Production of Sufu, a Traditional Chinese Fermented Soybean Food, by

Food Sci. Technol. Res., 15 (4), 347 352, 2009 Production of Sufu, a Traditional Chinese Fermented Soybean Food, by Fermentation with Mucor flavus at Low Temperature Yong-Qiang Cheng 1, Qing hu 1, Li-Te Li 1, Masayoshi Saito 2 and Li-Jun Yin 1* 1 College of Food Science and Nutritional Engineering, China Agricultural University, Qinghua East Road, Haidian District, Beijing, 100083, China 2 Food Science and Technology Division, Japan International Research Center for Agricultural Sciences, Owashi, Tsukuba, Ibaraki 305-8686, Japan Received July 20, 2008; Accepted March 4, 2009 This study evaluated biochemical changes, especially those related to protein degradation, occurring in sufu fermentation with Mucor flavus at low temperature. The effects of incubation temperature on M. flavus growth, biomass accumulation and protease production were described. Results indicated that M. flavus could grow well on the surface of soybean curd and produce considerable protease to degrade soybean proteins during sufu fermentation at lower temperature such as 15. Almost no subunits of protein could be observed after maturation for 8 weeks in M. flavus-type sufu. Post-fermentation was the main stage responsible for the hydrolysis of protein together with the increase in the content of amino-type nitrogen and free amino acid. Our results demonstrated that M. flavus had possible application as an alternative strain for sufu manufacturing at low temperature. Keywords: sufu, Mucor flavus, fermentation, protein degradation Introduction In Asian countries, soybean is consumed in many forms, including soymilk, soybean curd products, and fermented soybean food such as miso, soy sauce, tempeh, natto and sufu. Sufu is a traditional fermented soybean curd originating in China (Steinkraus, 1996). It is a soft cheese-like product with a spreadable creamy consistency, and it has been consumed widely as an appetizer for centuries in China (Zhang and Shi, 1993; Han, et al., 2001; Han, et al., 2003a). There is a similar product called tofuyo in Okinawa, Japan (Yasuda, 2000, 2001). Several types of sufu can be distinguished, according to different local processes or colour and flavour in China. Mould-fermented sufu is the most popular type due to its attractive colour and strong flavour (Han, et al., 2001). Sufu is produced by means of solid-state fermentation of soybean curd after inoculation with pure culture moulds. In commercial practice, Actinomucor spp., Mucor spp. and Rhizopus spp. are used for sufu preparation. Among fungal genera, Actinomucor elegans and Actinomucor taiwanensis *To whom correspondence should be addressed. E-mail: yinljmail@yahoo.com.cn seemed to be the most frequently used for commercial sufu production in China. However, these two mould species generally grow well at temperatures ranging from 25 to 30, hence making it difficult to produce sufu when the indoor temperature of the factory exceeds 35 during hot summer or below 20 during cold winter. Recently, the screening of alternative strains for the commonly used starter cultures in sufu production is receiving much attention. (Han, et al., 2003b; Hu, 2006; Hu, et al., 2008; Hu and Zhao, 1998a; Hu and Zhao, 1998b). Fermentation temperature greatly affects the growth of fungus and the variations in the enzyme activities produced by microorganisms, which are thought to have a close relation to sufu quality. Protein degradation is one of the important biochemical events during sufu fermentation. It had been reported that most of the soybean proteins were degraded into peptides and amino acids in well-qualified sufu. With the exception of the investigation of Chou et al. (1998) on the enzymes produced by A. taiwanensis on soybean curd, Han et al. (2003b) studied the optimal incubation conditions for protease production by A. elegans and R. oligosporus. It was reported that subunits of protein could not be detected

348 and the total free amino acids significantly increased in sufu fermented by Actinomucor elegans and Rhizopus oligosporus. An insufficient amount of enzyme or enzyme activity was found when A. elegans was used as starter culture at temperature exceeding 30 and below 25. However, R. oligosporus could accumulate more enzyme activities than A. elegans at higher temperature such as 35, and hence it was suggested to be a potential alternative starter to produce sufu of high quality during hot summer. Our previous study showed that β-glucosidase hydrolysis was the major contributor to the conversion of isoflavones, one of the most important active compounds in soybean, from glycoside to aglycone in sufu, which was greatly affected by fermentation temperature (Yin, et al., 2005). Hu and Zhao (1998a, 1998b) and Deng et al. (1996) also screened mutants and isolated several thermo-tolerant strains for sufu production. But so far, much less information describing sufu preparation at lower temperature such as those below 20 was available. As a matter of fact, Mucor flavus could grow well at low temperature and it had been isolated from commercial sufu for several decades (Fang, 1942; Zhang and Shi, 1993). It can be expected to be used as an alternative starter culture for sufu production during cold seasons. In the present study, the effects of incubation temperature on the M. flavus growth by measuring their biomass increment and its protease production on soybean curd were investigated. The changes in the chemical components during sufu preparation, especially those related to the degradation of protein, were analyzed in order to further evaluate the possibility of M. flavus being applied as an alternative strain for sufu manufacturing at low temperature. Y-Q. Cheng et al. Materials and Methods Microorganism and growth as determined by dry weight Mucor flavus CAU817 was originally isolated from commercial sufu in our laboratory (Hu, 2006; Hu et al., 2008). A pure culture inoculum was prepared by using a sterile inoculation loop to scrape off the sporulating mycelia from the surface of PDA (Potato Dextrose Agar) sub-culture slants. These were then transferred to the soy milk agar prepared with the addition of 0.05 wt% MgSO 4, 0.1 wt% KH 2 PO 4, 0.05 wt% (NH 4 ) 2 SO 4 and 0.25 wt% soluble starch to the concentrated soy milk, which were aerobically incubated at a 5 interval from 5 to 20. Dry weight determinations were performed according to Gibb and Walsh (1980). Triplicate flasks of each fungus were assayed for dry weight at 12 h intervals for a total 120 h. Preparation of sufu by using M. flavus as starter culture Processing of sufu was modified according to the method described by Yin et al. (2004). The steps and production conditions were as follows: (1) Soybean curd was prepared by CaSO 4 precipitation from boiled soymilk and was then cut into rectangular pieces, approximately 3.2 3.2 1.6 cm. (2) Pre-fermentation. Fresh soybean curd pieces were inoculated with Mucor flavus by spraying spore suspensions onto their surface of the tofu pieces, and incubated for 84 h under 90% relative humidity and air circulation. Semifinished products were called pehtze. (3) Post-fermentation. Pehtzes were piled up in a container and 15% salt was spread between the layers for 5 d. Twelve pieces of salted pehtzes were matured in glass bottles with a dressing mixture consisting of 4% koji red rice, 15% alcohol beverage, 5% sugar, 1.6% Chiang (wheat-based miso), and spices. Red sufu was obtained after ripening at room temperature (around 25 ) for 8 weeks. Chemical analysis of soybean curd, pehtze, salted pehtze and sufu The moisture, crude protein, crude lipid and NaCl contents of samples were determined by the official methods of AOAC (2000). Amino-type nitrogen contents were determined by the formol titration method described by Pike (1998). Free amino acids were determined according to the method described by Sarker et al. (1997). A 10 g sample was homogenized with 10 ml of 8 % sulphosalicylic acid solution. The slurry was then diluted to 50 ml with the same solution and centrifuged at 10,000 g for 20 min followed by filtration of the supernatant through a 0.45 μm membrane (Millipore, Milford, MA, USA). Amino acids were extracted based on the procedure of Abiodun et al. (1999) with slight modification. Eight milliliter of 6 N HCl was added to a 100 mg sample in a sealed tube, which was held for 24 h in an oil bath at 110. A Hitachi 835-50 Amino Acid Autoanalyser (Hitachi Co. Ltd, Japan) was used for separating the amino acids. Total free amino acid was the sum of the content of the individual amino acids. Assay of protease activity Protease activity was determined by the method of Wang et al. (2008). Briefly, 200 μl of the crude extract was added to the reaction mixture, containing 2% (w/v) casein (Sigma) in 200 μl of 50 mm Tris- HCl (ph 8). The mixture was incubated at 35 for 10 min. The reaction was stopped by the addition of 400 μl of 10 % trichloroacetic acid (TCA), followed by centrifugation at 9,000 g for 15 min. The supernatant were determined by the Folin-phenol reagent. One unit of protease activity was defined as the amount of enzyme that liberated 1 μmol tyrosine per min. A blank was run in the same manner, except for the addition of enzyme after the addition of 10 % TCA solution. Sodium dodecyl sulfate-polyacrylamide gel electro-

Low Temperature Fermentation of Sufu by Mucor flavus 349 phoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the Phastsystem (Amersham Pharmacia-LKB Biotechnology) with PhastGel Gradient 8-25% acrylamide gel. After eletrophoresis, the gels were stained with Coomassie Blue. The molecular weight was indicated by using a low molecular weight calibration kit (Pharmacia-LKB Biotechnology). Results and Discussion Effect of incubation temperature on fungal growth The mycelia dry weights of organism produced on soy milk agar at various incubation temperatures are shown in Fig. 1. It was shown that the biomass of Mucor flavus increased when soybean curd was incubated at both 10 and 15. An initial rapid increase was observed in the first 72 h of fermentation, followed by a gradual increase with a final microbial population reaching 1.41 g dry matter at 10 and 1.66 g dry matter at 15. Although M. flavus could produce substantial biomass at 20 in the first 24 h of incubation, the continuous incubation did not result in further accumulation of biomass. Almost no biomass was produced when M. flavus was incubated at 5. Visual observation confirmed that M. flavus was able to grow well on soybean curd at lower temperatures such as 10 and 15 (Table 1). From Table 1, it seemed that 15 was the best appropriate temperature for M. flavus to ferment soybean curd under the present investigation. Fungus incubated at 15 was observed to produce colonial morphologies which were thicker and denser as compared to those incubated at other temperatures. Although M. flavus was also capable of growth on the surface of soybean curd at 20, the accumulated mycelia tended to age after 48 h of incubation. Mycelial growth was invisible on the surface of soybean curd when incubated at 5. Effect of incubation temperature on protease activity expressed in pehtze The developed flavour and texture of sufu during maturation are determined by the enzymes produced by the mould in pehtze. Protease activity may play an important role on the hydrolysis of protein to liberate the free amino acid. We investigated protease production by Mucor flavus in pehtze, as presented in Fig. 2. The protease activities produced by M. flavus were fairly dependent on the con- Biomass dry weight (g) 2 1.6 1.2 0.8 0.4 5 C 10 C 15 C 20 C 0 0 24 48 72 96 120 Incubation time (h) Fig. 1. Mycelia dry weights of Mucor flavus produced on soy juice agar at various incubation temperatures. Values represent the mean ± SD (n = 3). Fig. 2. Proteolytic enzyme production by Mucor flavus at various incubation temperatures. Values represent the mean ± SD (n = 3). Table 1. Visual observation of mycelial growth a on soybean curd incubated by M. flavus at various temperatures (n = 3). Temperature ( C) Colonial morphologies at various incubation time (h) 24 36 48 60 72 84 96 5 NG NG NG NG = = = 10 NG NG + ++ +++ +++ +++ 15 NG = + ++ +++ +++ +++ 20 = + ++ AW AW AW AW a NG: no growth; =: slight growth; +: visible growth of mycelial (~2mm length); ++: fully covered with mycelial (~5mm length); +++: thick and dense growth of mycelial (~10mm); AW: become aging and withering

350 dition of processing including temperature and incubation time. Within the conditions investigated, M. flavus yielded the highest protease activity after 84 h of incubation at 15. Although M. flavus could also grow well on the surface of soybean curd at 10, longer incubation time was needed to achieve similar levels of protease activities as that at 15. Only few protease activities accumulated were observed when soybean curd was incubation at 5 or 20. These results were in agreement with the influence of incubation temperature on the biomass growth of M. flavus on soybean curd. Therefore, the continuous experiment was carried out to prepare sufu by the fermentation of soybean curd with M. flavus at 15. Degradation of soybean protein during sufu fermentation Since proteolysis is considered to be the most important biochemical reactions which is beneficial for the formation of characteristics texture, flavour, taste and consumer acceptance of sufu (McSweeney and Fox, 1997; Han, et al., 2003c), the degradation of soybean protein during sufu fermentation was evaluated from several viewpoints in this work. Fig. 3 shows the SDS-PAGE profiles of soybean curd, pehtze and salted pehtze as well as sufu at various ripening stages. From Fig 3, it was shown that the major subunits of protein in soybean curd were different from that of fermented soybean curd, suggesting that the degradation of soybean protein took place during sufu fermentation as function of microorganism, which altered the subunit patterns of protein quickly. Bands of soybean β-conglycinin (7S) and glycinin (11S) can be clearly identified in pehtze and salted pehtze, 97 67 43 30 20.1 14.4 Lane 1 2 3 4 5 6 7 8 Marker Tofu Pehtze Salted Ripening time (week) pehtze 2 4 6 8 Fig. 3. SDS-PAGE profiles during sufu preparation by Mucor flavus. Y-Q. Cheng et al. however, almost all of the protein subunits were invisible in sufu after 8 weeks of maturation. It seemed that the main components of sufu were peptides with a molecular weight below 14 kda. In an earlier literature, a similar pattern was reported as the major protein subunits were clearly identified in salted pehtze and in sufu after 10 days of ripening but disappeared in sufu after 60 days of ripening in sufu fermented by A. elegans (Han, et al., 2003b). Our results suggested that soybean protein of M. flavus-type sufu degraded by the comparable speed as that of A. elegans-type sufu. Secondly, the increase of amino-type nitrogen gives further indication of the hydrolysis of protein. The changes in amino-type nitrogen during sufu preparation are shown in Table 2. From Table 2, the content of amino-type nitrogen significantly increased in pehtze, salted pehtze and sufu even though the amino-type nitrogen was rarely included in soybean curd. Particularly, the amino-type nitrogen increased from 0.39 g/100g dry matter in pehtze to 1.61 g/100g dry matter in sufu, suggesting that protein hydrolysis occurred mainly in the stage of post-fermentation. Yu et al. (2001) reported that the value of amino-type nitrogen in sufu should be up to 1.5 g/100g dry matter in order to produce sufu with good flavour, implying that the content of amino-type nitrogen could be used as a standard for monitoring the maturation of sufu. From this viewpoint, M. flavus could be a suitable starter culture for sufu preparation at 15. In addition, it had been reported that the liberated free amino acids (FAAs) were important flavour enhancing compounds in sufu (Ho et al., 1989). It had been reported that many different flavour compounds in sufu were the results of volatile compounds formed from decarboxylation, deamination, transamination and other transformations of amino acids, which made substantial contributions to sufu flavour (Hwan and Chou, 1999). In the present study, changes in the total FAAs in soybean curd, pehtze, salted pehtze and sufu are summarized separately in Table 3. The total content of FAAs in pehtze, salted pehtze and sufu was remarkably higher than soybean curd. The salt addition increased the composition of dry matter, which may possibly cause the lower FAA concentration in salted pehtze as compared with pehtze. It is evident that most of the FAAs were formed during the ripening stage of post-fermentation since the FAAs content in sufu increased greatly compared to that in salted pehtze. Hydrophilic amino acid, especially acidic amino acid dominated the proportion of total free amino acid. Glutamic acid was the most abundant acid, followed by aspartic acid, together representing around 30% of total FAA. These results were in agreement with those results obtained from the works of Chou and Hwan (1994) and Han (2003d). It had been reported that Glutamic acid in combination with salt

Low Temperature Fermentation of Sufu by Mucor flavus 351 Step Moisture (g/100 g fresh weight) ph Crude protein (g/100 g dry matter) Crude fat (g/100 g dry matter) Salt (g/100 g dry matter) Amino-type nitrogen (mg/100 g dry matter) Soybean curd 70.54±0.46 6.85±0.30 38.1±0.2 25.57±0.32 ND a 0.12±0.01 Pehtze 69.59±0.82 7.02±0.17 36.4±0.4 25.53±0.22 ND a 0.39±0.09 Saleted pehtze 52.03±1.54 6.96±0.42 29.9±0.2 21.27±0.14 34.67±0.85 0.86±0.07 Sufu 59.24±0.32 6.61±0.22 26.1±0.3 20.42±0.93 25.71±0.61 1.61±0.05 Value represent the mean ± SD ( n = 3) a ND: not detected. Table 2. Chemical parameters of soybean curd, pehtze, salted pehtze and sufu. FAA tofu pehtze salted pehtze Red Sufu 1 Red Sufu 2 White Sufu 3 Grey Sufu 4 Asp 0.04 0.65 0.39 5.14 5.20 7.90 0.70 Thr 0.03 0.71 0.57 1.63 2.40 4.70 0.10 Ser 0.05 1.15 0.97 2.95 2.60 0.20 0.20 Glu 0.19 1.90 1.25 16.64 14.40 18.50 0.10 Pro ND 0.46 0.32 2.36 2.90 3.70 0.00 Gly 0.02 0.28 0.15 2.19 2.20 5.20 0.00 Ala 0.06 0.68 0.58 3.4 4.90 7.90 8.90 Val 0.04 0.80 0.67 3.33 3.50 6.50 6.80 Met 0.03 0.29 0.26 1.69 1.00 1.70 0.00 Ile 0.01 0.66 0.42 3.11 3.70 7.30 7.10 Leu 0.02 1.36 1.27 5.72 6.10 10.30 9.60 Tyr 0.12 0.84 0.71 3.11 4.10 0.90 3.70 Phe 0.13 1.33 1.63 3.89 4.80 7.40 6.50 Lys 0.04 0.88 0.66 3.94 4.00 7.50 0.20 His 0.02 0.22 0.16 0.93 1.00 1.80 0.05 Arg 0.13 0.19 0.37 0.45 0.20 0.00 0.00 Total 0.62 12.4 10.38 69.88 63.00 91.50 43.95 Mean of duplicate 1 Red Sufu prepared by the present work. Table 3. Free amino acid (FAA) changes during sufu preparation (mg/g sufu dry matter). 2-4 Sufu with 11% salt content prepared by Han (2003 d). (NaCl) contributes to the flavor and hedonic characteristics of foods (Halpern, 2000), also referred to as the unami taste. Table 3 also compared the FAA composition of M. flavustype sufu with typical Actinomucor elegans-type sufu products, including red sufu, white sufu and grey sufu, which were fermented with controlled temperature (around 25 ). Although total FAA of M. flavus-type sufu obtained in the present work was lower than that of white sufu fermented by A. elegans under common temperature, it was comparable to the level of total FAA of A. elegans-type red sufu and significantly higher than that of grey sufu (Han, 2003d). Properties of soybean curd, pehtze, salted pehtze and sufu The chemical parameters of soybean curd, pehtze, salted pehtze and sufu are shown in Table 2. Considerable differences at each stage of sufu fermentation were observed. The contents of crude protein and crude lipid decreased in salted pehtze. This was mainly the results from the added salt which has a great impact on the composition of the dry matter, since NaCl content increased up to 34.67 g / 100 g dry matter in salted pehtze. Crude protein continuously decreased by 3.8 g / 100 g dry matter in sufu during ripening, which might be attributed to the diffusion of soluble protein from sufu into the dressing mixture. No significant changes were observed in the content of crude lipid during sufu postfermentation. In conclusion, findings of this study suggested that Mucor flavus was able to produce considerable levels of biomass and proteolytic enzyme to degrade protein in soybean curd at lower temperature, which were influenced by the temperature of incubation. M. flavus could be considered as an alternative starter for sufu production at low temperature such as 15. It remains to be established as to whether flavour compounds and texture of sufu produced with M. flavus are comparable to sufu fermented by other commercially used starter at common temperatures. Acknowledgments This study was conducted within the framework of the collaborative research project between Japan and China titled Development of sustainable production and utilization of major food resources in China, supported by the Japan Interna-

352 tional Research Center for Agricultural Sciences (JIRCAS). This work was supported by a fund (No. 20776151) from the National Natural Science Foundation of China (NSFC) and a fund of the National Key-technologies R&D Program (2006BAD27B09) of the 11 th 5-year Plan of the People s Republic of China. References Abiodun, S., Anthony, O., & Omolara, T. I. (1999). Biochemical composition of infant weaning food fabricated from fermented blends of cereal and soybean. Food Chem., 65, 35-39. AOAC. (2000). Official Methods of Analysis of International 17 th edition, Association of Official Analytical Chemists. Chou, C. C., Ho, F. M., & Tsai, C. S. (1988). Effects of temperature and relative humidity on the growth of and enzyme production by Actinomucor taiwanensis during sufu pehtze preparation. Appl. Environ. Microb., 54, 688-692. Chou, C. C., & Hwan, C. H. (1994). Effect of ethanol on the hydrolysis of protein and lipid during the ageing on a Chinese fermented soya bean curd-sufu. J. Sci. 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