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Effect of γ-polyglutamate on the rheological properties and microstructure of tofu
Food Hydrocolloids 25 (2011) 1034e1040
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Effect of g-polyglutamate on the rheological properties and microstructure of tofu Chiung-Yuan Lee, Meng-I Kuo* Department of Food Science, Fu-Jen Catholic University, 510 Chung Cheng Rd., Taipei 24205, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2009 Accepted 5 October 2010
The effects of g-polyglutamates with different concentrations (0.1%, 0.15%, 0.2%) and molecular weights (high, medium, low) addition on the rheological properties, microstructure, and syneresis of tofu were studied. The addition of g-polyglutamate increased the gelation time, and decreased the storage modulus (G0 ) and the loss modulus (G00 ) of tofu. The molecular weight and concentration of g-polyglutamate effectively changed the rheological properties of tofu. The network of tofu without g-polyglutamate addition was constructed by fine strands in a dense arrangement as seen by using scanning electron microscope. However, the addition of g-polyglutamate reduced the thickness of the strands in tofu network. Tofu syneresis was also reduced by the addition of g-polyglutamate. Increase the concentration of g-polyglutamate significantly decreased the syneresis of tofu. This trend was more evident on the tofu with high molecular weight g-polyglutamate. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Tofu g-Polyglutamate Rheological property Microstructure Syneresis
1. Introduction Soybeans contain 35e40% of protein (on a dry wet basis) and have been processed to make various forms of soy foods. About 90% of soybean proteins are consumed in the form of tofu in Asia. Tofu is usually recognized as a salt- or acid-coagulated soy protein gel containing water, soy lipid, and other constituents entrapped inside its network. Glycinin (11S globulin) and b-conglycinin (7S globulin) are two major proteins in soybeans, which account for about 65e80% (by weight) of the total seed proteins present (Liu, 1999). In commercial tofu production, the 11S and 7S globulins can be induced to form gels by heat and coagulant addition. According to Kohyama, Sano, and Doi (1995), heat-induced denaturation resulted in the exposure of the hydrophobic regions of the soy proteins. These denatured soy proteins were negatively charged (Kohyama & Nishinari, 1993). Coagulant addition neutralized the net charge of denatured soy proteins. Consequently, the hydrophobic interaction induced the random aggregation of denatured soy proteins, leading to the gel formation of tofu (deMan, deMan, & Gupta, 1986). Rearrangement of the gel network during storage was still in progress, resulting in the increase of syneresis in tofu (Lee, 2007; Shen, 2008). Several macromolecules have been applied as food additives in tofu preparation to modify its texture, or to extend its shelflife (Chang, Lin, & Chen, 2003; Karim, Sulebele, Azhar, & Ping, 1999; Kim & Han, 2003). * Corresponding author. Tel.: þ886 2 2905 2019; fax: þ886 2 2209 3271. E-mail address: [email protected] (M.-I. Kuo). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.10.001
The g-polyglutamic acid (g-polyglutamate, H form) and the g2þ 2þ forms) are non-toxic polyglutamates (Naþ, Kþ, NHþ 4 , Ca , and Mg polypeptides produced by Bacillus subtitlis through a fermentation process (Ho, Yang, & Yang, 2006). They are consisted of numerous glutamic acid units connected by a-amino and g-carboxyl groups. These polypeptides usually have a molecular weight ranging from 5000 up to 900,000. The g-polyglutamates not only possess excellent water absorption property but also have good capacity in coordination of metal ions (Ho et al., 2006). Those properties allow it to have a broad range of industrial applications, including the use of nutrition supplements and food additives. Although gpolyglutamate has been reported to be used as an antifreeze agent, or the carrier for encapsulation (Chiu et al., 2007; Mitsuiki, Mizuno, Tanimoto, & Motoki, 1998), its water adsorption capacity have not been widely studied in the food systems. Understanding the mechanism involving in the interactions between soy proteins and macromolecules is important for exploring its potential in developing a novel gel texture. Dynamic rheological analysis along with small amplitude oscillatory tests has been used to give a general indication of the structure change in soy protein gel under non-destructive condition (Apichartsrangkoon, 2003; Kohyama et al., 1995; Kohyama, Yoshida, & Nishinar, 1992; Maltais, Remondetto, & Subirade, 2008). Scanning electron microscope (SEM) has been used to provide a stereoscopic image of the fine structure of tofu (Kao, Su, & Lee, 2003; Shen, 2008). The objective of this study was to investigate the effect of g-polyglutamate addition on the microstructure and the rheological properties of tofu.
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2. Materials and methods 2.1. Materials Non-GMO soybeans were purchased from a local supplier. The
g-polyglutamates in Naþ form of low molecular weight (low MW, 200e400 kDa), medium molecular weight (medium MW, 600e800 kDa), and high molecular weight (high MW,1000e1500 kDa) were provided by Vedan Enterprise Co (Taichung, Taiwan). Glucono-dlactone (GDL) was obtained from SigmaeAldrich Co (St. Louis, USA). 2.2. Preparation of tofu Tofu was prepared according to the method as in Liu and Chang (2003) with a few modifications. Soybeans (1000 g) were washed and soaked in a tank of 3000 mL distilled water at 25 C. After 8 h of soaking, another 3000 mL of distilled water was added and soybeans were ground with water in a food grinder (CL-010, Great Yen Electric Food Grinder Co. Ltd., Taoyuan, Taiwan). Subsequently, soymilk was strained through a 120 mesh sieve, and was heated to 95 C for 5 min with regular stirring. The cooked soymilk was cooled in the refrigerator to 10 C and mixed with 0.3% w/w GDL alone, or together with g-polyglutamate at 0.1%, 0.15%, and 0.2% concentrations. The mixture was then transferred to a container (10 8 cm) and was heated in the water bath at 80 C for 30 min. The pH of mixture was recorded during heating. After the tofu was cooled down to room temperature, it was stored for 1 day at 4 C for further analysis. Tofu that was prepared with GDL alone was used as control.
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the box at 4 C for 24 h. The total liquid exuded during the 24 h storage period was weighted. Syneresis was then expressed as the percentage of exuded liquid weight to the sliced tofu sample weight. 2.5. Microstructure analysis Scanning electron microscope (S-3000N, Hitachi High-Technologies Co., Japan) was used to examine the microstructure of tofu. Tofu was cut into 5 5 5 mm cubes. Samples were frozen in liquid nitrogen and freeze-dried. Dried samples were placed on the aluminum stub and were fixed by using double-sided adhesive carbon-tapes. The nature-broken side of each cubic sample was faced on the top. Samples were then sputter-coated with gold. The voltage of microscope was operated at 15 kV for observation. 2.6. Statistical analysis The Statistical Analysis System (SAS Institute Inc., Cary North, USA) software was used for data analysis. Effects of g-polyglutamates addition were determined by using the Analysis of Variance (ANOVA), whereas Duncan’s multiple range test was used for multiple compression among the means of the samples. 3. Results and discussion 3.1. Gelation Fig. 1 shows the pH of soymilk containing 0.3% GDL and 0.2%
g-polyglutamates in different molecular weights during heating at 2.3. Rheological measurement A controlled stress dynamic rheometer (AR2000 ex, TA Instruments, Inc., New Castle, USA) was used to investigate the dynamic viscoelastic properties of samples. Stress amplitude sweeps were performed firstly to ensure that all measurements were carried out within the linear viscoelastic region (data not shown). According to the results, the stress amplitude of 1 Pa was chosen for further analysis. The gelation process of tofu was observed based on the method of Maltais et al. (2008) with a few modifications. After the addition of GDL or g-polyglutamate, cooked soymilk was immediately loaded onto the rheometer. A cone and plate geometry (40 mm diameter) with a cone angle of 2 was used. An oscillating stress was applied at the frequency of 1 Hz. Temperature sweep of sample was performed from 25 C to 80 C at the heating rate of 5 C/min. Time sweep of sample was then carried out at 80 C for 30 min. Storage modulus (G0 ) and loss modulus (G00 ) were recorded as a function of time. The rheological properties of tofu were measured according to the method of Molina, Puppo, and Wagner (2004). Tofu was cut into cylinders with 40 mm in diameter and 2 mm in thickness. The parallel plate geometry (40 mm diameter) with a gap of 2 mm was used. Frequency sweep was conducted on the sample at 4 C from 0.01 to 10 Hz. All the rheological measurements were carried out in triplicates.
80 C for 30 min. The initial pH of soymilks was between 6.0 and 6.5. The pH of soymilk did not change significantly during 30 min heating. However, the pH of control soymilk containing GDL decreased gradually during heating. Similar changes in pH of soymilks containing GDL and 0.2% g-polyglutamates in different molecular weights were observed. The final pH of soymilks was between 4.5 and 5.0. These results indicated that the addition of g-polyglutamates did not affect the pH change of soymilk containing GDL during heating. Fig. 2 illustrates the viscoelastic behavior of soymilk with 0.3% GDL during non-isothermal heating from 25 C to 80 C at 5 C/min followed by isothermal heating at 80 C for 30 min Fig. 2A shows the gelation curve of soymilk as a function of temperature. The values of G0 were not stable around 0.01 Pa, and the values of G00 decreased
2.4. Syneresis analysis The syneresis of tofu was measured according to the method proposed by Amstrong, Hill, Schrooyen, and Mitchell (1994) with modifications. Tofu was cut into slices with 15 mm in diameter and 5 mm in thickness. A group of six pieces of sliced sample were weighted and put on the stainless steel mesh inside a plastic box. The mesh was lifted by small sticks. Thus, the exuded liquid could drip away from the sliced tofu samples. The box was sealed with parafilm to prevent the evaporation of free water. The sample was stored in
Fig. 1. The pH of soymilk containing 0.3% (w/w) GDL and 0.2% g-polyglutamates in different molecular weights during heating at 80 C for 30 min.
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10
A
G′ & G″ (Pa)
1
0.1
0.01
0.001 20
30
40
50
60
70
80
90
o
Temperature ( C) 1000
protons in the solution leading to a small drop in the pH value (Fig. 1). Based on the DLVO (Derjaguin-Landau-Verwey- Overbeek) theory, the release of protons by adding GDL neutralized the surface charges of soluble aggregates in heated soymilk. Consequently, the van der Waals attraction and hydrophobic interaction of neutralized soluble aggregates became predominant, and induced the coagulation, leading to the gel formation (deMan et al., 1986; Kohyama et al., 1995; Maltais et al., 2008). Fig. 3 shows the effect of high MW g-polyglutamate addition on tofu gelation. The addition of g-polyglutamate significantly increased the heating temperature and the time that were needed for reaching the gelation point (the point at which G0 was higher than G00 ) of tofu. The gelation time of tofu increased significantly when the concentration of high MW g-polyglutamate increased from 0.10% to 0.20%. Table 1 shows the viscoelastic properties of tofu prepared with GDL and g-polyglutamate, and the viscoelastic properties of a control tofu prepared with single use of GDL during heat-induced gelation.
B
A
100
100
G′ & G″ (Pa)
G′ & G″ (Pa)
1000
10
10 1 0.1 0.01
1 0
5
10
15
20
25
30
0.001
35
1000
Time (min)
G′ & G″ (Pa)
slightly as temperature increased from 25 C to 75 C. The G00 values were higher than the G0 values, indicating that the soymilk behaviors as a viscous solution. When the temperature was further increased from 75 C to 80 C, both G0 and G00 values increased significantly. The values of G0 were higher than that of G00 as the temperature above 76.9 C, suggests that the phase transition of viscous soymilk to the viscoelastic soy curd (tofu) occurred. Fig. 2B shows the gelation curve of tofu as a function of time at 80 C. Although the values of G0 and G00 both increased with time, the change in G0 values was more evident than G00 values. The G0 and G00 were 331 Pa and 21 Pa, respectively, after heating at 80 C for 30 min. Nagano, Hirotsuka, Mori, Kohyama, and Nishinari (1992) found that the denaturation process was a prerequisite for the gelation of globular proteins. In the present study, soymilk was heated at 95 C for 5 min before the addition of GDL. Thus, the denaturation of soy proteins in soymilk was completed during heating. The quaternary structure of soy proteins was dissociated into subunits during the heating process. Subsequently, they were associated into soluble aggregates with the hydrophobic regions and negative charges exposed outside by electrostatic attraction and disulfide linkage (Kohyama & Nishinari, 1993; Kohyama et al., 1995; Koshiyama, Hamano, & Fukushima, 1981; Mori, Nakamura, & Utsumi, 1981; Utsumi, Damodaran, & Kinsella, 1984). The addition of GDL with non-isothermal heating resulted in gradually cleaving GDL into gluconic acid, and in generating
10 1 0.1 0.01 0.001 1000
C
100
G′ & G″ (Pa)
Fig. 2. Heat-induced gelation curves of tofu with 0.3% (w/w) GDL as a function of temperature (25e80 C, at 5 C/min) (A) and time (30 min, at 80 C) (B) at a stress amplitude of 1 Pa (C) Storage modulus G0 ; (B) Loss modulus G00 .
B
100
10 1 0.1 0.01 0.001 10
20
30
40
50
Time (min) Fig. 3. Heat-induced gelation curves of tofu with 0.3% (w/w) GDL and high molecular weight g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), 0.20% (C) (w/w) as a function of time (30 min, at 80 C) at a stress amplitude of 1 Pa (C) Storage modulus G0 ; (B) loss modulus G00 .
C.-Y. Lee, M.-I. Kuo / Food Hydrocolloids 25 (2011) 1034e1040 Table 1 The viscoelastic properties of tofu containing 0.3% (w/w) GDL and g-polyglutamates in different molecular weights and concentrations during heat-induced gelation.a
gepolyglutamate
G*gelc/(Tgel)c (Pa)/(min)
G0 maxc (Pa)
G00
High MW 0.10% 0.15% 0.20%
0.56 0.02E/8.3 0.9A 0.74 0.01F/12.6 0.1C 0.87 0.04G/12.6 0.1C
248.7 18.1G 200.5 10.5F 112.1 1.7D
19.6 0.2G 16.5 1.1F 8.5 0.1BC
Medium MW 0.10% 0.15% 0.20%
0.20 0.04A/9.9 0.6B 0.38 0.08C/11.0 0.7B 0.41 0.03CD/14.7 0.7D
196.1 14.2F 139.1 5.8E 44.9 1.4A
14.7 1.1E 10.7 1.0D 3.5 0.1A
Low MW 0.10% 0.15% 0.20% Control
0.18 0.30 0.46 0.37
139.2 0.9E 86.3 4.2C 64.4 0.6B 331.8 0.8H
9.5 0.7CD 7. 3 2.1B 6.9 0.2B 21.8 0.8H
0.04A/10.8 0.7B 0.02B/13.8 1.5CD 0.08D/17.1 0.9E 0.04C/eb
c
10000
(Pa)
G′ & G″ (Pa)
max
1037
1000
100
10 0.01
The G*gel (G* at gelation point) of tofu was increased and Tgel (time to reach the gelation point at 80 C) was decreased as the molecular weight of added g-polyglutamate increased. Increase the concentration of added g-polyglutamate increased both G*gel and Tgel of tofu. Also, the G0 max and G00 max of tofu increased with increasing the molecular weight of added g-polyglutamate. However, both G0 max and G00 max of tofu decreased with increasing the concentration of added gpolyglutamate. Tofu prepared with g-polyglutamate showed a significantly lower G0 max and G00 max values than the control tofu. Since the g-polyglutamate contains carboxyl group, it exists as a negatively charged polymer in the aqueous solution. The addition of g-polyglutamate in heated soymilk would increase the amount of negative charges. When GDL was added, the neutralization on the surface charge of soluble aggregates was slow and the surface charge of soluble aggregates might not be totally screened due to the presence of g-polyglutamate in the heated soymilk, leading to the retardation of tofu gelation and the reduction of van der Waals attraction and hydrophobic interaction between soluble aggregates.
10000
10
A
1000
100 0.01
0.1
1
10
Frequency (Hz)
3.2. Rheological properties
1000
G′′ (Pa)
After gelation, tofu was further moved to store at 4 C for 1 d, and the rheological properties, syneresis, and microstructure of tofu were evaluated. A typical plot of frequency sweep for control tofu with GDL is shown in Fig. 4. Both G0 and G00 increased slightly as the frequency increased. G0 was higher than G00 indicating that the tofu had formed a continuous network structure (Steffe, 1996). Fig. 5 shows the G0 and G00 of tofu with high MW g-polyglutamate as a function of frequency. The G0 and G00 of tofu with g-polyglutamate also increased slightly with the increase of frequency. Yoo, Figueiredo, and Rao (1994) suggested that frequency dependent property of G0 and G00 might be due to a faster molecular mobility of solid particles at higher frequencies. The G0 , G00 , and tand values at the frequency of 1 Hz of tofu containing GDL and g-polyglutamates with various molecular weights and concentrations were summarized in Table 2. The G0 and G00 of tofu decreased significantly with the decrease of the molecular weight of added g-polyglutamate. The increase of the concentration of high MW g-polyglutamate significantly decreased the G0 and G00 of tofu. Changes in the G0 of tofu containing medium MW g-polyglutamate in different concentrations showed a similar trend. However, the G0 and G00 of tofu were not affected by the concentration of the low MW g-polyglutamate. Generally, the molecular weight and concentration
1
Fig. 4. The rheological parameters of tofu with 0.3% (w/w) GDL as a function of frequency (0.01e10 Hz) at the stress amplitude of 1 Pa (C) Storage modulus G0 ; (B) loss modulus G00 .
G′ (Pa)
Values are mean of triplicates. Mean values with different superscript uppercase letters within a column indicate significant difference (P < 0.05). b Tofu gelation at 76.9 C. c G*gel ¼ G* at gelation point; Tgel ¼ Time to reach the gelation point at 80 C; G0 max ¼ Maximum value of G0 ; G00 max ¼ Maximum value of G00 .
0.1
Frequency (Hz)
a
B
100
10 0.01
0.1
1
10
Frequency (Hz) Fig. 5. The storage modulus (G0 ) and loss modulus (G00 ) of tofu with 0.3% (w/w) GDL and high molecular weight g-polyglutamate at concentrations of 0.10% (C), 0.15% (B), and 0.20% (;) (w/w) as a function of frequency (0.01e10 Hz) at a stress amplitude of 1 Pa.
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Table 2 The viscoelastic parameters at frequency 1 Hz of tofu containing 0.3% (w/w) GDL and g-polyglutamates in different molecular weights and concentrations.a
g-polyglutamate
G0 (Pa)
G00 (Pa)
tand
High MW 0.10% 0.15% 0.20%
1003.4 28.1D 793.3 71.9C 517.2 33.6A
172.3 6.0A 136.7 12.9B 91.5 4.9C
0.172 0.005BC 0.172 0.001BC 0.177 0.003BC
Medium MW 0.10% 0.15% 0.20%
811.5 118.2C 694.8 50.2B 626.8 17.0B
143.5 28.7B 123.1 7.7B 126.8 11.5B
0.177 0.001BC 0.177 0.010BC 0.202 0.002AB
Low MW 0.10% 0.15% 0.20% Control
458.3 24.4A 499.2 11.5A 451.1 17.0A 1030.7 1.4D
86.2 6.2C 78.9 17.0C 96.5 17.0C 176.0 1.7A
0.188 0.158 0.213 0.171
0.001ABC 0.027C 0.030A 0.002BC
a Values are mean of triplicates. Mean values with different superscript uppercase letters within a column indicate significant difference (P < 0.05).
of added g-polyglutamate did not influence the tand of tofu. These results suggested that although differences in the moduli values of tofu containing GDL and g-polyglutamates in different molecular weights and concentrations were observed, the rheological systems were similar. The tand of tofu was between 0.158 and 0.213 and was much smaller than unity, indicating a typical weak gel structure (Clark & Ross-Murphy, 1987). According to Ferry (1980), the concept of entanglement coupling plays a crucial role in illustrating the viscoelastic properties of undiluted system with high molecular weight polymers (molecular weight above 20,000). High MW g-polyglutamates apparently interacted with soy proteins in tofu at a higher extent. More entanglements might take place between high MW g-polyglutamate and protein molecules. This could result in a more stable gel network and a firmer texture of tofu with high MW g-polyglutamate, when it was compared to the tofu containing with low MW g-polyglutamate. However, the electrostatic repulsion caused by g-polyglutamate reduced the hydrophobic interactions between soy proteins, resulting in a weaker tofu structure as the increase of the concentration of g-polyglutamate. 3.3. Microstructure The microstructure of control tofu is presented in Fig. 6. Control tofu revealed a honeycomb-like structure with pores of relatively
Fig. 7. SEM micrographs of tofu with 0.3% (w/w) GDL and high MW g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), and 0.20% (C) (w/w). Scale bars represent 100 mm.
Fig. 6. SEM micrographs of tofu formed at 0.3% (w/w) GDL. Scale bar represents 100 mm.
even size. The network of tofu was constructed by fine strands in a dense arrangement. Doi (1993) demonstrated two models of globular protein network: random aggregation and a string of beads structure. Gelation of denatured soy proteins at low cation concentration was preferential along linear direction since the surface charges of denatured soy proteins were screened gradually. Thus, the interaction among the hydrophobic regions of soy
C.-Y. Lee, M.-I. Kuo / Food Hydrocolloids 25 (2011) 1034e1040
Fig. 8. SEM micrographs of tofu with 0.3% (w/w) GDL and medium MW g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), and 0.20% (C) (w/w). Scale bars represent 100 mm.
proteins was favored, resulting in a filamentous network (Hermansson, 1985; Maltais et al., 2008; Remondetto & Subirade, 2003). In the present study, the surface charges of the soluble aggregates in the heated soymilk were diminished gradually by protons, promoting the gelation of tofu with filamentous structure. The microstructure of tofu prepared with high MW and medium MW g-polyglutamate are shown in Figs. 7 and 8, respectively.
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Fig. 9. SEM micrographs of tofu with 0.3% (w/w) GDL and low MW g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), and 0.20% (C) (w/w). Scale bars represent 100 mm.
The structure of tofu with high MW g-polyglutamate showed a discontinuous network with small pores uniformly distributed. The addition of g-polyglutamate decreased the strands thickness of tofu network. The structure of tofu with medium MW g-polyglutamate also showed a discontinuous network. However, the pores of network were larger in tofu with medium MW gpolyglutamate than those in tofu with high MW g-polyglutamate. Increase of the concentration of added g-polyglutamate increased
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C.-Y. Lee, M.-I. Kuo / Food Hydrocolloids 25 (2011) 1034e1040
to the project. Special thanks to Dr. Shyh-Forng Guo for critical comment on the manuscript.
10 9
ef
f
ef
8
de cd
Syneresis(%)
7 6 5 ab
4 a
3 2 1 0
0.10% HMW
0.15% HMW
References
bc
bc
0.20% HMW
0.10% MMW
0.15% MMW
0.20% MMW
0.10% LMW
0.15% LMW
0.20% LMW
γ-polyglutamate
Fig. 10. The syneresis of tofu containing 0.3% (w/w) GDL and g-polyglutamates in different molecular weights and concentrations. The standard error is represented by the length of the bars. abcd Different letters indicate significant difference (P < 0.05) between the means of syneresis values. HMW ¼ high molecular weight; MMW ¼ medium molecular weight; LMW ¼ low molecular weight.
the pore size of tofu network. The microstructure of tofu with low MW g-polyglutamate is presented in Fig. 9. The strand thickness of tofu network with low MW g-polyglutamate was less than that of tofu with high MW or medium MW g-polyglutamate. There were many discontinuous fragments and irregular pores in the network of tofu with low MW g-polyglutamate. These might be due to the decrease of interaction strength between the soluble aggregates in tofu network. The concentration of added low MW g-polyglutamate did not affect the microstructure of tofu. 3.4. Syneresis Fig. 10 presents the syneresis of tofu prepared with GDL and
g-polyglutamates in different molecular weights and concentrations. The syneresis of control tofu was 17.11%. Sun and Breene (1991) mentioned that the syneresis of protein gel during storage period was caused by the increase in the cross-linking between protein molecules via various interactions, which forced the exudation of water entrapped within the gel. The addition of g-polyglutamate decreased the syneresis of tofu to below 10%, indicating that the water-holding capacity of tofu was increased by the addition of gpolyglutamate. Increase the concentration of added g-polyglutamate significantly decreased the syneresis of tofu. This trend was more pronounced on the tofu with high MW g-polyglutamate. Changes in the rheological properties, microstructure, and syneresis of tofu by g-polyglutamate were observed in this study. The g-polyglutamate did not serve as coagulant. Rather, it worked as water-binding agent to improve the syneresis in the preparation of tofu. However, excess negative charges induced by gpolyglutamate in the heated soymilk inhibited the coagulation of soluble aggregates, causing the changes in the rheological properties and the microstructure of tofu. However, the entanglement between high MW g-polyglutamates and protein molecules might stabilize the tofu structure. Therefore, the structure and rheological properties between tofu with high MW g-polyglutamate and that with low MW g-polyglutamate were different, leading to a different extent of tofu syneresis. From these results, it is concluded that gpolyglutamate could be a practical food additive to improve the shelf-life, and to modify the texture of tofu, though further research on sensory evaluation is needed. Acknowledgments The authors express grateful thanks to the National Science Council of Taiwan (NSC) for its financial support (95-2313-B-030-003)
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Texture and microstructure of soybean curd (tofu) as affected by different coagulants. Food Microstructure, 5, 83e89. Doi, E. (1993). Gels and gelling of globular proteins. Trends in Food Science and Technology, 4, 1e5. Ferry, J. D. (1980). Viscoelastic properties of polymers (3rd ed.). New York: John Wiley & Sons, Inc. Hermansson, A. (1985). Structure of soya glycinin and conglycinin gels. Journal of the Science of Food and Agriculture, 36, 822e832. Ho, G. H., Yang, T. H., & Yang, J. (2006). Dietary products comprising one or more of gamma-polyglutamic acid (gamma-polyglutamate, H form) and gamma-polyglutamates for use as nutrition supplements. United State Patent, No. US 2006/ 0257468 A1. Kao, F. J., Su, N. W., & Lee, M. H. (2003). Effect of calcium sulfate concentration in soymilk on the microstructure of firm tofu and the protein constitutions in tofu whey. Journal of Agriculture and Food Chemistry, 51, 6211e6216. Karim, A. A., Sulebele, G. A., Azhar, M. E., & Ping, C. Y. (1999). Effect of carrageenan on yield and properties of tofu. Food Chemistry, 66, 159e165. Kim, M., & Han, J. S. (2003). Evaluation of physico-chemical characteristics and microstructure of tofu containing high viscosity chitosan. International Journal of Food Science and Technology, 37, 277e283. Kohyama, K., & Nishinari, K. (1993). Rheological studies on the gelation process of soybean 7S and 11S proteins in the presence of glucono-d-lactone. Journal of Agricultural and Food Chemistry, 41, 8e14. Kohyama, K., Sano, Y., & Doi, E. (1995). Rheological characteristics and gelation mechanism of tofu (soybean curd). Journal of Agricultural and Food Chemistry, 43, 1808e1812. Kohyama, K., Yoshida, M., & Nishinar, K. (1992). Rheological study on gelation of soybean 11S protein by Glucono-d-lactone. Journal of Agricultural and Food Chemistry, 40, 740e744. Koshiyama, I., Hamano, M., & Fukushima, D. (1981). A heat denaturation study of the 11S globulin in soybean seeds. Food Chemistry, 6, 309e322. Lee, C. Y. (2007). Investigation on the physicochemical properties of packed tofu. MS Thesis, Taiwan. Liu, K. S. (1999). Soybeans chemistry, technology and utilization. Gaithersburg, Maryland: Aspen Publishers. Liu, Z. S., & Chang, S. K. C. (2003). Development of a rapid titration method for predicting optimal coagulant concentration for filled tofu. Journal of Agriculture and Food Chemistry, 51, 5214e5221. Maltais, A., Remondetto, G. E., & Subirade, M. (2008). Mechanisms involves in the formation and structure of soya protein cold-set gels: a molecular and supramolecular investigation. Food Hydrocolloids, 22, 550e559. Mitsuiki, M., Mizuno, A., Tanimoto, H., & Motoki, M. (1998). Relationship between the antifreeze activities and the chemical structures of oligo- and poly(glutamic acid)s. Journal of Agricultural and Food Chemistry, 46, 891e895. Molina, O. S. E., Puppo, M. C., & Wagner, J. R. (2004). Relationship between structural changes and functional properties of soy protein isolates-carrageenan systems. Food Hydrocolloids, 18, 1045e1053. Mori, T., Nakamura, T., & Utsumi, S. (1981). Gelation mechanism of soybean 11S globulin: formation of soluble aggregates as transient intermediates. Journal of Food Science, 47, 26e30. Nagano, T., Hirotsuka, M., Mori, H., Kohyama, K., & Nishinari, K. (1992). Dynamic viscoelastic study on the gelation of 7S globulin from soybeans. Journal of Agricultural and Food Chemistry, 40, 941e944. Remondetto, G. E., & Subirade, M. (2003). Molecular mechanisms of Fe2þ-induced b-lactoglobulin cold gelation. Biopolymers, 69, 461e469. Shen, Y. R. (2008). Investigation on the state of protein and water of calcium sulfate tofu. MS thesis, Taiwan. Steffe, J. F. (1996). Rheological methods in food process engineering (2nd ed.). Michigan: Freeman Press. Sun, N., & Breene, W. M. (1991). Calcium sulphate concentration influence on yield and quality of tofu from five soybean varieties. Journal of Food Science, 56, 1604e1607. Utsumi, S., Damodaran, S., & Kinsella, J. E. (1984). Heat-induced interactions between soybean proteins: preferential association of 11S basic subunits and b subunits of 7S. Journal of Agricultural and Food Chemistry, 32, 1406e1412. Yoo, B., Figueiredo, A. A., & Rao, M. A. (1994). Rheological properties of mesquite seed gum in steady and dynamic shear. LWT-Food Science and Technology, 27, 151e157.
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Effect of g-polyglutamate on the rheological properties and microstructure of tofu Chiung-Yuan Lee, Meng-I Kuo* Department of Food Science, Fu-Jen Catholic University, 510 Chung Cheng Rd., Taipei 24205, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2009 Accepted 5 October 2010
The effects of g-polyglutamates with different concentrations (0.1%, 0.15%, 0.2%) and molecular weights (high, medium, low) addition on the rheological properties, microstructure, and syneresis of tofu were studied. The addition of g-polyglutamate increased the gelation time, and decreased the storage modulus (G0 ) and the loss modulus (G00 ) of tofu. The molecular weight and concentration of g-polyglutamate effectively changed the rheological properties of tofu. The network of tofu without g-polyglutamate addition was constructed by fine strands in a dense arrangement as seen by using scanning electron microscope. However, the addition of g-polyglutamate reduced the thickness of the strands in tofu network. Tofu syneresis was also reduced by the addition of g-polyglutamate. Increase the concentration of g-polyglutamate significantly decreased the syneresis of tofu. This trend was more evident on the tofu with high molecular weight g-polyglutamate. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Tofu g-Polyglutamate Rheological property Microstructure Syneresis
1. Introduction Soybeans contain 35e40% of protein (on a dry wet basis) and have been processed to make various forms of soy foods. About 90% of soybean proteins are consumed in the form of tofu in Asia. Tofu is usually recognized as a salt- or acid-coagulated soy protein gel containing water, soy lipid, and other constituents entrapped inside its network. Glycinin (11S globulin) and b-conglycinin (7S globulin) are two major proteins in soybeans, which account for about 65e80% (by weight) of the total seed proteins present (Liu, 1999). In commercial tofu production, the 11S and 7S globulins can be induced to form gels by heat and coagulant addition. According to Kohyama, Sano, and Doi (1995), heat-induced denaturation resulted in the exposure of the hydrophobic regions of the soy proteins. These denatured soy proteins were negatively charged (Kohyama & Nishinari, 1993). Coagulant addition neutralized the net charge of denatured soy proteins. Consequently, the hydrophobic interaction induced the random aggregation of denatured soy proteins, leading to the gel formation of tofu (deMan, deMan, & Gupta, 1986). Rearrangement of the gel network during storage was still in progress, resulting in the increase of syneresis in tofu (Lee, 2007; Shen, 2008). Several macromolecules have been applied as food additives in tofu preparation to modify its texture, or to extend its shelflife (Chang, Lin, & Chen, 2003; Karim, Sulebele, Azhar, & Ping, 1999; Kim & Han, 2003). * Corresponding author. Tel.: þ886 2 2905 2019; fax: þ886 2 2209 3271. E-mail address: [email protected] (M.-I. Kuo). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.10.001
The g-polyglutamic acid (g-polyglutamate, H form) and the g2þ 2þ forms) are non-toxic polyglutamates (Naþ, Kþ, NHþ 4 , Ca , and Mg polypeptides produced by Bacillus subtitlis through a fermentation process (Ho, Yang, & Yang, 2006). They are consisted of numerous glutamic acid units connected by a-amino and g-carboxyl groups. These polypeptides usually have a molecular weight ranging from 5000 up to 900,000. The g-polyglutamates not only possess excellent water absorption property but also have good capacity in coordination of metal ions (Ho et al., 2006). Those properties allow it to have a broad range of industrial applications, including the use of nutrition supplements and food additives. Although gpolyglutamate has been reported to be used as an antifreeze agent, or the carrier for encapsulation (Chiu et al., 2007; Mitsuiki, Mizuno, Tanimoto, & Motoki, 1998), its water adsorption capacity have not been widely studied in the food systems. Understanding the mechanism involving in the interactions between soy proteins and macromolecules is important for exploring its potential in developing a novel gel texture. Dynamic rheological analysis along with small amplitude oscillatory tests has been used to give a general indication of the structure change in soy protein gel under non-destructive condition (Apichartsrangkoon, 2003; Kohyama et al., 1995; Kohyama, Yoshida, & Nishinar, 1992; Maltais, Remondetto, & Subirade, 2008). Scanning electron microscope (SEM) has been used to provide a stereoscopic image of the fine structure of tofu (Kao, Su, & Lee, 2003; Shen, 2008). The objective of this study was to investigate the effect of g-polyglutamate addition on the microstructure and the rheological properties of tofu.
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2. Materials and methods 2.1. Materials Non-GMO soybeans were purchased from a local supplier. The
g-polyglutamates in Naþ form of low molecular weight (low MW, 200e400 kDa), medium molecular weight (medium MW, 600e800 kDa), and high molecular weight (high MW,1000e1500 kDa) were provided by Vedan Enterprise Co (Taichung, Taiwan). Glucono-dlactone (GDL) was obtained from SigmaeAldrich Co (St. Louis, USA). 2.2. Preparation of tofu Tofu was prepared according to the method as in Liu and Chang (2003) with a few modifications. Soybeans (1000 g) were washed and soaked in a tank of 3000 mL distilled water at 25 C. After 8 h of soaking, another 3000 mL of distilled water was added and soybeans were ground with water in a food grinder (CL-010, Great Yen Electric Food Grinder Co. Ltd., Taoyuan, Taiwan). Subsequently, soymilk was strained through a 120 mesh sieve, and was heated to 95 C for 5 min with regular stirring. The cooked soymilk was cooled in the refrigerator to 10 C and mixed with 0.3% w/w GDL alone, or together with g-polyglutamate at 0.1%, 0.15%, and 0.2% concentrations. The mixture was then transferred to a container (10 8 cm) and was heated in the water bath at 80 C for 30 min. The pH of mixture was recorded during heating. After the tofu was cooled down to room temperature, it was stored for 1 day at 4 C for further analysis. Tofu that was prepared with GDL alone was used as control.
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the box at 4 C for 24 h. The total liquid exuded during the 24 h storage period was weighted. Syneresis was then expressed as the percentage of exuded liquid weight to the sliced tofu sample weight. 2.5. Microstructure analysis Scanning electron microscope (S-3000N, Hitachi High-Technologies Co., Japan) was used to examine the microstructure of tofu. Tofu was cut into 5 5 5 mm cubes. Samples were frozen in liquid nitrogen and freeze-dried. Dried samples were placed on the aluminum stub and were fixed by using double-sided adhesive carbon-tapes. The nature-broken side of each cubic sample was faced on the top. Samples were then sputter-coated with gold. The voltage of microscope was operated at 15 kV for observation. 2.6. Statistical analysis The Statistical Analysis System (SAS Institute Inc., Cary North, USA) software was used for data analysis. Effects of g-polyglutamates addition were determined by using the Analysis of Variance (ANOVA), whereas Duncan’s multiple range test was used for multiple compression among the means of the samples. 3. Results and discussion 3.1. Gelation Fig. 1 shows the pH of soymilk containing 0.3% GDL and 0.2%
g-polyglutamates in different molecular weights during heating at 2.3. Rheological measurement A controlled stress dynamic rheometer (AR2000 ex, TA Instruments, Inc., New Castle, USA) was used to investigate the dynamic viscoelastic properties of samples. Stress amplitude sweeps were performed firstly to ensure that all measurements were carried out within the linear viscoelastic region (data not shown). According to the results, the stress amplitude of 1 Pa was chosen for further analysis. The gelation process of tofu was observed based on the method of Maltais et al. (2008) with a few modifications. After the addition of GDL or g-polyglutamate, cooked soymilk was immediately loaded onto the rheometer. A cone and plate geometry (40 mm diameter) with a cone angle of 2 was used. An oscillating stress was applied at the frequency of 1 Hz. Temperature sweep of sample was performed from 25 C to 80 C at the heating rate of 5 C/min. Time sweep of sample was then carried out at 80 C for 30 min. Storage modulus (G0 ) and loss modulus (G00 ) were recorded as a function of time. The rheological properties of tofu were measured according to the method of Molina, Puppo, and Wagner (2004). Tofu was cut into cylinders with 40 mm in diameter and 2 mm in thickness. The parallel plate geometry (40 mm diameter) with a gap of 2 mm was used. Frequency sweep was conducted on the sample at 4 C from 0.01 to 10 Hz. All the rheological measurements were carried out in triplicates.
80 C for 30 min. The initial pH of soymilks was between 6.0 and 6.5. The pH of soymilk did not change significantly during 30 min heating. However, the pH of control soymilk containing GDL decreased gradually during heating. Similar changes in pH of soymilks containing GDL and 0.2% g-polyglutamates in different molecular weights were observed. The final pH of soymilks was between 4.5 and 5.0. These results indicated that the addition of g-polyglutamates did not affect the pH change of soymilk containing GDL during heating. Fig. 2 illustrates the viscoelastic behavior of soymilk with 0.3% GDL during non-isothermal heating from 25 C to 80 C at 5 C/min followed by isothermal heating at 80 C for 30 min Fig. 2A shows the gelation curve of soymilk as a function of temperature. The values of G0 were not stable around 0.01 Pa, and the values of G00 decreased
2.4. Syneresis analysis The syneresis of tofu was measured according to the method proposed by Amstrong, Hill, Schrooyen, and Mitchell (1994) with modifications. Tofu was cut into slices with 15 mm in diameter and 5 mm in thickness. A group of six pieces of sliced sample were weighted and put on the stainless steel mesh inside a plastic box. The mesh was lifted by small sticks. Thus, the exuded liquid could drip away from the sliced tofu samples. The box was sealed with parafilm to prevent the evaporation of free water. The sample was stored in
Fig. 1. The pH of soymilk containing 0.3% (w/w) GDL and 0.2% g-polyglutamates in different molecular weights during heating at 80 C for 30 min.
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10
A
G′ & G″ (Pa)
1
0.1
0.01
0.001 20
30
40
50
60
70
80
90
o
Temperature ( C) 1000
protons in the solution leading to a small drop in the pH value (Fig. 1). Based on the DLVO (Derjaguin-Landau-Verwey- Overbeek) theory, the release of protons by adding GDL neutralized the surface charges of soluble aggregates in heated soymilk. Consequently, the van der Waals attraction and hydrophobic interaction of neutralized soluble aggregates became predominant, and induced the coagulation, leading to the gel formation (deMan et al., 1986; Kohyama et al., 1995; Maltais et al., 2008). Fig. 3 shows the effect of high MW g-polyglutamate addition on tofu gelation. The addition of g-polyglutamate significantly increased the heating temperature and the time that were needed for reaching the gelation point (the point at which G0 was higher than G00 ) of tofu. The gelation time of tofu increased significantly when the concentration of high MW g-polyglutamate increased from 0.10% to 0.20%. Table 1 shows the viscoelastic properties of tofu prepared with GDL and g-polyglutamate, and the viscoelastic properties of a control tofu prepared with single use of GDL during heat-induced gelation.
B
A
100
100
G′ & G″ (Pa)
G′ & G″ (Pa)
1000
10
10 1 0.1 0.01
1 0
5
10
15
20
25
30
0.001
35
1000
Time (min)
G′ & G″ (Pa)
slightly as temperature increased from 25 C to 75 C. The G00 values were higher than the G0 values, indicating that the soymilk behaviors as a viscous solution. When the temperature was further increased from 75 C to 80 C, both G0 and G00 values increased significantly. The values of G0 were higher than that of G00 as the temperature above 76.9 C, suggests that the phase transition of viscous soymilk to the viscoelastic soy curd (tofu) occurred. Fig. 2B shows the gelation curve of tofu as a function of time at 80 C. Although the values of G0 and G00 both increased with time, the change in G0 values was more evident than G00 values. The G0 and G00 were 331 Pa and 21 Pa, respectively, after heating at 80 C for 30 min. Nagano, Hirotsuka, Mori, Kohyama, and Nishinari (1992) found that the denaturation process was a prerequisite for the gelation of globular proteins. In the present study, soymilk was heated at 95 C for 5 min before the addition of GDL. Thus, the denaturation of soy proteins in soymilk was completed during heating. The quaternary structure of soy proteins was dissociated into subunits during the heating process. Subsequently, they were associated into soluble aggregates with the hydrophobic regions and negative charges exposed outside by electrostatic attraction and disulfide linkage (Kohyama & Nishinari, 1993; Kohyama et al., 1995; Koshiyama, Hamano, & Fukushima, 1981; Mori, Nakamura, & Utsumi, 1981; Utsumi, Damodaran, & Kinsella, 1984). The addition of GDL with non-isothermal heating resulted in gradually cleaving GDL into gluconic acid, and in generating
10 1 0.1 0.01 0.001 1000
C
100
G′ & G″ (Pa)
Fig. 2. Heat-induced gelation curves of tofu with 0.3% (w/w) GDL as a function of temperature (25e80 C, at 5 C/min) (A) and time (30 min, at 80 C) (B) at a stress amplitude of 1 Pa (C) Storage modulus G0 ; (B) Loss modulus G00 .
B
100
10 1 0.1 0.01 0.001 10
20
30
40
50
Time (min) Fig. 3. Heat-induced gelation curves of tofu with 0.3% (w/w) GDL and high molecular weight g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), 0.20% (C) (w/w) as a function of time (30 min, at 80 C) at a stress amplitude of 1 Pa (C) Storage modulus G0 ; (B) loss modulus G00 .
C.-Y. Lee, M.-I. Kuo / Food Hydrocolloids 25 (2011) 1034e1040 Table 1 The viscoelastic properties of tofu containing 0.3% (w/w) GDL and g-polyglutamates in different molecular weights and concentrations during heat-induced gelation.a
gepolyglutamate
G*gelc/(Tgel)c (Pa)/(min)
G0 maxc (Pa)
G00
High MW 0.10% 0.15% 0.20%
0.56 0.02E/8.3 0.9A 0.74 0.01F/12.6 0.1C 0.87 0.04G/12.6 0.1C
248.7 18.1G 200.5 10.5F 112.1 1.7D
19.6 0.2G 16.5 1.1F 8.5 0.1BC
Medium MW 0.10% 0.15% 0.20%
0.20 0.04A/9.9 0.6B 0.38 0.08C/11.0 0.7B 0.41 0.03CD/14.7 0.7D
196.1 14.2F 139.1 5.8E 44.9 1.4A
14.7 1.1E 10.7 1.0D 3.5 0.1A
Low MW 0.10% 0.15% 0.20% Control
0.18 0.30 0.46 0.37
139.2 0.9E 86.3 4.2C 64.4 0.6B 331.8 0.8H
9.5 0.7CD 7. 3 2.1B 6.9 0.2B 21.8 0.8H
0.04A/10.8 0.7B 0.02B/13.8 1.5CD 0.08D/17.1 0.9E 0.04C/eb
c
10000
(Pa)
G′ & G″ (Pa)
max
1037
1000
100
10 0.01
The G*gel (G* at gelation point) of tofu was increased and Tgel (time to reach the gelation point at 80 C) was decreased as the molecular weight of added g-polyglutamate increased. Increase the concentration of added g-polyglutamate increased both G*gel and Tgel of tofu. Also, the G0 max and G00 max of tofu increased with increasing the molecular weight of added g-polyglutamate. However, both G0 max and G00 max of tofu decreased with increasing the concentration of added gpolyglutamate. Tofu prepared with g-polyglutamate showed a significantly lower G0 max and G00 max values than the control tofu. Since the g-polyglutamate contains carboxyl group, it exists as a negatively charged polymer in the aqueous solution. The addition of g-polyglutamate in heated soymilk would increase the amount of negative charges. When GDL was added, the neutralization on the surface charge of soluble aggregates was slow and the surface charge of soluble aggregates might not be totally screened due to the presence of g-polyglutamate in the heated soymilk, leading to the retardation of tofu gelation and the reduction of van der Waals attraction and hydrophobic interaction between soluble aggregates.
10000
10
A
1000
100 0.01
0.1
1
10
Frequency (Hz)
3.2. Rheological properties
1000
G′′ (Pa)
After gelation, tofu was further moved to store at 4 C for 1 d, and the rheological properties, syneresis, and microstructure of tofu were evaluated. A typical plot of frequency sweep for control tofu with GDL is shown in Fig. 4. Both G0 and G00 increased slightly as the frequency increased. G0 was higher than G00 indicating that the tofu had formed a continuous network structure (Steffe, 1996). Fig. 5 shows the G0 and G00 of tofu with high MW g-polyglutamate as a function of frequency. The G0 and G00 of tofu with g-polyglutamate also increased slightly with the increase of frequency. Yoo, Figueiredo, and Rao (1994) suggested that frequency dependent property of G0 and G00 might be due to a faster molecular mobility of solid particles at higher frequencies. The G0 , G00 , and tand values at the frequency of 1 Hz of tofu containing GDL and g-polyglutamates with various molecular weights and concentrations were summarized in Table 2. The G0 and G00 of tofu decreased significantly with the decrease of the molecular weight of added g-polyglutamate. The increase of the concentration of high MW g-polyglutamate significantly decreased the G0 and G00 of tofu. Changes in the G0 of tofu containing medium MW g-polyglutamate in different concentrations showed a similar trend. However, the G0 and G00 of tofu were not affected by the concentration of the low MW g-polyglutamate. Generally, the molecular weight and concentration
1
Fig. 4. The rheological parameters of tofu with 0.3% (w/w) GDL as a function of frequency (0.01e10 Hz) at the stress amplitude of 1 Pa (C) Storage modulus G0 ; (B) loss modulus G00 .
G′ (Pa)
Values are mean of triplicates. Mean values with different superscript uppercase letters within a column indicate significant difference (P < 0.05). b Tofu gelation at 76.9 C. c G*gel ¼ G* at gelation point; Tgel ¼ Time to reach the gelation point at 80 C; G0 max ¼ Maximum value of G0 ; G00 max ¼ Maximum value of G00 .
0.1
Frequency (Hz)
a
B
100
10 0.01
0.1
1
10
Frequency (Hz) Fig. 5. The storage modulus (G0 ) and loss modulus (G00 ) of tofu with 0.3% (w/w) GDL and high molecular weight g-polyglutamate at concentrations of 0.10% (C), 0.15% (B), and 0.20% (;) (w/w) as a function of frequency (0.01e10 Hz) at a stress amplitude of 1 Pa.
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Table 2 The viscoelastic parameters at frequency 1 Hz of tofu containing 0.3% (w/w) GDL and g-polyglutamates in different molecular weights and concentrations.a
g-polyglutamate
G0 (Pa)
G00 (Pa)
tand
High MW 0.10% 0.15% 0.20%
1003.4 28.1D 793.3 71.9C 517.2 33.6A
172.3 6.0A 136.7 12.9B 91.5 4.9C
0.172 0.005BC 0.172 0.001BC 0.177 0.003BC
Medium MW 0.10% 0.15% 0.20%
811.5 118.2C 694.8 50.2B 626.8 17.0B
143.5 28.7B 123.1 7.7B 126.8 11.5B
0.177 0.001BC 0.177 0.010BC 0.202 0.002AB
Low MW 0.10% 0.15% 0.20% Control
458.3 24.4A 499.2 11.5A 451.1 17.0A 1030.7 1.4D
86.2 6.2C 78.9 17.0C 96.5 17.0C 176.0 1.7A
0.188 0.158 0.213 0.171
0.001ABC 0.027C 0.030A 0.002BC
a Values are mean of triplicates. Mean values with different superscript uppercase letters within a column indicate significant difference (P < 0.05).
of added g-polyglutamate did not influence the tand of tofu. These results suggested that although differences in the moduli values of tofu containing GDL and g-polyglutamates in different molecular weights and concentrations were observed, the rheological systems were similar. The tand of tofu was between 0.158 and 0.213 and was much smaller than unity, indicating a typical weak gel structure (Clark & Ross-Murphy, 1987). According to Ferry (1980), the concept of entanglement coupling plays a crucial role in illustrating the viscoelastic properties of undiluted system with high molecular weight polymers (molecular weight above 20,000). High MW g-polyglutamates apparently interacted with soy proteins in tofu at a higher extent. More entanglements might take place between high MW g-polyglutamate and protein molecules. This could result in a more stable gel network and a firmer texture of tofu with high MW g-polyglutamate, when it was compared to the tofu containing with low MW g-polyglutamate. However, the electrostatic repulsion caused by g-polyglutamate reduced the hydrophobic interactions between soy proteins, resulting in a weaker tofu structure as the increase of the concentration of g-polyglutamate. 3.3. Microstructure The microstructure of control tofu is presented in Fig. 6. Control tofu revealed a honeycomb-like structure with pores of relatively
Fig. 7. SEM micrographs of tofu with 0.3% (w/w) GDL and high MW g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), and 0.20% (C) (w/w). Scale bars represent 100 mm.
Fig. 6. SEM micrographs of tofu formed at 0.3% (w/w) GDL. Scale bar represents 100 mm.
even size. The network of tofu was constructed by fine strands in a dense arrangement. Doi (1993) demonstrated two models of globular protein network: random aggregation and a string of beads structure. Gelation of denatured soy proteins at low cation concentration was preferential along linear direction since the surface charges of denatured soy proteins were screened gradually. Thus, the interaction among the hydrophobic regions of soy
C.-Y. Lee, M.-I. Kuo / Food Hydrocolloids 25 (2011) 1034e1040
Fig. 8. SEM micrographs of tofu with 0.3% (w/w) GDL and medium MW g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), and 0.20% (C) (w/w). Scale bars represent 100 mm.
proteins was favored, resulting in a filamentous network (Hermansson, 1985; Maltais et al., 2008; Remondetto & Subirade, 2003). In the present study, the surface charges of the soluble aggregates in the heated soymilk were diminished gradually by protons, promoting the gelation of tofu with filamentous structure. The microstructure of tofu prepared with high MW and medium MW g-polyglutamate are shown in Figs. 7 and 8, respectively.
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Fig. 9. SEM micrographs of tofu with 0.3% (w/w) GDL and low MW g-polyglutamate at concentrations of 0.10% (A), 0.15% (B), and 0.20% (C) (w/w). Scale bars represent 100 mm.
The structure of tofu with high MW g-polyglutamate showed a discontinuous network with small pores uniformly distributed. The addition of g-polyglutamate decreased the strands thickness of tofu network. The structure of tofu with medium MW g-polyglutamate also showed a discontinuous network. However, the pores of network were larger in tofu with medium MW gpolyglutamate than those in tofu with high MW g-polyglutamate. Increase of the concentration of added g-polyglutamate increased
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to the project. Special thanks to Dr. Shyh-Forng Guo for critical comment on the manuscript.
10 9
ef
f
ef
8
de cd
Syneresis(%)
7 6 5 ab
4 a
3 2 1 0
0.10% HMW
0.15% HMW
References
bc
bc
0.20% HMW
0.10% MMW
0.15% MMW
0.20% MMW
0.10% LMW
0.15% LMW
0.20% LMW
γ-polyglutamate
Fig. 10. The syneresis of tofu containing 0.3% (w/w) GDL and g-polyglutamates in different molecular weights and concentrations. The standard error is represented by the length of the bars. abcd Different letters indicate significant difference (P < 0.05) between the means of syneresis values. HMW ¼ high molecular weight; MMW ¼ medium molecular weight; LMW ¼ low molecular weight.
the pore size of tofu network. The microstructure of tofu with low MW g-polyglutamate is presented in Fig. 9. The strand thickness of tofu network with low MW g-polyglutamate was less than that of tofu with high MW or medium MW g-polyglutamate. There were many discontinuous fragments and irregular pores in the network of tofu with low MW g-polyglutamate. These might be due to the decrease of interaction strength between the soluble aggregates in tofu network. The concentration of added low MW g-polyglutamate did not affect the microstructure of tofu. 3.4. Syneresis Fig. 10 presents the syneresis of tofu prepared with GDL and
g-polyglutamates in different molecular weights and concentrations. The syneresis of control tofu was 17.11%. Sun and Breene (1991) mentioned that the syneresis of protein gel during storage period was caused by the increase in the cross-linking between protein molecules via various interactions, which forced the exudation of water entrapped within the gel. The addition of g-polyglutamate decreased the syneresis of tofu to below 10%, indicating that the water-holding capacity of tofu was increased by the addition of gpolyglutamate. Increase the concentration of added g-polyglutamate significantly decreased the syneresis of tofu. This trend was more pronounced on the tofu with high MW g-polyglutamate. Changes in the rheological properties, microstructure, and syneresis of tofu by g-polyglutamate were observed in this study. The g-polyglutamate did not serve as coagulant. Rather, it worked as water-binding agent to improve the syneresis in the preparation of tofu. However, excess negative charges induced by gpolyglutamate in the heated soymilk inhibited the coagulation of soluble aggregates, causing the changes in the rheological properties and the microstructure of tofu. However, the entanglement between high MW g-polyglutamates and protein molecules might stabilize the tofu structure. Therefore, the structure and rheological properties between tofu with high MW g-polyglutamate and that with low MW g-polyglutamate were different, leading to a different extent of tofu syneresis. From these results, it is concluded that gpolyglutamate could be a practical food additive to improve the shelf-life, and to modify the texture of tofu, though further research on sensory evaluation is needed. Acknowledgments The authors express grateful thanks to the National Science Council of Taiwan (NSC) for its financial support (95-2313-B-030-003)
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