavajhyala et al., 1997; Kim et al., 2002), UV irradiation (Gennadios et al., 1998; Rhim et al., 1999a), ␥-irradiation (Lacroix et al., 2002), sodium alginate or propylene glycol alginate alkylation (Rhim et al., 1999b), sodium dodecyl sulfate treatment (Rhim et al., 2002), aldehydes cross-linking (Ghorpade et al., 1995; Rhim et al., 1998, 2000) and enzymatic cross-linking (Motoki et al., 1987; Stuchell and Krochta, 1994; Yildirim and Hettiarachchy, 1997; Mariniello et al., 2003). It is obvious that the chemical or enzymatic cross-linking methods are effective to improve the mechanical films of soy proteins. From the consideration of safety, the enzymatic cross-linking methods seem to be more popular and advantageous than the chemical cross-linking ones. However, to the date, only a few studies have been reported on the effects of enzymatic cross-linking on the properties of soy proteins films. Transglutaminase (TGase, E.C. 2.3.2.13) is a kind of enzyme, which can catalyze the cross-linking reactions between proteins, and thus modify the several selective properties of protein films. The effects of TGase treatment on the film properties have been studied for many proteins, such as ␣s1 -casein (Motoki et al., 1987), whey proteins (Mahmoud and Savello, 1993; Yildirim and Hettiarachchy, 1997), 11S globulin (Yildirim and Hettiarachchy, 1997), egg white proteins (Lim et al., 1998), demidated gluten (Larr´e et al., 2000) and pectin-soy flour (Mariniello et al., 2003). Gener-
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ally, the cross-linkage by TGase increases the tensile strength and strain, and decreases the solubility of the constitutive proteins of the film, indicating the potential application of TGase for improving the properties of protein films. To our knowledge, there is no literature directly concerning the effects of TGase treatment on the properties of SPI films. Furthermore, the applied enzyme amount of TGase in most reports is high and excessive. Therefore, there is a necessity to study the effect of the treatment of TGase (especially at low enzyme level) on the mechanical or other properties of SPI films. Thus, our aim was to investigate the TGase treatment on the properties and microstructures of SPI films cast with various plasticizers (glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol). The effects of different plasticizers on the properties of TGase-treated SPI films were also evaluated.
2. Materials and methods 2.1. Materials N␣-CBZ-GLN-GLY and l-glutamic acid ␥monohydroxamate were purchased from the Sigma Chemical Co. Commercial SPI was obtained from Wonderful Tech. Co. (Shandong Province, China), containing (on dry basis) 6.5% moisture, 1.0% ash, 0.2% lipid, 90.2% protein (determined by Kjeldahl method, N × 6.25). The commercial microbial transglutminase (MTGase) was gifted from Chanshou Biological Co. Ltd. (Jiashu Province, China), and stored in the freezer (−20 ◦ C) before use. Other chemical reagents used in this study were of analytical or better grade. 2.2. Treatment of MTGase and its activity determination The enzyme powder was completely dissolved in 0.05 mol L−1 Tris–HCl (pH 6.0), placed at 4 ◦ C for 4 h, and then centrifuged at 1000 × g for 15 min (at 4 ◦ C) to remove most of aggregates or precipitates composed of impure proteins. The supernatant was stored at 4 ◦ C, and the enzyme activity (near 20 U mL−1 ) kept steady at least for 2 weeks. The enzymatic activity of TGase was measured by the colorimetric procedure using
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N␣-CBZ-GLN-GLY as the substrate (Folk and Cole, 1965).
were taken on each WVP specimen, one at the center and four around the perimeter, and the mean values were used in WVP calculations.
2.3. Film preparation 2.3.1. Heat cast films (control) The film-forming solutions were prepared by slowly dissolving 5 g SPI in a constantly stirred 0.05 mol L−1 Tris–HCl buffer (pH 8.0) containing 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and a 1:1 mixture thereof. After heated at 70 ◦ C for 20 min, the solutions were centrifuged at low speed (100 × g) to remove air bubbles, then cooled to room temperature (25 ± 1 ◦ C) and cast onto leveled glass plates (21 cm × 35 cm). The film thickness was controlled by casting the same volume solution (80 mL) on each plate. The castings were air-dried at room temperature (25 ± 1 ◦ C) for 24 h. The dried films were peeled off the plates and various specimens for property testing were cut. Specimens of 2.5 cm × 10 cm rectangular strips were for tensile testing, circles of 7 cm diameter for WVP testing, 2 cm × 2 cm squares for MC and TSM testing, circles of 4 cm diameter for lipid barrier property testing, and 1.2 cm × 4 cm rectangular strips for transparency testing. 2.3.2. MTGase-treated films The film-forming solutions with various plasticizers were prepared and heat-treated as the control. Prior to casting, 4 units per SPI (U g−1 ) of MTGase was added into the film-forming solutions pre-cooled to room temperature, and mixed well, then followed by the same process as the control.
2.6. Tensile strength (TS) and percentage elongation at break (E) A TA-XT2i texture analyzer (SMS Co. Ltd., England) was used to assess the TS and E of films. Initial grip separation and cross-head speed were set to 50 mm and 1 mm s−1 , respectively. The TS value was calculated by dividing the maximum load by the initial cross-sectional area of the specimen. The E value was calculated as the percentage of change of the initial gage length of a specimen (50 mm) at the point of a sample failure. 2.7. Water vapor permeability (WVP) The WVP of films was measured using the ASTM method (ASTM, E96-93, 1993). Circular plastic cups with diameter of 3 cm and depth of 5 cm were used. Three grams of CaCl2 were placed in each cup, and the cups were covered with circular films with diameter of 7 cm. Sealed cups were pre-weighed with their contents and placed in a desiccator kept at 25 ◦ C. One liter of pure water was placed in the bottom for providing 100% RH at 25 ◦ C. Then, the cups were weighed every 12 h for a week. The WVP of films was measured from the weight gain of the cups. The WVP (g mm m−2 h−1 kPa−1 ) was calculated as Eq. (1): WVP =
WVTR · L P
(1)
All film specimens were conditioned at 25 ◦ C for 2 days in a desiccator with 50% RH before testing (ASTM, D618-61, 1995).
where WVTR was the measured water vapor transmission rate (g m−2 h−1 ) through the film specimen; L the mean film thickness (mm); P was the partial water vapor pressure difference (kPa) across the two sides of the film specimen (the vapor pressure of pure water at 25 ◦ C = 3.1671 kPa).
2.5. Thickness
2.8. Moisture content (MC)
Film thickness was measured with a hand-held micrometer to the nearest 0.001 mm. Five thickness measurements were taken on each tensile testing specimen along the length of the strip with the mean value used in TS calculations. Similarly, five measurements
Pre-weighed film samples (±0.0001 g) were put into weighing bottles and dried in an air-circulating oven at 105 ◦ C for 24 h. The MC value was determined as the percentage of initial film weight lost during drying and reported on wet basis.
2.4. Conditioning
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2.9. Total soluble matter (TSM)
2.12. Surface hydrophobicity
Two kinds of methods for TSM determination were used and compared (Rhim et al., 1998). The first method: three pre-weighted and dried specimens from a cast film were placed in 30 mL of distilled water, and stored in an environmental chamber at 25 ◦ C for 24 h with occasional gentle stirring. The insoluble dry matter was measured by removing the film pieces from the beakers, gently rinsing them with distilled water, and then drying them in an air-circulating oven (at 105 ◦ C for 24 h). The weight of soluble dry matter was calculated by subtracting the weight of insoluble dry matter from the initial weight of dry matter. The second method: three specimens were directly immersed in water (at 25 ◦ C for 24 h) as described above and subsequently dried in an oven (at 105 ◦ C for 24 h) to determine soluble dry matter. Initial dry matter values needed for TSM calculations were those obtained from MC measurements for the same film.
Surface hydrophobicity was assessed by measuring contact angle (ODG 20 AMP, Dataphiscis Instruments GmbH, Germany). A 4-L drop of de-ionized water was placed on the surface of the film with an automatic piston syringe and photographed. An image analyzer was used to measure the angle formed between the base, constituted of the surface of the film in contact with the drop of water, and the tangent to the drop of water.
2.10. Lipid barrier property
2.13.1. Statistical analysis Microcal Origin V.6.1 software (Microcal software, Northampton, USA) was used for statistical analysis of means and standard deviations. Duncan’s Multiple Range Test (P < 0.05) using SAS procedures (Release 6.08, SAS Institute Inc., Cary, NC) was used to detect significant difference in different mean values.
The permeability coefficient of oil (PO) was used as the indication for lipid barrier property of films. Tubules with 5 mL salad oil were sealed with circular film specimen with diameter of 4 cm and upside down on filter papers, and then stored in an chamber at 40% RH and 25 ◦ C with filter papers together. The weight of filter papers was recorded every day for a week. The PO value was calculated from Eq. (2): PO =
W × FT ST
(2)
where W was the weight variance with time (g); FT the mean film thickness (mm); S the film area covered in the mouth of tubules (mm−2 ); T was the time (d). Slopes of the steady state (linear) portion of weight gain versus time curves were used to estimate W/T. 2.11. Transparency Film strips cut in 1.2 cm × 4 cm were attached to one side of a colorimetric cup. The relative transparency of films was measured at 500 nm, while the empty colorimetric cup was used as the control.
2.13. Scanning electron microscopy (SEM) The samples were dried in a desiccator with silica gel for a week, and then the samples were cut into various kinds of specimens. After gold-coated, the specimens were examined in a model XL 30 environment scanning electron microscopy (Philips Electromics, Mahwah, NJ).
3. Results and discussion 3.1. Effect of transglutaminase treatment on the properties of cast SPI films 3.1.1. Tensile strength and elongation at break TS and EB of control and MTGase-treated films of SPI cast with various plasticizers are shown in Table 1. Mean TS values of cast SPI films with 0.6 plasticizer per SPI (g g−1 ) of glycerol, a mixture of glycerol and sorbitol (1:1) and sorbitol, increase by 17, 20 and 8%, respectively, after treated by four units per SPI (U g−1 ) of MTGase (Table 1). Increases in TS or other mechanical properties of films treated by guinea pig liver or microbial transglutaminase have also been reported for other proteins, such as ␣s1 -casein (Motoki et al., 1987), whey proteins (Mahmoud and Savello, 1993), 11S globulins, or a mixture of whey proteins
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Table 1 Tensile strength and elongation at break for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol, and 1:1 mixture of glycerol and sorbitol, respectivelya Plasticizers
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Control
MTGase-treated
Thickness (mm)
Tensile strength (TS) (MPa)
Elongation at break (EB) (%)
Thickness (mm)
Tensile strength (TS) (MPa)
Elongation at break (EB) (%)
0.094 ± 0.005 0.095 ± 0.005 0.094 ± 0.004
2.21 ± 0.25ch 2.58 ± 0.11cg 4.16 ± 0.04bf
159.87 ± 9.20df 102.04 ± 13.68dg 101.77 ± 15.60dg
0.105 ± 0.003 0.103 ± 0.001 0.110 ± 0.010
2.58 ± 0.28bh 3.10 ± 0.17bg 4.48 ± 0.35bf
105.88 ± 9.20ef 80.04 ± 5.40eg 27.33 ± 3.61eh
Superscript letters (b and c) indicate significant (P ≤ 0.05) difference between TS means in same row. Superscript letters (d and e) indicate significant (P ≤ 0.05) difference between EB means in same row. Superscript letters (f, g and h) indicate significant (P ≤ 0.05) differences of TS and EB within a column. a Data are means of five replicates ± standard deviations.
and 11S globulins (Yildirim and Hettiarachchy, 1997), and demidated gluten (Larr´e et al., 2000). The TGaseinduced TS increase of cast protein films has been attributed to the occurrence of cross-linking within film structures, or the formation of high molecular weight (MW) biopolymers (Motoki et al., 1987; Mahmoud and Savello, 1993; Yildirim and Hettiarachchy, 1997; Larr´e et al., 2000). In some of those earlier reports, the TS values of cast films increased by TGase treatment much higher than that in the present study. This seems to be due to the differences of enzyme amount applied, incubation time or action mode. In both control and MTGase-treated films, the TS values of SPI films with various plasticizers at same level increase in the order: glycerol < 1:1 mixture of glycerol and sorbitol < sorbitol (P ≤ 0.05) (Table 1). In usual, plasticizers are added to a polymeric matrix before drying, just to overcome the brittleness. Plasticizers increase film flexibility due to their ability to reduce internal hydrogen bonding between polymer chains while increasing spacing (Lieberman and Gilbert, 1973). A plasticizer’s composition, size and shape influence its ability to disrupt protein-chain hydrogen bonding, including its ability to attract water to the plasticized protein system. Water is an effective plasticizer for protein films. Thus, attracted water influences the plasticizing ability of an added plasticizer (Sothornvit and Krochta, 2001). Although glycerol (MW, 92.09) and sorbitol (MW, 182.17) have similar structures, the former is a smaller MW plasticizer and is more hygroscopic (at constant RH from adsorption and desorption isotherms) (Leung, 1986). Thus, the smaller size of glycerol and its greater amount of related water increase its effectiveness as a plasticizer. Similar results
have been obtained in whey protein films, fish myofibrillar protein-based films, gelatin-soluble starch films, casein-based films and sodium caseinate-soluble starch films (Sothornvit and Krochta, 2001). However, it is well known that, films with sorbitol are very brittle and fragile, and easily break during peeling. Mean EB values of cast SPI films with glycerol, a mixture of glycerol and sorbitol (1:1) and sorbitol decline by 34, 22 and 73%, respectively, after treated by MTGase (Table 1), suggesting the development of a more compact and less elastic film structure after the MTGase treatment. A similar result was reported in edible pectin-soy flour films obtained in the presence of TGase (Mariniello et al., 2003), while a diverse result was observed in deamidated gluten films crosslinked by MTGase (Larr´e et al., 2000). Larr´e et al. (2000) reported that the action of TGase induced a simultaneous increase in TS and EB, and explained that the formation of covalent linkages by MTGase were flexible enough to permit a gain in elongation. However, in many other cross-linking cases, such as formaldehyde-treated pea protein films (Gu´eguen et al., 1998), glutenin-rich films cross-linked by aldehydes (Hern´andez-Mu˜noz et al., 2004), ultraviolet-irradiated wheat gluten, corn zein, egg albumin and sodium caseinate films (Rhim et al., 1999a), the increases in TS of films are often accompanied by the decreases in EB. 3.1.2. Water vapor transmission rate and water vapor permeability The permeability of films to water vapor was measured at 25 ◦ C, with a RH gradient of 0–100%. Slight but not significant (P > 0.05) differences of WVTR are observed between control and MTGase-treated films
Superscript letter (b) indicates not significant (P > 0.05) difference between WVTR means in same row. Superscript letters (c and d) indicate significant (P ≤ 0.05) difference between WVP means in same row. Superscript letters (e, f and g) indicate significant (P ≤ 0.05) differences of WVTR and WVP within a column. a Data are means of five replicates ± standard deviations.
1.31 ± 0.00ce 35.53 ± 0.80be 0.117 ± 0.003 36.73 ± 0.50be 0.101 ± 0.003
1.17 ± 0.05df
1.33 ± 0.05ce 1.36 ± 0.01ce 36.51 ± 0.24be 34.96 ± 0.20be 0.115 ± 0.003 0.123 ± 0.001 37.29 ± 0.10be 36.09 ± 0.50be 0.105 ± 0.001 0.093 ± 0.003
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Water vapor transmission rate (WVTR) (g h−1 m−2 )
1.24 ± 0.01de 1.06 ± 0.05dg
Water vapor permeability (WVP) (g mm k−1 Pa−1 h−1 m−2 ) Water vapor transmission rate (WVTR) (g h−1 m−2 ) Thickness (mm)
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Thickness (mm)
Water vapor permeability (WVP) (g mm k−1 Pa−1 h−1 m−2 )
MTGase-treated Control
3.1.3. Moisture content and total soluble mass The MC of cast SPI films after conditioning at 50% RH and 25 ◦ C for 2 days decreases slightly, but not significantly (P > 0.05), after treated by MTGase (Table 3). The cross-linking induced by TGase seems to decrease moisture uptake by proteins, since protein amino groups (especially those of lysinyl residues) cross-linked are not available to bind water by hydrogen bonding. Several similar results have been reported for glutaraldehyde-treated collagen films (Weadock et al., 1984), formaldedyde-treated SPI and wheat gluten molded plastics (Jane et al., 1993). In both control and MTGase-treated cases, the MC of films cast with glycerol is much higher than that with sorbitol or its mixture with glycerol (1:1) (Table 3). This is because the glycerol molecules have more hydroxyls to adsorb or trap more water molecules as compared to sorbitol molecules (at the same weight level). Two methods mentioned in Section 2 were carried out to measure the TSM of control and MTGase-treated films. The TSM of SPI films measured by two different methods decreases significantly (P ≤ 0.05), after treated by MTGase (Table 3). There is an exception for films cast with sorbitol, and measured by the second method. In this case, the TSM of control and
Plasticizers
of SPI, though the mean thickness of various films is different (Table 2), suggesting the SPI-based films are poor barriers of water vapor. However, the WVP of MTGase-treated films are significantly (P ≤ 0.05) higher than that of control films (Table 2). The WVP is related with the film thickness and the partial water vapor pressure difference across the two sides of the film specimen (see Eq (1)). Therefore, the film thickness differences between control and MTGase-treated films (Table 2) may account for the differences of WVP values. However, the significant decreases in WVP due to the cross-linking have been reported in UV-treated egg albumin films (Rhim et al., 1999a) and SPI-based films treated by ␥-irradiation (Lacroix et al., 2002). The differences of cross-linking mode or extent in different cases may account for the diversity of WVP results. In addition, the effects of various plasticizers on the WVTR of control and MTGase-treated films of SPI are also not significant (P > 0.05) (Table 2). The significant differences (P ≤ 0.05) of WVP with different plasticizers can also be attributed to the differences of both film thickness and molecule nature.
Table 2 Water vapor transmission rate and water vapor permeability for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectivelya
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Table 3 Moisture content and total soluble mass for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectivelya Plasticizers
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Control
MTGase-treated
Moisture content (MC) (%)
Total soluble massb TSM) (%)
Total soluble massc (TSM) (%)
Moisture content (MC) (%)
Total soluble massb (TSM) (%)
Total soluble massc (TSM) (%)
27.34 ± 2.62bh 16.35 ± 0.06bi 12.61 ± 0.11bj
37.29 ± 0.16dj 44.14 ± 0.12di 52.16 ± 0.03dh
40.12 ± 3.45fi 49.31 ± 1.97fh 50.12 ± 1.20fh
25.01 ± 3.43bh 14.23 ± 0.81bi 10.97 ± 0.20bcj
33.39 ± 0.93ej 40.32 ± 0.89ei 48.1 ± 0.20eh
31.44 ± 1.50gj 39.76 ± 0.77gi 49.43 ± 0.13fh
Superscript letters (b and c) indicate significant (P ≤ 0.05) difference between MC means in same row. Superscript letters (d and e) indicate significant (P ≤ 0.05) difference between TSM means (the first method) in same row. Superscript letters (f and g) indicate significant (P ≤ 0.05) difference between TSM means (the second method) in same row. Superscript letters (h, i and j) indicate significant (P ≤ 0.05) differences of MC and TSM within a column. a Data are means of five replicates ± standard deviations. b Indicate the first method mentioned in Section 2.8. c Indicate the second method mentioned in Section 2.8.
MTGase-treated films are not significant (P > 0.05). Similar to TS increases, reduced TSM values imply the occurrence of cross-linking in MTGase-treated films of SPI. Similar results were reported in ultraviolettreated collagen and gelatin films (Tomihata et al., 1992), ultraviolet-treated corn zein films and egg albumin films (Rhim et al., 1999a) and heat-cured soy protein films (Rangavajhyala et al., 1997). For example, Rangavajhyala et al. (1997) working with the solubility of heat-cured SPI films pointed out that heat-induced aggregation of proteins during film formation increased MW of proteins, and consequently decreased film solubility. In contrast with the MC case, the TSM of control and MTGase-treated films obtained with glycerol is lowest in all cases (Table 3). The lower MC values of films seem to be as a whole accompanied by higher TSM values, suggesting that the water adsorption and film swelling of films may be important for the MC and TSM. Since the films with sorbitol contain less moisture content (compared to those with glycerol), these films will more easily swell after being added into the water, due to water adsorption of protein and sorbitol molecules within the films. Thus, more total soluble mass will leak out of the films. A similar result was obtained in SPI-dialdehyde starch films (Rhim et al., 1998). 3.1.4. Lipid barrier property There was nearly no variance in lipid barrier property between control and MTGase-treated films (data
not shown). Protein films usually have good lipid barrier property and poor water barrier property due to their inherent hydrophilicity. This is consistent with many previous reports (Gennadios et al., 1994; Krochta and De Mulder-Johnston, 1997). 3.1.5. Transparency The mean transparency of SPI films cast with 0.6 plasticizer per SPI of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, decline by 12, 9 and 8%, respectively, after treated by MTGase (Table 4). The decrease in transparency by MTGase treatment is probably due to the cross-linking or aggregation induced by MTGase, which can make the film-forming dispersions more turbid. The transparency of control films cast with glycerol is significantly (P ≤ 0.05) higher than that with sorbitol, or 1:1 mixture of glycerol and sorbitol. However, there is no significant (P > 0.05) variance of transparency among MTGase-treated films cast with various pasticizers (Table 4). 3.1.6. Surface hydrophobicity The enzymatic treatment of the SPI films cast with various plasticizers increases the mean contact angle by 5–16◦ (Table 4), indicating the surface hydrophobicity of SPI films are remarkably modified by MTGase treatment. This is probably accounted for the more exposure of hydrophobic cores or groups of SPI, after treated by MTGase. In a previous study about the MTGaseinduced coagulation and gelation of SPI, we showed that the hydrophobic interactions of basic subunits of
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Table 4 Transparency and contact angle for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectivelya Plasticizers
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Control
MTGase-treated
Transparency (%)
Contact angle with water
77.3 ± 1.5bf 74.4 ± 2.1bg 73.1 ± 1.2bg
25.5 ± 5.6eg 26.6 ± 6.2eg 33.6 ± 6.7ef
(◦ )
Transparency (%)
Contact angle with water (◦ )
68.4 ± 1.7cf 67.5 ± 1.4cf 67.2 ± 1.4cf
35.3 ± 4.7dg 41.5 ± 7.6df 39.4 ± 7.3df
Superscript letters (b and c) indicate significant (P ≤ 0.05) difference between transparency means in same row. Superscript letters (d and e) indicate significant (P ≤ 0.05) difference between contact angle means in same row. Superscript letters (f and g) indicate significant (P ≤ 0.05) differences of transparency and contact angle within a column. a Data are means of five replicates ± standard deviations.
glycinin and -subunit of conglycinin mainly account for the formation of aggregates or gels (Tang et al., in press). However, a negative result was obtained in MTGase-cross-linked deamidated gluten films (Larr´e et al., 2000). Larr´e et al. (2000) reported that the action of TGase tended to decrease the contact angle of deamidated gluten films, and explained this was due to a variation of the protein orientation after cross-linking or to a modification of the number of pore size. Thus, it can be seen that the modification of surface hydrophobicity of protein films by TGase maybe be related with the type and nature of target protein. In addition, the increasing extent of contact angle of SPI films after MTGase treatment is dependent upon the plasticizer type applied. For example, the contact angle of SPI cast with a mixture of glycerol and sorbitol (1:1) increased by 56%, after MTGase treatment and that obtained with sorbitol only by 17% (Table 4).
containing glycerol cases, the moisture loss rate is most evidently affected by the treatment of MTGase. This result is consistent with the changes of contact angle (Table 4), suggesting that higher surface hydrophobicity of protein films be associated with slower moisture loss rate of film-forming solutions. The solid matter concentration of film-forming solutions close to surface is highest, due to the incessant evaporation of water, therefore, the moisture loss rate of film-forming solutions lies on the diffusivity of moisture from the interior to the surface of solution. Results obtained in this study showed that MTGase treatment seems to decrease the diffusivity of moisture of film-forming solutions of SPI.
3.2. Effect of transglutaminase treatment on the drying of film-forming solutions The effects of MTGase treatment on the drying process of film-forming solutions of SPI with various plasticizers (glycerol, sorbitol or a mixture thereof) at room temperature are shown in Fig. 1. In all cases, the moisture loss rate is highest during prior drying (∼6 h), then slightly slows down with further drying process (6–9 h) (Fig. 1). This is in accordance with the decrease of free moisture or the increase of concentration of film-forming solutions with increasing drying time. The moisture loss rate of film-forming solutions of SPI with various plasticizers are decreased by the MTGase treatment (Fig. 1). In the glycerol or a mixture
Fig. 1. Moisture loss profiles of control and MTGase-treated filmforming solutions with 0.6 plasticizer per SPI (g g−1 ) of glycerol, 1:1 mixture of glycerol and sorbitol, and sorbitol, respectively, during drying process. Each film-forming solution (40 mL) is cast on 21 cm × 18 cm surface of a glass plate.
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3.3. Scanning electron microscopy The control and MTGase-treated films of SPI, cast with various plasticizers (glycerol, sorbitol and a mixture thereof), were analyzed by SEM. SEM of film surfaces (Fig. 2) shows that the surface microstructures of MTGase-treated films of SPI are more rough
and uneven than those of control films. There are more ripples or protruding strips on the surfaces of MTGasetreated films (Fig. 2, panel B). This result seems to be conformable with that of surface hydrophobicity (Table 4) and moisture loss rate of film-forming solutions (Fig. 1), and can be accounted for the aggregation of exposed hydrophobic groups of SPI induced by
Fig. 2. SEM micrographs (at 400× magnification) of surfaces of control and MTGase-treated films of SPI. Panel A indicates the surfaces of control films of SPI, and panel B for those of MTGase-treated films. The number of 1, 2 and 3 represents the films cast with 0.6 g/g SPI of glycerol, a mixture of glycerol and sorbitol (1:1) and sorbitol, respectively.
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Fig. 3. SEM micrographs (at 3200× magnification) of cross-sections of control and MTGase-treated films of SPI, cast with 0.6 g/g SPI of glycerol. Panel A indicates the cross-section of control films, and panel B for that of MTGase-treated films.
MTGase. However, a contrary result was reported in pectin-soy flour films obtained in the presence of TGase (Mariniello et al., 2003), showing that the TGasetreated films of pectin-soy flour had a smoother surface and higher homogeneity. The presence of pectin or other components may account for it, because the interactions between pectin and proteins are obvious in this case. Furthermore, there are observable differences of surface microstructure of control and MTG-treated films cast with different plasticizers (Fig. 2). This is tightly related with the differences of water-holding ability of various plasticizers. The glycerol molecules have higher water-holding ability due to having more hydroxyls, therefore, the surfaces of films cast with glycerol are smoother. The SEM micrographs (at 3200× magnification) of cross-section of control and MTGase-treated SPI films cast with glycerol are shown in Fig. 3. The crosssection of MTGase-treated films shows a homogenous and compact microstructure (Fig. 3, panel A), and lacks the meshy or knitting-like structure shown in control films (Fig. 3, panel B). This result directly illuminates the higher TS and lower EB, TSM and transparency of MTGase-treated films comparable with the controls.
4. Conclusions The cross-linking treatment by transglutaminase was found to be an effective and practical method to improve properties of SPI films cast with var-
ious plasticizers (glycerol, sorbitol and a mixture thereof). The TS and surface hydrophobicity of SPI films increased significantly (P ≤ 0.05), and the E, MC, TSM and transparency decreased significantly (P ≤ 0.05), after treated by MTGase treatment. The MTG-treated films indicated coarse surfaces and more compact microstructure of cross-section, which directly accounted for their higher TS and lower EB and TSM compared to the controls.
Acknowledgements This work is part of the research projects of Chinese National Natural Science Fund (serial number: 20306008), sponsored by the NNSF of China. The authors gratefully acknowledge its financial support.
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Effect of transglutaminase treatment on the properties of cast films of soy protein isolates Chuan-He Tang a,b,∗,1 , Yan Jiang a,1 , Qi-Biao Wen a , Xiao-Quan Yang a,b b
a Department of Food Science and Technology, South China University of Technology, GuangZhou 510640, PR China Research and Development Center of Food Proteins, South China University of Technology, GuangZhou 510640, PR China
Received 27 December 2004; received in revised form 6 June 2005; accepted 16 June 2005
Abstract The objective of this work was to investigate the effect of microbial transglutaminase (MTGase) treatment on the properties and microstructures of soy protein isolate (SPI) films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectively. Tensile strength (TS), elongation at break (EB), water vapor transmission rate (WVTR) or water vapor permeability (WVP), moisture content (MC), total soluble matter (TSM), lipid barrier property and surface hydrophobicity of control and MTGase-treated films were evaluated after conditioning film specimens at 25 ◦ C and 50% relative humidity (RH) for 48 h. The treatment by 4 units per SPI (U g−1 ) of MTGase increased the TS and surface hydrophobicity by 10–20% and 17–56%, respectively, and simultaneously significantly (P ≤ 0.05) decreased the E, MC and transparency. The WVTR or TSM of SPI films seemed to be not significantly affected by enzymatic treatment (P > 0.05). The MTGase treatment also slowed down the moisture loss rate of film-forming solutions with various plasticizers during the drying process, which was consistent with the increase of surface hydrophobicity of SPI films. Microstructural analyses indicated that the MTGase-treated films of SPI had a rougher surface and more homogeneous or compact cross-section compared to the controls. These results suggested that the MTGase treatment of film-forming solutions of SPI prior to casting could greatly modify the properties and microstructures of SPI films. © 2005 Elsevier B.V. All rights reserved. Keywords: Edible films; Microbial tranglutaminase (MTGase); Soy protein isolates (SPI); Film property
1. Introduction
∗ Corresponding author. Tel.: +86 20 87114262; fax: +86 20 87114263. E-mail address: [email protected] (C.-H. Tang). 1 Contributed equally to this work.
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.06.020
Biodegradable films have received considerable research interest as selective barriers of gases, vapors and solutes as well as for mechanical protection. A lot of biopolymers, including polysaccharide, protein and other components have been applied to the fabri-
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cation of biodegradable films. The film-forming ability of several plant proteins have been widely investigated, including soy proteins (Gennadios and Weller, 1991; Gennadios et al., 1994; Stuchell and Krochta, 1994; Kunte et al., 1997; Rhim et al., 2000), corn zein (Yamada et al., 1995; Parris and Coffin, 1997), wheat proteins (Gennadios and Weller, 1990; Gennadios et al., 1993; Gontard et al., 1992, 1993; Sanchez et al., 1998), cotton seed proteins (Marqui´e et al., 1995), pea proteins (Gu´eguen et al., 1998), peanut protein (Jangchud and Chinnan, 1999), and sunflower proteins (Orliac et al., 2002, 2003). A number of studies have concentrated on the mechanical and barrier properties of soy protein isolates (SPI) films in particular. Numerous physical, chemical or enzymatic treatments have been used to modify the properties of soy protein films, especially those mechanical and surface properties. Such treatments include heat curing (Gennadios et al., 1996; Rangavajhyala et al., 1997; Kim et al., 2002), UV irradiation (Gennadios et al., 1998; Rhim et al., 1999a), ␥-irradiation (Lacroix et al., 2002), sodium alginate or propylene glycol alginate alkylation (Rhim et al., 1999b), sodium dodecyl sulfate treatment (Rhim et al., 2002), aldehydes cross-linking (Ghorpade et al., 1995; Rhim et al., 1998, 2000) and enzymatic cross-linking (Motoki et al., 1987; Stuchell and Krochta, 1994; Yildirim and Hettiarachchy, 1997; Mariniello et al., 2003). It is obvious that the chemical or enzymatic cross-linking methods are effective to improve the mechanical films of soy proteins. From the consideration of safety, the enzymatic cross-linking methods seem to be more popular and advantageous than the chemical cross-linking ones. However, to the date, only a few studies have been reported on the effects of enzymatic cross-linking on the properties of soy proteins films. Transglutaminase (TGase, E.C. 2.3.2.13) is a kind of enzyme, which can catalyze the cross-linking reactions between proteins, and thus modify the several selective properties of protein films. The effects of TGase treatment on the film properties have been studied for many proteins, such as ␣s1 -casein (Motoki et al., 1987), whey proteins (Mahmoud and Savello, 1993; Yildirim and Hettiarachchy, 1997), 11S globulin (Yildirim and Hettiarachchy, 1997), egg white proteins (Lim et al., 1998), demidated gluten (Larr´e et al., 2000) and pectin-soy flour (Mariniello et al., 2003). Gener-
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ally, the cross-linkage by TGase increases the tensile strength and strain, and decreases the solubility of the constitutive proteins of the film, indicating the potential application of TGase for improving the properties of protein films. To our knowledge, there is no literature directly concerning the effects of TGase treatment on the properties of SPI films. Furthermore, the applied enzyme amount of TGase in most reports is high and excessive. Therefore, there is a necessity to study the effect of the treatment of TGase (especially at low enzyme level) on the mechanical or other properties of SPI films. Thus, our aim was to investigate the TGase treatment on the properties and microstructures of SPI films cast with various plasticizers (glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol). The effects of different plasticizers on the properties of TGase-treated SPI films were also evaluated.
2. Materials and methods 2.1. Materials N␣-CBZ-GLN-GLY and l-glutamic acid ␥monohydroxamate were purchased from the Sigma Chemical Co. Commercial SPI was obtained from Wonderful Tech. Co. (Shandong Province, China), containing (on dry basis) 6.5% moisture, 1.0% ash, 0.2% lipid, 90.2% protein (determined by Kjeldahl method, N × 6.25). The commercial microbial transglutminase (MTGase) was gifted from Chanshou Biological Co. Ltd. (Jiashu Province, China), and stored in the freezer (−20 ◦ C) before use. Other chemical reagents used in this study were of analytical or better grade. 2.2. Treatment of MTGase and its activity determination The enzyme powder was completely dissolved in 0.05 mol L−1 Tris–HCl (pH 6.0), placed at 4 ◦ C for 4 h, and then centrifuged at 1000 × g for 15 min (at 4 ◦ C) to remove most of aggregates or precipitates composed of impure proteins. The supernatant was stored at 4 ◦ C, and the enzyme activity (near 20 U mL−1 ) kept steady at least for 2 weeks. The enzymatic activity of TGase was measured by the colorimetric procedure using
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N␣-CBZ-GLN-GLY as the substrate (Folk and Cole, 1965).
were taken on each WVP specimen, one at the center and four around the perimeter, and the mean values were used in WVP calculations.
2.3. Film preparation 2.3.1. Heat cast films (control) The film-forming solutions were prepared by slowly dissolving 5 g SPI in a constantly stirred 0.05 mol L−1 Tris–HCl buffer (pH 8.0) containing 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and a 1:1 mixture thereof. After heated at 70 ◦ C for 20 min, the solutions were centrifuged at low speed (100 × g) to remove air bubbles, then cooled to room temperature (25 ± 1 ◦ C) and cast onto leveled glass plates (21 cm × 35 cm). The film thickness was controlled by casting the same volume solution (80 mL) on each plate. The castings were air-dried at room temperature (25 ± 1 ◦ C) for 24 h. The dried films were peeled off the plates and various specimens for property testing were cut. Specimens of 2.5 cm × 10 cm rectangular strips were for tensile testing, circles of 7 cm diameter for WVP testing, 2 cm × 2 cm squares for MC and TSM testing, circles of 4 cm diameter for lipid barrier property testing, and 1.2 cm × 4 cm rectangular strips for transparency testing. 2.3.2. MTGase-treated films The film-forming solutions with various plasticizers were prepared and heat-treated as the control. Prior to casting, 4 units per SPI (U g−1 ) of MTGase was added into the film-forming solutions pre-cooled to room temperature, and mixed well, then followed by the same process as the control.
2.6. Tensile strength (TS) and percentage elongation at break (E) A TA-XT2i texture analyzer (SMS Co. Ltd., England) was used to assess the TS and E of films. Initial grip separation and cross-head speed were set to 50 mm and 1 mm s−1 , respectively. The TS value was calculated by dividing the maximum load by the initial cross-sectional area of the specimen. The E value was calculated as the percentage of change of the initial gage length of a specimen (50 mm) at the point of a sample failure. 2.7. Water vapor permeability (WVP) The WVP of films was measured using the ASTM method (ASTM, E96-93, 1993). Circular plastic cups with diameter of 3 cm and depth of 5 cm were used. Three grams of CaCl2 were placed in each cup, and the cups were covered with circular films with diameter of 7 cm. Sealed cups were pre-weighed with their contents and placed in a desiccator kept at 25 ◦ C. One liter of pure water was placed in the bottom for providing 100% RH at 25 ◦ C. Then, the cups were weighed every 12 h for a week. The WVP of films was measured from the weight gain of the cups. The WVP (g mm m−2 h−1 kPa−1 ) was calculated as Eq. (1): WVP =
WVTR · L P
(1)
All film specimens were conditioned at 25 ◦ C for 2 days in a desiccator with 50% RH before testing (ASTM, D618-61, 1995).
where WVTR was the measured water vapor transmission rate (g m−2 h−1 ) through the film specimen; L the mean film thickness (mm); P was the partial water vapor pressure difference (kPa) across the two sides of the film specimen (the vapor pressure of pure water at 25 ◦ C = 3.1671 kPa).
2.5. Thickness
2.8. Moisture content (MC)
Film thickness was measured with a hand-held micrometer to the nearest 0.001 mm. Five thickness measurements were taken on each tensile testing specimen along the length of the strip with the mean value used in TS calculations. Similarly, five measurements
Pre-weighed film samples (±0.0001 g) were put into weighing bottles and dried in an air-circulating oven at 105 ◦ C for 24 h. The MC value was determined as the percentage of initial film weight lost during drying and reported on wet basis.
2.4. Conditioning
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2.9. Total soluble matter (TSM)
2.12. Surface hydrophobicity
Two kinds of methods for TSM determination were used and compared (Rhim et al., 1998). The first method: three pre-weighted and dried specimens from a cast film were placed in 30 mL of distilled water, and stored in an environmental chamber at 25 ◦ C for 24 h with occasional gentle stirring. The insoluble dry matter was measured by removing the film pieces from the beakers, gently rinsing them with distilled water, and then drying them in an air-circulating oven (at 105 ◦ C for 24 h). The weight of soluble dry matter was calculated by subtracting the weight of insoluble dry matter from the initial weight of dry matter. The second method: three specimens were directly immersed in water (at 25 ◦ C for 24 h) as described above and subsequently dried in an oven (at 105 ◦ C for 24 h) to determine soluble dry matter. Initial dry matter values needed for TSM calculations were those obtained from MC measurements for the same film.
Surface hydrophobicity was assessed by measuring contact angle (ODG 20 AMP, Dataphiscis Instruments GmbH, Germany). A 4-L drop of de-ionized water was placed on the surface of the film with an automatic piston syringe and photographed. An image analyzer was used to measure the angle formed between the base, constituted of the surface of the film in contact with the drop of water, and the tangent to the drop of water.
2.10. Lipid barrier property
2.13.1. Statistical analysis Microcal Origin V.6.1 software (Microcal software, Northampton, USA) was used for statistical analysis of means and standard deviations. Duncan’s Multiple Range Test (P < 0.05) using SAS procedures (Release 6.08, SAS Institute Inc., Cary, NC) was used to detect significant difference in different mean values.
The permeability coefficient of oil (PO) was used as the indication for lipid barrier property of films. Tubules with 5 mL salad oil were sealed with circular film specimen with diameter of 4 cm and upside down on filter papers, and then stored in an chamber at 40% RH and 25 ◦ C with filter papers together. The weight of filter papers was recorded every day for a week. The PO value was calculated from Eq. (2): PO =
W × FT ST
(2)
where W was the weight variance with time (g); FT the mean film thickness (mm); S the film area covered in the mouth of tubules (mm−2 ); T was the time (d). Slopes of the steady state (linear) portion of weight gain versus time curves were used to estimate W/T. 2.11. Transparency Film strips cut in 1.2 cm × 4 cm were attached to one side of a colorimetric cup. The relative transparency of films was measured at 500 nm, while the empty colorimetric cup was used as the control.
2.13. Scanning electron microscopy (SEM) The samples were dried in a desiccator with silica gel for a week, and then the samples were cut into various kinds of specimens. After gold-coated, the specimens were examined in a model XL 30 environment scanning electron microscopy (Philips Electromics, Mahwah, NJ).
3. Results and discussion 3.1. Effect of transglutaminase treatment on the properties of cast SPI films 3.1.1. Tensile strength and elongation at break TS and EB of control and MTGase-treated films of SPI cast with various plasticizers are shown in Table 1. Mean TS values of cast SPI films with 0.6 plasticizer per SPI (g g−1 ) of glycerol, a mixture of glycerol and sorbitol (1:1) and sorbitol, increase by 17, 20 and 8%, respectively, after treated by four units per SPI (U g−1 ) of MTGase (Table 1). Increases in TS or other mechanical properties of films treated by guinea pig liver or microbial transglutaminase have also been reported for other proteins, such as ␣s1 -casein (Motoki et al., 1987), whey proteins (Mahmoud and Savello, 1993), 11S globulins, or a mixture of whey proteins
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Table 1 Tensile strength and elongation at break for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol, and 1:1 mixture of glycerol and sorbitol, respectivelya Plasticizers
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Control
MTGase-treated
Thickness (mm)
Tensile strength (TS) (MPa)
Elongation at break (EB) (%)
Thickness (mm)
Tensile strength (TS) (MPa)
Elongation at break (EB) (%)
0.094 ± 0.005 0.095 ± 0.005 0.094 ± 0.004
2.21 ± 0.25ch 2.58 ± 0.11cg 4.16 ± 0.04bf
159.87 ± 9.20df 102.04 ± 13.68dg 101.77 ± 15.60dg
0.105 ± 0.003 0.103 ± 0.001 0.110 ± 0.010
2.58 ± 0.28bh 3.10 ± 0.17bg 4.48 ± 0.35bf
105.88 ± 9.20ef 80.04 ± 5.40eg 27.33 ± 3.61eh
Superscript letters (b and c) indicate significant (P ≤ 0.05) difference between TS means in same row. Superscript letters (d and e) indicate significant (P ≤ 0.05) difference between EB means in same row. Superscript letters (f, g and h) indicate significant (P ≤ 0.05) differences of TS and EB within a column. a Data are means of five replicates ± standard deviations.
and 11S globulins (Yildirim and Hettiarachchy, 1997), and demidated gluten (Larr´e et al., 2000). The TGaseinduced TS increase of cast protein films has been attributed to the occurrence of cross-linking within film structures, or the formation of high molecular weight (MW) biopolymers (Motoki et al., 1987; Mahmoud and Savello, 1993; Yildirim and Hettiarachchy, 1997; Larr´e et al., 2000). In some of those earlier reports, the TS values of cast films increased by TGase treatment much higher than that in the present study. This seems to be due to the differences of enzyme amount applied, incubation time or action mode. In both control and MTGase-treated films, the TS values of SPI films with various plasticizers at same level increase in the order: glycerol < 1:1 mixture of glycerol and sorbitol < sorbitol (P ≤ 0.05) (Table 1). In usual, plasticizers are added to a polymeric matrix before drying, just to overcome the brittleness. Plasticizers increase film flexibility due to their ability to reduce internal hydrogen bonding between polymer chains while increasing spacing (Lieberman and Gilbert, 1973). A plasticizer’s composition, size and shape influence its ability to disrupt protein-chain hydrogen bonding, including its ability to attract water to the plasticized protein system. Water is an effective plasticizer for protein films. Thus, attracted water influences the plasticizing ability of an added plasticizer (Sothornvit and Krochta, 2001). Although glycerol (MW, 92.09) and sorbitol (MW, 182.17) have similar structures, the former is a smaller MW plasticizer and is more hygroscopic (at constant RH from adsorption and desorption isotherms) (Leung, 1986). Thus, the smaller size of glycerol and its greater amount of related water increase its effectiveness as a plasticizer. Similar results
have been obtained in whey protein films, fish myofibrillar protein-based films, gelatin-soluble starch films, casein-based films and sodium caseinate-soluble starch films (Sothornvit and Krochta, 2001). However, it is well known that, films with sorbitol are very brittle and fragile, and easily break during peeling. Mean EB values of cast SPI films with glycerol, a mixture of glycerol and sorbitol (1:1) and sorbitol decline by 34, 22 and 73%, respectively, after treated by MTGase (Table 1), suggesting the development of a more compact and less elastic film structure after the MTGase treatment. A similar result was reported in edible pectin-soy flour films obtained in the presence of TGase (Mariniello et al., 2003), while a diverse result was observed in deamidated gluten films crosslinked by MTGase (Larr´e et al., 2000). Larr´e et al. (2000) reported that the action of TGase induced a simultaneous increase in TS and EB, and explained that the formation of covalent linkages by MTGase were flexible enough to permit a gain in elongation. However, in many other cross-linking cases, such as formaldehyde-treated pea protein films (Gu´eguen et al., 1998), glutenin-rich films cross-linked by aldehydes (Hern´andez-Mu˜noz et al., 2004), ultraviolet-irradiated wheat gluten, corn zein, egg albumin and sodium caseinate films (Rhim et al., 1999a), the increases in TS of films are often accompanied by the decreases in EB. 3.1.2. Water vapor transmission rate and water vapor permeability The permeability of films to water vapor was measured at 25 ◦ C, with a RH gradient of 0–100%. Slight but not significant (P > 0.05) differences of WVTR are observed between control and MTGase-treated films
Superscript letter (b) indicates not significant (P > 0.05) difference between WVTR means in same row. Superscript letters (c and d) indicate significant (P ≤ 0.05) difference between WVP means in same row. Superscript letters (e, f and g) indicate significant (P ≤ 0.05) differences of WVTR and WVP within a column. a Data are means of five replicates ± standard deviations.
1.31 ± 0.00ce 35.53 ± 0.80be 0.117 ± 0.003 36.73 ± 0.50be 0.101 ± 0.003
1.17 ± 0.05df
1.33 ± 0.05ce 1.36 ± 0.01ce 36.51 ± 0.24be 34.96 ± 0.20be 0.115 ± 0.003 0.123 ± 0.001 37.29 ± 0.10be 36.09 ± 0.50be 0.105 ± 0.001 0.093 ± 0.003
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Water vapor transmission rate (WVTR) (g h−1 m−2 )
1.24 ± 0.01de 1.06 ± 0.05dg
Water vapor permeability (WVP) (g mm k−1 Pa−1 h−1 m−2 ) Water vapor transmission rate (WVTR) (g h−1 m−2 ) Thickness (mm)
301
Thickness (mm)
Water vapor permeability (WVP) (g mm k−1 Pa−1 h−1 m−2 )
MTGase-treated Control
3.1.3. Moisture content and total soluble mass The MC of cast SPI films after conditioning at 50% RH and 25 ◦ C for 2 days decreases slightly, but not significantly (P > 0.05), after treated by MTGase (Table 3). The cross-linking induced by TGase seems to decrease moisture uptake by proteins, since protein amino groups (especially those of lysinyl residues) cross-linked are not available to bind water by hydrogen bonding. Several similar results have been reported for glutaraldehyde-treated collagen films (Weadock et al., 1984), formaldedyde-treated SPI and wheat gluten molded plastics (Jane et al., 1993). In both control and MTGase-treated cases, the MC of films cast with glycerol is much higher than that with sorbitol or its mixture with glycerol (1:1) (Table 3). This is because the glycerol molecules have more hydroxyls to adsorb or trap more water molecules as compared to sorbitol molecules (at the same weight level). Two methods mentioned in Section 2 were carried out to measure the TSM of control and MTGase-treated films. The TSM of SPI films measured by two different methods decreases significantly (P ≤ 0.05), after treated by MTGase (Table 3). There is an exception for films cast with sorbitol, and measured by the second method. In this case, the TSM of control and
Plasticizers
of SPI, though the mean thickness of various films is different (Table 2), suggesting the SPI-based films are poor barriers of water vapor. However, the WVP of MTGase-treated films are significantly (P ≤ 0.05) higher than that of control films (Table 2). The WVP is related with the film thickness and the partial water vapor pressure difference across the two sides of the film specimen (see Eq (1)). Therefore, the film thickness differences between control and MTGase-treated films (Table 2) may account for the differences of WVP values. However, the significant decreases in WVP due to the cross-linking have been reported in UV-treated egg albumin films (Rhim et al., 1999a) and SPI-based films treated by ␥-irradiation (Lacroix et al., 2002). The differences of cross-linking mode or extent in different cases may account for the diversity of WVP results. In addition, the effects of various plasticizers on the WVTR of control and MTGase-treated films of SPI are also not significant (P > 0.05) (Table 2). The significant differences (P ≤ 0.05) of WVP with different plasticizers can also be attributed to the differences of both film thickness and molecule nature.
Table 2 Water vapor transmission rate and water vapor permeability for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectivelya
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Table 3 Moisture content and total soluble mass for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectivelya Plasticizers
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Control
MTGase-treated
Moisture content (MC) (%)
Total soluble massb TSM) (%)
Total soluble massc (TSM) (%)
Moisture content (MC) (%)
Total soluble massb (TSM) (%)
Total soluble massc (TSM) (%)
27.34 ± 2.62bh 16.35 ± 0.06bi 12.61 ± 0.11bj
37.29 ± 0.16dj 44.14 ± 0.12di 52.16 ± 0.03dh
40.12 ± 3.45fi 49.31 ± 1.97fh 50.12 ± 1.20fh
25.01 ± 3.43bh 14.23 ± 0.81bi 10.97 ± 0.20bcj
33.39 ± 0.93ej 40.32 ± 0.89ei 48.1 ± 0.20eh
31.44 ± 1.50gj 39.76 ± 0.77gi 49.43 ± 0.13fh
Superscript letters (b and c) indicate significant (P ≤ 0.05) difference between MC means in same row. Superscript letters (d and e) indicate significant (P ≤ 0.05) difference between TSM means (the first method) in same row. Superscript letters (f and g) indicate significant (P ≤ 0.05) difference between TSM means (the second method) in same row. Superscript letters (h, i and j) indicate significant (P ≤ 0.05) differences of MC and TSM within a column. a Data are means of five replicates ± standard deviations. b Indicate the first method mentioned in Section 2.8. c Indicate the second method mentioned in Section 2.8.
MTGase-treated films are not significant (P > 0.05). Similar to TS increases, reduced TSM values imply the occurrence of cross-linking in MTGase-treated films of SPI. Similar results were reported in ultraviolettreated collagen and gelatin films (Tomihata et al., 1992), ultraviolet-treated corn zein films and egg albumin films (Rhim et al., 1999a) and heat-cured soy protein films (Rangavajhyala et al., 1997). For example, Rangavajhyala et al. (1997) working with the solubility of heat-cured SPI films pointed out that heat-induced aggregation of proteins during film formation increased MW of proteins, and consequently decreased film solubility. In contrast with the MC case, the TSM of control and MTGase-treated films obtained with glycerol is lowest in all cases (Table 3). The lower MC values of films seem to be as a whole accompanied by higher TSM values, suggesting that the water adsorption and film swelling of films may be important for the MC and TSM. Since the films with sorbitol contain less moisture content (compared to those with glycerol), these films will more easily swell after being added into the water, due to water adsorption of protein and sorbitol molecules within the films. Thus, more total soluble mass will leak out of the films. A similar result was obtained in SPI-dialdehyde starch films (Rhim et al., 1998). 3.1.4. Lipid barrier property There was nearly no variance in lipid barrier property between control and MTGase-treated films (data
not shown). Protein films usually have good lipid barrier property and poor water barrier property due to their inherent hydrophilicity. This is consistent with many previous reports (Gennadios et al., 1994; Krochta and De Mulder-Johnston, 1997). 3.1.5. Transparency The mean transparency of SPI films cast with 0.6 plasticizer per SPI of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, decline by 12, 9 and 8%, respectively, after treated by MTGase (Table 4). The decrease in transparency by MTGase treatment is probably due to the cross-linking or aggregation induced by MTGase, which can make the film-forming dispersions more turbid. The transparency of control films cast with glycerol is significantly (P ≤ 0.05) higher than that with sorbitol, or 1:1 mixture of glycerol and sorbitol. However, there is no significant (P > 0.05) variance of transparency among MTGase-treated films cast with various pasticizers (Table 4). 3.1.6. Surface hydrophobicity The enzymatic treatment of the SPI films cast with various plasticizers increases the mean contact angle by 5–16◦ (Table 4), indicating the surface hydrophobicity of SPI films are remarkably modified by MTGase treatment. This is probably accounted for the more exposure of hydrophobic cores or groups of SPI, after treated by MTGase. In a previous study about the MTGaseinduced coagulation and gelation of SPI, we showed that the hydrophobic interactions of basic subunits of
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Table 4 Transparency and contact angle for control and MTGase-treated films cast with 0.6 plasticizer per SPI (g g−1 ) of glycerol, sorbitol and 1:1 mixture of glycerol and sorbitol, respectivelya Plasticizers
Glycerol Glycerol/sorbitol (1:1) Sorbitol
Control
MTGase-treated
Transparency (%)
Contact angle with water
77.3 ± 1.5bf 74.4 ± 2.1bg 73.1 ± 1.2bg
25.5 ± 5.6eg 26.6 ± 6.2eg 33.6 ± 6.7ef
(◦ )
Transparency (%)
Contact angle with water (◦ )
68.4 ± 1.7cf 67.5 ± 1.4cf 67.2 ± 1.4cf
35.3 ± 4.7dg 41.5 ± 7.6df 39.4 ± 7.3df
Superscript letters (b and c) indicate significant (P ≤ 0.05) difference between transparency means in same row. Superscript letters (d and e) indicate significant (P ≤ 0.05) difference between contact angle means in same row. Superscript letters (f and g) indicate significant (P ≤ 0.05) differences of transparency and contact angle within a column. a Data are means of five replicates ± standard deviations.
glycinin and -subunit of conglycinin mainly account for the formation of aggregates or gels (Tang et al., in press). However, a negative result was obtained in MTGase-cross-linked deamidated gluten films (Larr´e et al., 2000). Larr´e et al. (2000) reported that the action of TGase tended to decrease the contact angle of deamidated gluten films, and explained this was due to a variation of the protein orientation after cross-linking or to a modification of the number of pore size. Thus, it can be seen that the modification of surface hydrophobicity of protein films by TGase maybe be related with the type and nature of target protein. In addition, the increasing extent of contact angle of SPI films after MTGase treatment is dependent upon the plasticizer type applied. For example, the contact angle of SPI cast with a mixture of glycerol and sorbitol (1:1) increased by 56%, after MTGase treatment and that obtained with sorbitol only by 17% (Table 4).
containing glycerol cases, the moisture loss rate is most evidently affected by the treatment of MTGase. This result is consistent with the changes of contact angle (Table 4), suggesting that higher surface hydrophobicity of protein films be associated with slower moisture loss rate of film-forming solutions. The solid matter concentration of film-forming solutions close to surface is highest, due to the incessant evaporation of water, therefore, the moisture loss rate of film-forming solutions lies on the diffusivity of moisture from the interior to the surface of solution. Results obtained in this study showed that MTGase treatment seems to decrease the diffusivity of moisture of film-forming solutions of SPI.
3.2. Effect of transglutaminase treatment on the drying of film-forming solutions The effects of MTGase treatment on the drying process of film-forming solutions of SPI with various plasticizers (glycerol, sorbitol or a mixture thereof) at room temperature are shown in Fig. 1. In all cases, the moisture loss rate is highest during prior drying (∼6 h), then slightly slows down with further drying process (6–9 h) (Fig. 1). This is in accordance with the decrease of free moisture or the increase of concentration of film-forming solutions with increasing drying time. The moisture loss rate of film-forming solutions of SPI with various plasticizers are decreased by the MTGase treatment (Fig. 1). In the glycerol or a mixture
Fig. 1. Moisture loss profiles of control and MTGase-treated filmforming solutions with 0.6 plasticizer per SPI (g g−1 ) of glycerol, 1:1 mixture of glycerol and sorbitol, and sorbitol, respectively, during drying process. Each film-forming solution (40 mL) is cast on 21 cm × 18 cm surface of a glass plate.
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3.3. Scanning electron microscopy The control and MTGase-treated films of SPI, cast with various plasticizers (glycerol, sorbitol and a mixture thereof), were analyzed by SEM. SEM of film surfaces (Fig. 2) shows that the surface microstructures of MTGase-treated films of SPI are more rough
and uneven than those of control films. There are more ripples or protruding strips on the surfaces of MTGasetreated films (Fig. 2, panel B). This result seems to be conformable with that of surface hydrophobicity (Table 4) and moisture loss rate of film-forming solutions (Fig. 1), and can be accounted for the aggregation of exposed hydrophobic groups of SPI induced by
Fig. 2. SEM micrographs (at 400× magnification) of surfaces of control and MTGase-treated films of SPI. Panel A indicates the surfaces of control films of SPI, and panel B for those of MTGase-treated films. The number of 1, 2 and 3 represents the films cast with 0.6 g/g SPI of glycerol, a mixture of glycerol and sorbitol (1:1) and sorbitol, respectively.
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Fig. 3. SEM micrographs (at 3200× magnification) of cross-sections of control and MTGase-treated films of SPI, cast with 0.6 g/g SPI of glycerol. Panel A indicates the cross-section of control films, and panel B for that of MTGase-treated films.
MTGase. However, a contrary result was reported in pectin-soy flour films obtained in the presence of TGase (Mariniello et al., 2003), showing that the TGasetreated films of pectin-soy flour had a smoother surface and higher homogeneity. The presence of pectin or other components may account for it, because the interactions between pectin and proteins are obvious in this case. Furthermore, there are observable differences of surface microstructure of control and MTG-treated films cast with different plasticizers (Fig. 2). This is tightly related with the differences of water-holding ability of various plasticizers. The glycerol molecules have higher water-holding ability due to having more hydroxyls, therefore, the surfaces of films cast with glycerol are smoother. The SEM micrographs (at 3200× magnification) of cross-section of control and MTGase-treated SPI films cast with glycerol are shown in Fig. 3. The crosssection of MTGase-treated films shows a homogenous and compact microstructure (Fig. 3, panel A), and lacks the meshy or knitting-like structure shown in control films (Fig. 3, panel B). This result directly illuminates the higher TS and lower EB, TSM and transparency of MTGase-treated films comparable with the controls.
4. Conclusions The cross-linking treatment by transglutaminase was found to be an effective and practical method to improve properties of SPI films cast with var-
ious plasticizers (glycerol, sorbitol and a mixture thereof). The TS and surface hydrophobicity of SPI films increased significantly (P ≤ 0.05), and the E, MC, TSM and transparency decreased significantly (P ≤ 0.05), after treated by MTGase treatment. The MTG-treated films indicated coarse surfaces and more compact microstructure of cross-section, which directly accounted for their higher TS and lower EB and TSM compared to the controls.
Acknowledgements This work is part of the research projects of Chinese National Natural Science Fund (serial number: 20306008), sponsored by the NNSF of China. The authors gratefully acknowledge its financial support.
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