Changes in Soybean Phytate Content as a Result of Field Growing Conditions and Influence on Tofu Texture

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1 Biosci. Biotechnol. Biochem., 70 (4), , 2006 Changes in Soybean Phytate Content as a Result of Field Growing Conditions and Influence on Tofu Texture Takahiro ISHIGURO, 1 Tomotada ONO, 1;y Takahiro WADA, 1 Chigen TSUKAMOTO, 1 and Yuhi KONO 2 1 The United Graduate School of Agricultural Sciences, Iwate University, Ueda , Morioka , Japan 2 National Agricultural Research Center for the Tohoku Region, Kaminodai 297, Kariwano, Daisen, Akita , Japan Received September 8, 2005; Accepted December 24, 2005 It is known that tofu quality tends to vary among soybeans even of the same variety. Cultivation environments can affect the contents of the soybeans. Twentyseven soybean varieties were grown in a drained paddy field and an upland field, and then their protein and phytate contents were determined using the Fourier transfer infrared spectroscopy (FT-IR) method. The phytate contents of 12 varieties were higher in the drained paddy field than in the upland field. On the other hand, the environmental factor had little effect on the protein contents. In order to determine whether the difference in phytate content affected tofu texture, the hardness of the tofu made from phytate-added soymilk was measured. The tofu texture having more phytate became softer in the range of the common coagulant concentration. We concluded that the difference in the phytate content of the soybeans among the environmental conditions is a factor that causes fluctuation in tofu quality. Key words: soybean; soymilk; phytate; tofu; texture Tofu has been a popular food in some Asian countries since ancient times. Its consumption is now increasing as a health food all over the world. It is known that the quality of tofu made from the same soybean varieties cultivated in different area tends to vary, even if it are made using the same processes. Soybean cultivation is increasing in fields that have been converted from paddy fields for rice (called drained paddy field in this study) in Japan. However, drained paddy fields can be different from the primary soybean field (called upland field in this study) based on certain conditions such as soil properties. The differences in the cultivation environments can affect several characteristics of the soybean. Thus, they affect tofu quality. The quality of tofu is mainly evaluated on the basis of its texture properties. Many studies 1,2) have clarified that the protein content of soybean has an effect on tofu texture. Phytate is also considered to affect tofu texture by reacting with protein and coagulants such as calcium and magnesium salts. 3) It has been reported that the solubility of soy protein decreased in the presence of phytate. 4) Okubo et al. reported that glycinin and phytate form insoluble complexes between ph 2.5 and ) Also, in the presence of calcium ions, more phytate binds to glycinin. 6,7) Some reports, as described below, showed that phytate has an effect on tofu texture. Saio et al. reported that the tofu yield increased and its texture became soft when phytate was added to soymilk during the tofu-making process. 7) Hou and Chang also investigated the effect of the phytate concentration in soymilk on the yield and texture of pressed tofu. 8) They reported the pressed tofu yield increased and that the texture was softer when phytate was reduced with phytase. 8) The effect of phytate on tofu properties can be variable due to the conditions of tofu making. Soybean constituents such as protein and phytate are regarded as the quality parameters of tofu. A variation in these constituents in the soybean seed must cause variability in tofu quality. In this study, in order to determine the cause of tofu quality variation, we performed two experiments: (1) We cultivated 27 varieties of soybean in both a drained paddy field and an upland field, and then their protein and phytate contents were determined, and (2) we further investigated the influence of soybean phytate content on tofu texture, because the phytate content of soybeans was variable due to the differences in field conditions. Materials and Methods Materials. Twenty-seven Japanese varieties of soybeans (Table 1) were grown in a drained paddy field and an upland field in These soybeans were used to investigate differences in soybean contents based on field conditions. A variety of Touiku 233 (2001) was y To whom correspondence should be addressed. Tel/Fax: ; tomon@iwate-u.ac.jp Abbreviations: FT-IR, Fourier transfer infrared spectroscopy; ATR, attenuated total refraction

2 Table 1. Soybean Varieties Used in This Study Enrei Suzunone Tohoku 135 Hatayutaka Suzuyutaka Tohoku 137 Kosuzu Tachiyutaka Tohoku 139 Nanbushirome Tatinagaha Tohoku 140 Nemashirazu Tomoyutaka Tohoku 141 Okushirome Tohoku 124 Tohoku 143 Oosuzu Tohoku 126 Tohoku 146 ryuhoh Tohoku 132 Tohoku 147 Suzukari Tohoku 134 Tohoku 149 used to examine the influence of the phytate content on tofu texture. The soybeans were preserved in a refrigerator at 4 C until used as a sample and were then used for two years. Preparation of raw soymilk. Each soybean (about 10 g) was soaked in deionized water for 18 h at 4 C. The swollen bean was ground (11,400 rpm, 4 min) into a homogenate with 8 times the water vs. the soybean dry weight using an Oster blender (Oster, Milwaukee, USA), and the homogenate was then filtered through a defatted cotton sheet. The filtrate was designated raw soymilk. Determination of protein and phytate in raw soymilk. The protein content of the raw soymilk was measured by the FT-IR method of Nakasato et al. 9) The FT-IR spectra were measured using a Fourier transfer IR spectrophotometer (Spectrum 2000, Perkin-Elmer, Beaconsfield, England) equipped with a horizontal-atr 45 ZnSe crystal cell (mirror angle 45 ). The phytate content was measured by the method of Ishiguro et al. 10) In order to separate the phytate from the raw soymilk, the phytate was completely precipitated by the addition of calcium under alkaline conditions (ph 11.5). The precipitate was dissolved in citrate buffer (ph 6.0), and then the IR spectra were measured with the ATR accessory. The phytate content was determined by the absorbance at 1,070 cm 1. Change in Soybean Phytate Content and Tofu Texture 875 Preparation of tofu-making device. A tofu-making device was prepared using a 25 ml plastic syringe (Terumo SS-20ESZ, Terumo, Tokyo). The needle site of the plastic syringe was cut, and a rubber stopper for the syringe was placed at this site. This device had a 90 mm length and 20 mm diameter. Preparation of tofu. The soymilks were degassed by vacuum aspiration and cooled below 10 C. After a coagulant (CaSO 4 or GDL) was added to the soymilks, they were quickly stirred and then poured into the tofumaking device. The device was covered with a glass ball for a lid and placed in a water bath (Isotemp Fisher general purpose water bath, Fisher Scientific, Boston, USA) for 1 h at a temperature of 70 C for CaSO 4 or at 90 C for GDL. Measurement of tofu texture. The tofu prepared using the tofu-making device was placed in a refrigerator (4 C) for over 18 h. When the curd texture was measured, the tofu curd was at room temperature. The tofu was pushed out of the device and the surface portion was discarded. The next section was pushed out to a 13 mm height and cut with a thin copper wire. The four sections as thick disks of tofu were made from one device, as shown in Fig. 1. The tofu sample had a 13 mm height and 20 mm diameter, and was placed on a measuring plate. A compression test was carried out at a compression rate of 2 cm/min using a Rheotech Fudoh Rheometer (Rheotech, Tokyo) with a 25 mm diameter cylindrical plunger. Breaking stress and breaking strain values of a curd were expressed as the mean value of four measurements. Measurement of tofu ph. Crushed tofu samples were centrifuged at 1;000 g for 10 min. The supernatant was used to measure the ph of the tofu. Preparation of soymilk for tofu making. The soybeans were soaked in deionized water for 18 h at 4 C. Each swollen bean was ground (11,400 rpm, 4 min) into a homogenate with 6 times the water vs. the soybean weight using an Oster blender (Oster, Milwaukee, USA), then an antifoam-emulsion (Antifoam-AF-Emulsion, Nakarai Tesque, Kyoto) was added (about 80 mg for 200 ml of soymilk) during the grinding. The homogenate was filtered through a defatted cotton sheet. The filtrate is the raw soymilk. In order to prepare soymilks having different phytate contents (0.20, 0.30, and ), a potassium phytate solution (ph 7.0) was added to the raw soymilk made from Touiku 233 (the phytate content was ). These raw soymilks were heated above 95 C for 5 min (the temperature was held at 99:0 0:3 C at least for 4 min), and then quickly cooled to room temperature Fig. 1. A Tofu-Making Device Made from a Syringe, and Four Sections of Tofu for Compression Test. A plastic syringe was used for tofu making. Four parts of tofu were taken.

3 876 T. ISHIGURO et al. Measurement of protein solubility in soymilk at various concentrations of calcium chloride. Soymilks containing various concentrations of CaCl 2 were prepared as follows: Concentrations of 0 to 200 mm of 0.1 ml CaCl 2 were added to 0.9 ml of soymilk and stirred. The mixtures were produced in a series containing 0 to 20 mm CaCl 2, and kept at room temperature for 30 min. The protein solubility of the soymilks in the presence of Ca ion was determined by measuring the protein content of the supernatants after centrifugation at 8;000 g for 3 min. The protein concentration was determined by the Bradford method. 11) Statistical analyses. Significant differences between group means were analyzed by a t-test (p > 0:05) using the WinSTAT program. Results and Discussion Effect of field difference on soybean components The uneven quality of tofu made with soybeans of the same variety from different districts must be caused by variations in the components of the seed. Twenty-seven soybean varieties were cultivated in both the drained paddy field and the upland field. The protein and phytate contents of these raw soymilk are shown in Fig. 2. The difference in the protein contents between these fields was smaller than that among the bean varieties. This result shows that the influence of environmental factors on protein contents is smaller than that of the hereditary factor. On the other hand, 12 soybean varieties grown in the drained paddy field contained significantly more phytate than those in the upland field. The phytate contents of the other 14 varieties showed no significant difference between the drained paddy and the upland field. Only one variety of soybean grown in the upland field contained more phytate than that from the drained paddy field. Variation in phytate contents was also observed among the different varieties. These results show that the phytate content is affected by both environmental and hereditary factors. This environmental variation in phytate might have caused the scatter in tofu quality. The Enrei and Oosuzu varieties are known to be wellsuited to tofu making. On the contrary, the Hatayutaka and Tachinagaha varieties are known to be difficult for tofu making. Based on the phytate contents (Fig. 2B), Enrei and Oosuzu show no difference in phytate contents between the drained paddy field and upland field. For Hatayutaka and Tachinagaha, the beans grown in the drained paddy field contained more phytate than those grown in the upland field. The lower variation in phytate contents in Enrei and Oosuzu might make possible stable tofu making. The effects of phytate contents on tofu properties are discussed in a later section of this report. Effect of phytate on the texture of tofu The phytate contents of some soybean varieties varied due to environmental factors. Hence the effects of phytate concentration in the soymilk on the tofu texture were investigated. Two kinds of coagulants, the salt type such as calcium or magnesium salt and the acid type such as GDL, are used in tofu making. Hence we investigated the texture of tofu made using these two kinds of coagulant. Effect of phytate on the texture of calcium tofu Calcium sulfate was used as a representative of the salt-type coagulants. Soymilks containing 0.20, 0.30, and phytate were prepared by adding phytate (potassium type, ph 7.0) to raw soymilk (Touiku 233), and then heated. A series of tofus was prepared from each soymilk using elevated concentrations of calcium sulfate. A compression test was performed to measure the breaking stress and breaking strain of the tofu. Breaking stress values are shown in Fig. 3. At less than 20 mm calcium sulfate as the coagulant, the breaking stress of the tofu decreased with the increasing phytate concentration in the soymilk. At more than 20 mm calcium sulfate, the phytate concentration had little effect on tofu breaking stress. At more than 50 mm calcium sulfate, the soymilk containing phytate did not form tofu, because the soymilk coagulated immediately, as soon as the coagulant was added, before mixing. These results show that the effect of phytate on the tofu texture was different under the various coagulant concentrations. On the other hand, breaking strain values of the tofu are shown in Fig. 4. At less than 20 mm coagulant, increasing phytate concentrations in soymilk decreased breaking strain of the tofu. On the other hand, at more than 20 mm coagulant, phytate have little effect on the breaking strain. In summary of effect of the phytate on calcium tofu texture: At less than 20 mm coagulant, soymilk having much phytate formed a soft tofu. This effect of phytate is consistent with the results of Saio et al., who reported that tofu texture became soft when phytate was added to the soymilk. 7) Between 20 and 50 mm coagulant, the phytate had little effect on the tofu texture. This result is consistent with that of Hou and Chang, who reported that tofu texture was not affected by additional phytate. 8) These results indicate that the effect of phytate on tofu texture can be variable in relation to the coagulant concentration. Since the calcium sulfate concentration used in tofu making is generally between 15 and 20 mm (0.25 to ), the phytate content of the soymilk must have an effect on tofu texture. Effect of phytate in soymilk on protein solubility change by the addition of calcium chloride The phytate concentration in the soymilk influenced the tofu texture for the commercial range of the calcium coagulant (15 to 20 mm). Coagulants cause protein

4 Change in Soybean Phytate Content and Tofu Texture A % B Kosuzu Tohoku 149 Hatayutaka Tohoku 141 Tohoku 137 Tachiyutaka Tohoku 147 Okushirome Tohoku 134 Tohoku 135 Tatiganaha Nemashirazu Nanbushirome Suzuyutaka Suzunone Tohoku 139 Tohoku 132 Tomoyutaka Tohoku 124 Enrei ryuhoh Osuzu Tohoku 126 Suzukari Tohoku 140 Tohoku 146 Tohoku 143 Fig. 2. Protein and Phytate Contents of Soymilks Made from Soybeans Grown in Drained Paddy Field and Upland Field. A, Protein; B, Phytate. Twenty-seven soybean varieties were grown in a drained paddy field, and an upland field,. Raw soymilk was prepared from each type of soybean to measure the contents. Protein contents were measured using the method of Nakasato et al. 9) Phytate content was measured using the method of Ishiguro et al. 10) 10 Breaking stress (kpa) CaSO4(mM) added as coagulant Fig. 3. Effect of Soymilk Phytate Concentration on CaSO 4 Tofu Hardness. Soymilks containing 0.20, 0.30, and phytate were prepared by adding potassium phytate (ph 7.0) in soymilk preparation. Tofus were made from these soymilks using various concentrations of CaSO 4, and then breaking stress was measured. solubility to decrease and the soymilk to curdle. The protein solubility of soymilks having various phytate contents was measured on the addition of calcium chloride, and shown in Fig. 5. The protein solubility in soymilks having low (), middle (), and high () phytate levels decreased at 5 to 8, 6 to 10, and 9

5 878 T. ISHIGURO et al Breaking strain ( ) CaSO4 (mm) added as coagulant Fig. 4. Effect of Phytate Concentration in Soymilk on Breaking Strain of CaSO 4 Tofu. Soymilks containing 0.20, 0.30, and phytate were prepared by adding potassium phytate (ph 7.0) in soymilk preparation. Tofus were made from these soymilks using various concentrations of CaSO 4, and then breaking strain was measured. Protein solubility (%) CaCl 2 (mm) added Fig. 5. Effect of Phytate Concentration in Soymilk on Its Protein Solubility on the Addition of Calcium Chloride. Calcium chloride of various concentrations was added to soymilks containing 0.20, 0.30, and phytate, and then the soymilks were centrifuged. The protein contents of the supernatants were measured by the Bradford method. 11) to 14 mm calcium chloride, respectively. This shows that the increase in phytate content in the soymilk required more calcium chloride to precipitate the protein. Liu and Chang (2004) 12) reported a similar result, that the phytate concentration in the soymilk correlates with the optimal coagulant concentration for tofu making. These results explain the fact that soymilk having much phytate did not form tofu curd below 10 mm calcium, but formed tofu curd above 20 mm (Fig. 3). On the other hand, low phytate soymilk with a high calcium coagulant did not form tofu due to rapid coagulation before mixing. When commercial tofu making is done on a large scale, partial coagulation may occur in soymilk mixtures with low phytate contents. Therefore, homogeneous tofu curd formation from soymilk containing less phytate might be more difficult than with more phytate. Breaking stress (kpa) GDL (%) added as coagulant Fig. 6. Effect of Soymilk Phytate Concentration on GDL Tofu Hardness. Soymilks containing 0.20, 0.30, and phytate were prepared by adding potassium phytate (ph 7.0) in soymilk preparation. Tofus were made from these soymilks using various concentrations of GDL, and then hardness was measured. Effect of phytate on GDL tofu texture GDL causes coagulation of soymilk as a result of decreased ph. GDL is commonly used to make a packed tofu. A series of tofus was prepared from soymilks containing 0.20, 0.30, or phytate at elevated concentrations of GDL. A compression test was performed to measure the breaking stress and breaking strain of the tofu. The breaking stress of the GDL tofu (Fig. 6) decreased with the phytate concentration in all ranges of the GDL concentration, and increased with the GDL concentration in each phytate concentration. The tofu prepared from a high phytate soymilk was softer than that from a low phytate one. The breaking strain of the GDL tofu (Fig. 7) tended to decrease with increasing phytate concentration as well as the breaking stress. Because breaking strain expresses the strength of the curd, phytate has the potential to affect tofu strength. More GDL was needed to get a constant curd strain in soymilk containing more phytate.

6 Change in Soybean Phytate Content and Tofu Texture Breaking strain ( ) GDL (%) added as coagulant Fig. 7. Effect of Phytate Concentration in Soymilk on Breaking Strain of GDL Tofu. Soymilks containing 0.20, 0.30, and phytate were prepared by adding potassium phytate (ph 7.0) in soymilk preparation. Tofus were made from these soymilks using various concentrations of GDL, and then breaking strain was measured. 7 ph GDL(%) added Fig. 8. Effect of Phytate Concentration in Soymilk on ph of GDL Tofu. Soymilks containing 0.20, 0.30, and phytate were prepared by adding potassium phytate (ph 7.0) in soymilk preparation. Tofus were made from the soymilks using various concentrations of GDL, and then the tofus were crushed and centrifuged. The supernatant was used for measurement of tofu ph. Curd formation occurs by a ph decrease, and therefore the GDL tofu texture must be affected by ph. Hence the ph of each tofu was measured, as shown in Fig. 8. The ph of the tofu decreased with increasing GDL, and higher concentrations of phytate in the soymilk introduced the higher phs. This means that phytate in soymilk has a ph buffering action against added GDL. These results show that variation in the phytate content cause an uneven GDL tofu texture, as well as in calcium tofu. In conclusion, some soybean varieties grown in the drained paddy field have higher phytate contents than those from the upland field. The phytate content influences the tofu texture for the general coagulant concentration used for tofu processing. Therefore, the variation in the phytate contents due to environmental factors should be regarded as one of the reasons causing variations in tofu quality. Acknowledgments Part of this work was supported by financial aid from the research project Selective Breeding for High-Quality Products and Development of New Products of the Ministry of Agriculture, Forestry and Fisheries of Japan. References 1) Shen, C. F., deman, L., Buzzell, R. I., and deman, J. M., Yield and quality of tofu as affected by soybean and soymilk characteristics: gulucono--lactone coagulant. J. Food Sci., 56, (1991). 2) Wang, H. L., Swain, E. W., and Kwolek, W. F., Effect of soybean varieties on the yield and quality of tofu. Cereal Chem., 60, (1983). 3) Toda, K., Ono, T., Kitamura, K., Hajika, M., Takahashi, K., and Nakamura, Y., Seed protein content and consistency of tofu prepared with different magnesium chloride concentrations in six Japanese soybean vari-

7 880 T. ISHIGURO et al. eties. Breeding Science, 53, (2003). 4) Saio, K., Koyama, E., and Watanabe, T., Proteincalcium-phytic acid relationships in soybean. I. Effects of calcium and phosphorus on solubility characteristics of soybean meal protein. Agric. Biol. Chem., 31, (1967). 5) Okubo, K., Myers, D. V., and Iacobucci, G. A., Binding of phytic acid to glycinin. Cereal Chem., 53, (1976). 6) Saio, K., Koyama, E., and Watanabe, T., Proteincalcium-phytic acid Relationships in soybean. II. Effects of phytic acid on combination of calcium with soybean meal protein. Agric. Biol. Chem., 32, (1968). 7) Saio, K., Koyama, E., Yamazaki, S., and Watanabe, T., Protein-calcium-phytic acid relationships in soybean. III. Effect of phytic acid on coagulative reaction in tofumaking. Agric. Biol. Chem., 33, (1969). 8) Hou, H. J., and Chang, K. C., Yield and textual properties of tofu as affected by the changes of phytate content during soybean storage. J. Food Sci., 68, (2003). 9) Nakasato, K., Ono, T., Ishiguro, T., Takamatsu, M., Tsukamoto, C., and Mikami, M., Rapid quantitative analysis of the major components in soymilk using Fourier-transform infrared spectroscopy (FT-IR). Food Sci. Technol. Res., 10, (2004). 10) Ishiguro, T., Ono, T., Nakasato, K., Tsukamoto, C., and Shimada, S., Rapid measurement of phytate in raw soymilk by mid-infrared spectroscopy. Biosci. Biotechnol. Biochem., 67, (2003). 11) Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, (1976). 12) Liu, Z. S., and Chang, K. C., Effect of soy milk characteristics and cooking conditions on coagulant requirements for making filled tofu. J. Agric. Food Chem., 52, (2004).