Characterization of Tofuyo (Fermented Tofu) Department of Bioscience and Biotechnology, University of the Ryukyus,

Transcription

1 I-12 Characterization of Tofuyo (Fermented Tofu) Masaaki YASUDA Department of Bioscience and Biotechnology, University of the Ryukyus, 1, Senbaru, Nishihara-cho, Okinawa , JAPAN Tofuyo is the excellent vegetable protein food made from tofu by the action of microorganisms in Okinawa, Japan. It is nutritiously rich with good protein, fat, and other nutrients. This food is a creamy cheese-type product with a mild flavor, fine texture, and good taste. Tofuyo is very similar to the foods called sufu, tou-fu-ju and furu in China or tau-hu-yee in Thailand and its root is considered as sufu in China. It was brought to Okinawa from Fujian, China and has been introduced since at the period of the Ryukyu dynasty in the 18 th century. Since the product was brined and had a strong taste, formerly it was not accepted directly by the people. Therefore, it was recreated by cooks in the dynasty for an accepted taste which was of milder flavor. As the food characteristic properties of smooth texture like as a cheese, sources of valuable protein, improvement of the circulation of blood by ethyl alcohol of awamori (traditional liquor in Okinawa) and suitability for one s taste, it has been treasured as nutritious food for health to be taken after an illness or as a side dish at such a time (1). Nowadays, attention has been paid to this food as vegetable cheese, which is cholesterol free food. In this paper, the author describes production and characterization of tofuyo prepared by Monascus fungus. Introduction Tofuyo is the excellent vegetable protein food made from tofu by the action of microorganisms in Okinawa, Japan. It is nutritiously rich with good protein, fat, and other nutrients. This food is a creamy cheese-type product with a mild flavor, fine texture, and good taste. Tofuyo is very similar to the foods called sufu, tou-fu-ju and furu in China or tau-hu-yee

2 in Thailand and its root is considered as sufu in China. It was brought to Okinawa from Fujian, China and has been introduced since at the period of the Ryukyu dynasty in the 18 th century. Since the product was brined and had a strong taste, formerly it was not accepted directly by the people. Therefore, it was recreated by cooks in the dynasty for an accepted taste which was of milder flavor. As the food characteristic properties of smooth texture like as a cheese, sources of valuable protein, improvement of the circulation of blood by ethyl alcohol of awamori (traditional liquor in Okinawa) and suitability for one s taste, it has been treasured as nutritious food for health to be taken after an illness or as a side dish at such a time (1). Nowadays, attention has been paid to this food as vegetable cheese, which is cholesterol free food. In this paper, the author describes production and characterization of tofuyo prepared by Monascus fungus. Process for making tofuyo Three steps are normally involved in making tofuyo; preparing tofu and dehydrated tofu, making koji, and maturing. The flow sheet for the preparation of tofuyo is shown in Fig Process for making tofu and dehydrated tofu (2) To make tofu, soybean was first washed, soaked at 25 o C for 8 h, and ground with 1 times volume of water for 2 min.the ground mixture was heated at 97 o C for 5 min, and strained through a coarse cloth to separate the soybean milk from the insoluble residue. The soymilk was then heated to 75 o C and calcium sulfate was added to coagulate the protein. The coagulated milk was then transferred into a cloth lined wooden box, and pressed with weight on top for 15 min so as to remove the whey. In this way tofu was formed. To prepare dehydrated tofu, the tofu was cut into 2 cm cubes. The cubes were dehydrated at room temperature for from 25 to 48 h. The dehydrated cubes were washed with awamori, and were used as the material for

3 tofuyo. Physical properties of the dehydrated tofu are important factors determining the quality of tofuyo. If the dehydrated tofu-cubes are too soft, it will fall into pieces during maturation. On the other hand, if it is too hard, it will be difficult to give a good texture and taste of tofuyo. During the dehydration, microorganisms such as bacteria grew on the surface of the cubes. In this process the bacteria play an important role in pre-fermentation which affects the quality of tofuyo by degrading soybean protein of tofu to some extent (3). 2. Process for making koji (4), (5) Rice koji was prepared from polished rice since it was essential that mold quickly penetrated into the rice kernel. Polished rice was soaked in water overnight. Excess water was drained off. The swollen rice was cooked with steam at atmospheric pressure for 6 min, cooled at 35 o C, and inoculated with starter inoculums of Monascus. The incubated rice was placed in wooden trays. During the development of the koji, temperature, moisture, and aeration are extremely important factors and must be rigidly controlled. After incubation at 32 o C for 7 days the red-koji was harvested (4). In the case of yellow-koji, the starter inoculums of Aspergillus oryzae was inoculated the steamed rice. After incubation at 3 o C for 2-3 days the yellow-koji was harvested (5). Koji is an important material for tofuyo making. It is a source of enzyme for converting starch into fermentable sugars as well as proteins into peptides and amino acids. Conditions of the koji for tofuyo making are as follows. One is that it has high activities of enzymes such as proteases, α- amylase and glucoamylase. Another is the provision of a good flavor and taste of the product. We examined the effect of steaming methods and rice varieties on the production of the enzymes and pigment of the koji (Table 1). Enzyme activities of the varieties of koji were indicated against relative activity of the koji prepared by using non-glutinous rice by steaming under atmospheric pressure. Monascus-pigment was also indicated.

4 In the system of steaming under atmospheric pressure, production of the enzymes and pigment of the koji prepared with glutinous rice was more abundant than that of non-glutinous rice. Traditionally, glutinous rice is used as raw material of the koji. α-amylase and protease of the koji prepared with broken rice of Thailand (a raw material for awamori making, Indica -type) was same as non-glutinous rice but the production of glucoamylase and pigment showed low levels. While, in the system of steaming under high pressure, production of the enzymes and pigment of the koji prepared with non-glutinous rice was in general, more abundant than that of broken rice or glutinous rice. Thus, it was found that the highest production of enzymes and pigment of the koji was obtained by employing autoclaved non-glutinous rice. These phenomena were also observed in the case of yellow-koji with Aspergillus oryzae (5). 3. Soaking and ripening (6)-(8) The last step in making tofuyo was soaking and ripening. To make the moromi, red-koji, and/or yellow-koji, awamori (35% of ethyl alcohol) and a small amount of salt were mixed, and kept at 4 o C for 24 h. The mixture was then ground. The dehydrated cubes were immersed in the moromi and allowed to ripen at 25 to 3 o C for 5 months. Ethyl alcohol concentration of the moromi was around 2% and the ph value was from 5.6 to 6.. It is unique and characteristic that the ripening was carried out under the presence of high alcohol concentration. The quality of the product was affected by varieties of the koji and liquor in the moromi. Chemical and physical characterization of tofuyo 1. Chemical changes during the ripening period (6) The chemical compositions of the tofuyo prepared by red-koji during the ripening period are shown in Table 2. Crude protein and crude fat contents of tofuyo decreased, but reducing sugar contents increased during the ripening period. Crude fiber was not detected. Sodium chloride content of the product was considerably constant (around 3%) during the ripening

5 period. Sodium chloride content of tofuyo was lower than that of Chinese sufu (9). In order to know the ripening of tofuyo, it is important to investigate the changes in enzyme activities such as of proteases and amylases during the ripening period. Protease is considered to be concerned with ripening, and amylases are concerned with providing the taste of the product. The ethyl alcohol concentration of the awamori was 2% as described above. Protease activity decreased markedly to 37% for the initial 15-day ripening, its value was almost maintained at around 3% until the end of 5-month ripening. It was considered from some of the results mentioned above that the protease activity was controlled by the presence of ethyl alcohol in the awamori. On the other hand, glucoamylase had higher activity throughought the ripening period than that of protease. α-amylase activity had a similar profile. The content of the reducing sugars of tofuyo and moromi increased during the ripening period. It was understood that glucose was produced by amylase action on starch during ripening. In this way, sweetness of the product was generated. Protein of the product was degraded slightly during the ripening. This may result in developing a good texture of tofuyo. It is likely that the hydrolytic products of proteins and lipids provide the principal constituents of a mild characteristic flavor of tofuyo. Digestion of soybean protein during fermentation was examined by a slab polyacrylamide gel electrophoresis. Some protein bands of soybean globulin such as α -, α- and β-subunits in β-conglycinin and acidic subunit in glycinin in the water-insoluble fraction of tofuyo disappeared after the 3-month ripening but that of basic subunit in glycinin still remained. It was found that the maturation of tofuyo was carried out by the phenomenon that soybean protein was hydrolyzed limitedly by protease in the moromi. And thus, it was found that the main components, which formed the body of tofuyo, consisted of basic subunit of glycinin and other polypeptides (Mr. 55 kda, kda). Some polypeptide bands in the water-soluble fraction of tofuyo, which have 31, 25, 23, 21 and 12-1 kda of molecular mass, were disappeared during the ripening period (-3 months). The nitrogen components of tofuyo during the ripening period are shown in Fig. 2. The

6 ratio of water-soluble nitrogen to the total nitrogen (called as protein solubility ratio or ripening ratio) reached to 36.3% after the 3-month ripening. This value was similar to that of white miso (39%) but was lower than that of red miso (61%) (1). The ratio of 4%-trichloroaceticacid-soluble nitrogen to the total nitrogen reached to 35.% after the 3- month ripening. The soybean proteins are digested by the proteases produced by the mold into peptides and amino acids. Free amino acids, which were good taste constituents such as glutamic acid, aspartic acid, glycin etc., were found in the water-soluble fraction. 2. Physical changes of tofuyo during the ripening period (11) The changes of physical properties of tofuyo during the 3-month fermentation were investigated. As shown in Table 3, the values of breaking stress and breaking energy of tofuyo decreased during the ripening period. It was found electron microscopically that the structure of soybean protein which formed the body of tofuyo changed from fibriform to small particle which connected each other during fermentation. Physiological function of tofuyo Recently, much attention has been paid to the physiological function in foods. The angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxy peptidase associated with the regulation of blood pressure. It converts angiotensin I to the potent presser peptide, angiotensin II, and also degrades depressor peptide bradykinin. ACE inhibitors from various foods have been recently studied in terms of their ability to prevent alleviate hypertension. ACE inhibitory activity was observed in a tofuyo extract. Some of them were isolated to homogeneity from the extract, and one was identified to be Tryp-Leu (IC 5 value, 29.9 M). The inhibitory activity of the peptide was completely preserved after a treatment with pepsin, chymotrypsin or trypsin (12).

7 Characterization of tofuyo-making The major characteristics in tofuyo making are that we do not have a process of molding on tofu-cubes and brining as done for sufu (9). Red-koji and awamori are used in our process. Red-koji may contribute to its natural color and the unique taste and aroma of the product. In fact, the results of our investigations showed that the product using red-koji was better than that of yellow-koji in color, taste, and aroma. Moreover, it is important that ripening is carried our under the presence of ethyl alcohol of awamori. Awamori contributes to the preservation of the product during the ripening period. Only a few percentages of salt is needed for seasoning the product. Awamori also contributes to form a good flavor of the product. Based on the results mentioned above, it can be inferred that the protease activity was controlled by the presence of awamori containing about 2% of ethyl alcohol concentration in the moromi. The protein of the product was degraded slightly during ripening. This may result in developing a good texture of tofuyo. It is likely that the hydrolytic products of protein and lipids provide the principal constitutions of mild characteristic flavor of tofuyo. The soybean proteins are digested by proteases produced by the mold into peptides and amino acids. Free glutamic acid and aspartic acid may contribute to the tastes of tofuyo. And released peptides (e.g. Tryp-Leu) contribute to the physiological function of the food such as angiotensin-i converting enzyme inhibitory activities that are concerned with improving hypertension. The soybean lipids are digested to some extent into fatty acids by lipase of the koji in the moromi. The added alcohol of awamori reacts with fatty acids chemically or enzymatically to form esters, some of which provide the pleasant aroma of tofuyo. Ethyl esters are characterized as the aroma components of tofuyo. Traditionally, tofuyo is consumed directly as relish with liquor such as awamori or as cakes served with tea. Recently it has been used in sushi, cracker-spread, and many kinds of hors-d oeuvre in the restaurants. Because tofuyo is a cream cheese-type product and has a mild flavor it would be expected to be suitable for use in western countries due to cholesterol free and low salt vegetable food for health.

8 References (1) Yasuda, M., Tofuyo and red koji. J. Brew. Soc. Japan (in Japanese), 77, , (1983). (2) Yasuda, M., Hokama, I., Production of soybean curd for tofuyomanufacturing. Nippon Shokuhin Kogyo Gakkaishi (in Japanese), 31, (1984). (3) Yasuda, M., Sakaguchi, M., Otonari, H., Okuhama, S., Kinjyo, S., Hongo, F., and Toyama, S., The role of microorganisms during dehydration of tofu cubes. Nippon Shokuhin Kogyo Gakkaishi (in Japanese), 39, (1992). (4) Yasuda, M., Uechi, G., and Miyazato, K., Production of koji with Monascus sp. for tofuyo-manufacturing. Nippon Shokuhin Kogyo Gakkaishi (in Japanese), 3, (1983). (5) Yasuda, M., Uechi, G., and Miyazato, K., On the production of koji with Aspergillus oryzae for tofuyo-manufacturing. Sci. Bull. Coll. Agr. Univ. Ryukyus (in Japanese), 28, (1981). (6) Yasuda, M. Matsumoto, T., Sakaguchi, M., and Kobamoto, N., Changes in chemical components of tofuyo prepared by Monascus fungus during fermentation. Nippon Shokuhin Kogyo Gakkaishi (in Japanese), 4, (1993). (7) Yasuda, M. Matsumoto, T., Sakaguchi, M., and Kinjyo, S., Changes in protein and nitrogen compounds of tofuyo prepared by Aspergilus oryzae during fermentation. Nippon Shokuhin Kogyo Gakkaishi (in Japanese), 41, (1994). (8) Yasuda, M. Matsumoto, T., Sakaguchi, M., and Kinjyo, S., Production of tofuyo using the combination of red and yellow kojis. Nippon Shokuhin Kagaku Kogaku Kaishi (in Japanese), 42, (1995).

9 (9) Wang, H.L., and Hesseltine, C.W., Sufu and Lao-Chao. J. Agr. Food Chem., 18, (197). (1) Brewing Society of Japan (editing), An outline of the components in fermented foods. Brewing Society of Japan (in Japansese), Tokyo, pp 337 (1977). (11) Yasuda, M., Kinjyo, S., and Miki, E., Changes in breaking characteristics, creep behavior and microstructure of tofuyo during fermentation, Nippon Shokuhin Kagaku Kogaku Kaishi (in Japanese), 43, (1996). (12) Kuba, M., Tanaka, K., Tawata, S., Takeda, Y., and Yasuda, M., Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented soybean food. Biosci. Biotechnol. Biochem., 67, (23).

10 Table 1. Effect of steaming methods and varieties of rice on the production of enzymes and pigment of red koji Relative activity (%) Streaming under atmospheric Streaming under A B C A B C α-amylase Glucoamylase Protease Pigment A Non-glutinous rice B Broken rice C Glutinous rice

11 Table 2. Changes in chemical composition of tofuyo prepared by Monascus fungus during the ripening period Ripening Crude Crude Crude Crude Reducing NaCl period protein fat ash fiber sugars (month) (%) (%) (%) (%) (%) Raw tofu Dehydrated Tofu Tofuyo

12 Table 3. Changes in breaking chracteristics of tofuyo during the ripening period Breakingstrain Breaking stress Breaking energy Sample ε(b) (cm/cm) P (b) ( 1 5 Pa) E (N) ( 1 3 J) day 15 days 3 days 6 days 9 days. 361± ± ± ±.2.371± ± ± ± ± ± ± ± ± ± ±.9 Koji, Awamori Salt Tofu (Soybean curd) Mixing Dehydrating in the room Homogenizing Washing with Awamori Moromi Dehydrated tofu Soaking Ripening TOFUYO

13 Fig. 1. Process for making tofuyo.

14 4 protein solubility ratio (%) Ripening period (month) Fig. 2. Changes in protein solubility ratio of tofuyo during the ripening period Protein solubility ratio was expressed with a water soluble nitrogen content per the total nitrogen content. These nitrogen contents were shown as oven dried basis.