1 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /1, No. /, * (,+) 181,+ Scientific Aspects of the Fermented Soybean Food, Tofuyo Masaaki Yasuda De

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1 1 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /1, No. /, * (,*+*) 181,+ Scientific Aspects of the Fermented Soybean Food, Tofuyo Masaaki Yasuda Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, + Senbaru, Nishihara-cho, Okinawa 3*- *,+- Scientific aspects of tofuyo, low-salt fermented tofu, has yet to be fully clarified. The present study describes chemical characteristics of tofuyo and roles of fungus Monascus in food production. Fermentation of tofuyo is unique with respect to its soybean proteins, which undergo limited hydrolysis by proteinases in the presence of ethyl alcohol originating from awamori (distilled liquor).the main components forming the body of tofuyo consist of basic subunit of glycinin and other polypeptides (Mr. // kda, ++ +/ kda). The soybean proteins were digested into peptides and amino acids during maturation. The amount of free glutamic acid and aspartic acid was greatly related to good taste ( umami-taste) of the product. Some of the liberated peptides (IFL and WL) inhibited angiotensin I-converting enzyme activity that produces the vasopressor peptide. As WL was completely preserved after treatment with gastrointestinal proteases, it was expected to have an antihypertensive e# ect. Homogeneous preparation of proteinase from Monascus was characterized as an aspartic enzyme, a key enzyme for ripening, chemical and physical properties, and formation of bioactive peptides of the product. Carboxypeptidases of this fungus were characterized as serine peptidases, which contribute to the release of amino acids from soybean proteins and their related peptides and to the removal of bitterness during hydrolysis of soybean proteins. We established a production method of a new type of tofu using an alkaline serine proteinase with high soybean-milk-coagulating activity isolated from Bacillus pumilus TYO- 01. We prepared a new fermented foodstu# using soybean protein isolates. This method has the potential benefit for improving nutrition and adding physiologically functional properties to soybean foods. (Accepted Mar. -,,*+*) Keywords : fermented soybean food, tofuyo, physiologically functional properties, genus Monascus, proteases : Monascus + andor +2 3*- *,+- + Corresponding author yasuda@agr.u-ryukyu.ac.jp +

2 182 /1 /,*+* / 2,,. / Fig. + Fig Asper- Monascus anka No. -.* M. purpureus No. -.*- SDS-PAGE Fig., / bo gillus oryzae 0 - : 12 +* *** +/ ***, SDS-PAGE * - +, +- +/ +0 - Process for making tofuyo 3 TCA 3 +* * , 0

$, SDS-PAGE pattern of the water-insoluble fraction of tofuyo$ prepared by red koji at various ripening stages Fig.

3 3 : 183 Fig., SDS-PAGE pattern of the water-insoluble fraction of tofuyo prepared by red koji at various ripening stages Fig. - SEM photographs showing the changes of tofuyo prepared by red koji during ripening period ao Fig ,3.,,+., 1.. *,.., -,.

4 184 /1 /,*+* / 4 Table + Changes in the free amino acids of tofuyo prepared by red koji during ripening period Amino acids Dehydrated Ripening period (month) Raw tofu (mg/ +** g) tofu *4/ + - / Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total amino acids +/,3 -+1 / ,-, * /- 12,*3.+*.2+,* /0,2-0* / *,.,, /.-,. 3*, *, * * - / 0 3 +/ / +,. *.,- 0* /+./ - +/.* 2/ +, / ++1,.0.*-.11 * +* / /- +/* +. /* 0/ +,2,** +1-1 / +1, /,,+0 +, *3 +1. / ,* /+/. GC GC-MS Table + -,. +*,*,+ Lioe Yasuda L L L L,, - L L,- ACE,/,0 VY VPP IPP,1 Kuba Yasuda

5 5 : 185 ACE ACE ACE HPLC, IFL Ile- Phe-Leu WL Trp-Leu SWISS- PROT bo ao bo -+ -, ICR --,1 ACE inhibition Digestion None Pepsin Chymotrypsin Trypsin Pepsin chymotrypsin and trypsin ACE ACE - ACE Table, WL ACE IFL TG HDL ACE DPPH ++, -diphenyl-,-picrylhydrazyl 1* DPPH,2,3 in /,. vivo -,.,3 IFL WL. Bacillus Table, Digestive stability of IFL and WL toward -* 2 Monascus purpureus Aspergillus oryzae +. Bacillus 12 :,/ 1* +,. M. purpureus A. oryzae ACE ACE go IC IFL /* ( g/ml) WL +2 +* +2 +*,3 ++,. +*.1 -/

6 186 /1 /,*+* / 6 SPYP WL ACE WL -. SPYP ACE Monascus LAIPVNKP -/ M. sp. No. -.*. M. purpureus ACE +,.* *** ph -*. Ki ;,0 /+--. nm +/ IFO * CPase M. purpureus +-, ***, * +*,* TCA TCA CPase N SDS-PAGE Glu Tyr CPase bo M. purpureus M. pilosus IFO , M. purpureus No. -.*- ACE bo M. purpureus No. -.*- -1.*.+ -3 Endo ACE bo M. ruber LAIPVNKP LPHF, --hydroxy---methyl-glutaryl (HMG)-CoA

7 7 : 187.,.- M. pilosus go GABA Kuba.. Yasuda GABA ACE ACE./.0 IY, VVY, VF VW Aniya M. anka.1 dimermic acid ICR M. purpureus - Fig.. The fermented soybean protein gel foods The products in the right hand, that in the center and left hands side were made by the red-koji, mixed-koji and yellow-koji, respectively. / Soybean-Milk-Coagulat- ing Enzyme ; SMCE Bacillus pumilus Fig. / TYO-01 +* +. min SPI Fig. / Changes in the ratio of the precipitate nitrogen to the total nitrogen in soybean milk treated with SMCE from Bacillus pumilus TYO /+ /+ /, Fig bp,2- /- /, /* /. ph 3*. /* ph 0*. 0* -* *** APRP reductase E.C monakolin K /+

8 188 /1 /,*+* / 8 +. min 00 /, // /0 + pp.,3/ , -1.*-.*3 +33* pp. 0/ +*-. pp. 30 +*/,**. / -* , , * -3 21* , 3 Monascus.* * Aspergillus oryzae * , / +, Yasuda, M., Soeishi, K. and Miyahira, M., Purification and properties of acid protease from Monascus sp. No. -.*-. Agric. Bio +. Chem.,.2, ( +32. ). +- Yasuda, M. and Sakaguchi, M., Degradation of soybean protein by an acid proteinase from Monascus anka. Food Sci. Technol. Int. Tokyo,., 02 ( +332). +., ,, / Liu, F., Tachibana, S., Taira, T., Ishihara, M. and Yasuda, M., Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus. J. Ind. Microbiol. Biotechnol., -+,,-,2 (,**.). +0 Liu, F., Tachibana, S., Taira, T., Ishihara, M. and Yasuda, M., Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus. J.

9 9 : 189 Ind. Microbiol. Biotechnol., -+, /1, /2* (,**.). pp. +, ,**. -/ Yasuda, M, Shimabukuro, M. and Kikuchi, S., Production,.- -,, -, purification and properties of acid porteinase from +2 Yasuda, M., Kuwae, M. and Matsushita, H., Purification genus Monascus. Nippon Shokuhin Kogyo Gakkaishi, -2, and properties of two forms of glucoamylase from 3/. 30+ ( +33+ ). Monasucs sp. No. -.*-. Agric. Biol. Chem., /-,,.1,.3-0 Fujita, H., Yokoyama, K. and Yoshikawa, M., Classific- ( +323). ation and antihypertensive activity of angiotensin I- +3 Tachibana, S. and Yasuda, M., Purification and charac- converting enzyme inhibitory peptides derived from terization of heterogeneous glucoamylases from Monascus food proteins. J. Food Sci., 0/, /0. /03 (,***). purpureus. Biosci. Biotechnol. Biochem., 1+,,/1-,/10 (,**1). -1 Kuba, M., Tana, C., Tawata, S. and Yasuda, M., Produc-,* Lioe, H.N., Apiyantono, A., Takara, K., Wada, K., Naoki, tion of angiotensin I-converting enzyme inhibitory pep- H. and Yasuda, M., Low molecular weight compounds tides from soybean protein with Monascus purpureus responsible for savory taste of Indonesian soy sauce. J. acid proteinase. Process Biochem.,.*,,+3+,+30 (,**/). Agric. Food Chem., /,, /3/* /3/0 (,**.). -2 Liu, F. and Yasuda, M., Debittering e# ect of Monascus,+ Lioe, H.N., Wada, K., Aoki, T. and Yasuda, M., Chemical carboxypeptidase during the hydrolysis of soybean and sensory characteristics of low molecular weight protein, J. Ind. Microbiol. Biotechnol., -,, (,**/). fractions obtained from three types of Japanese soy -3 Wang, T.H. and Lin, T.F., Monascus rice products. Adv. sauce (shoyu)-koikuchi, tamari and shiro shoyu. Food Food Nutr. Res., /-, +,- +,/3 (,**1). Chemistry, +**, (,**1)..* Endo, A. and Monacolin, K, a new hypocholesterolemic,, Lioe, H.N., Apiyantono, A., Takara, K., Wada, K. and agent produced by Monascus species. J. Antibiot, -,, 2/, Yasuda, M., Umami taste enhancement of MSG/NaCl 2/. ( +313). mixtures by subthreshhold L-a-aromatic amino acids. J..+ Endo, A, Biological and pharmacological activity of Food Sci., 1*, S.*+ S.*/ (,**/). inhibitors of --hydroxy---methylglutaryl coenzyme A,- Lioe H.N., reductase. Trends Biochem. Sci., January, pp +* +- ( +32+ ). /-., 23,*1,++ p. +,1,**0 +33.,. Yasuda, M. and Kobayashi, A., Preparation and characin.- terization of tofuyo (fermented soybean curd). In Trends Food Biotechnology, edited by Ghee, H.A, (Singa- /-,0-,1+ +33/ pore Institute of Food Science and Technology, Singa-.. Kuba, M., Tanaka, K., Sesoko, M., Inoue, F. and Yasuda, pore) pp. 2, 20 ( +323). M., Angiotensin I-converting enzyme inhibitory pep-,/ Kawasaki, K., Seki, E., Osajima, K., Yoshida, M., Asada, tides in red-mold rice made by Monascus purpureus. K., Matsui, T. and Osajima, Y., Antihypertensive e# ect Process Biochem.,.., (,**3). of Valyl-Tyrosine, a short chain peptide derived from./ Aniya, Y., Yokomakura, T., Yonamine, M., Shimada, K., sardine muscle hydrolyzate, on mild hypertensive sub- Nagamine, T., Shimabukuro, M. and Gibo, H., Screening jects. J. Human Hypertens, +., /+3 /,- (,***). of antioxidant action of various molds and protection of,0 Takano, T., Milk derived peptides and hypertension Monascus anka against experimentally induced liver reduction. Int. Dairy Journal, 2, -1/ -2+ ( +332). injuries of rats. Gen. Pharmacol., -,,,,/,-+ ( +333).,1 Kuba, M., Tanaka, K., Tawata, S., Takeda, Y. and Yasuda,.0 Aniya, Y., Ohtani, I., Higa, T., Miyagi, C., Gibo, H., M., Angiotensin I-converting enzyme inhibitory peptides isolated from Tofuyo fermented soybean food. Biosci. Biotechnol. and Biochem., 01, +,12 +,2- (,**-). Shimabukuro, M., Nakanishi, H. and Taria, J., Dimermic acid as an antioxidant of the mold, Monascus anka. Free Radic. Biol. Med.,,2, 333 +**. (,***).,2 Craft, I.L., Geddes, D., Hyde, C.W., Wise, I. J. and.1 Matthews, D.M., Absorption and malabsorption of glycine +.3 and glycine peptides in man. Gut, 3,.,/.-1 ( +302)...3./,,**/,3 Chun, H., Sasaki, M., Fujiyama, Y. and Bamba, T., E# ect.2 Yasuda, M. and Nagamatsu, K., Preparation of soybean of peptide chain length on absorption and intact transport protein gel for a novel fermented protein food. The of hydrolyzed soybean peptide in rat intestinal everted sac. J. Clin. Biochem. Nutr.,,+, * ( +330). Science Bulletin of the Faculty of Agriculture, University of the Ryukyus,..,,3+,31 ( +331). -* Kuba, M., Shinjo, S. and Yasuda, M., Antihypertensive.3 Yasuda, M., Morikawa, S., Sakaguchi, M. and Yasuda, S., and hypocholesterolemic e# ects of Tofuyo in spontane- ously hypertensive rats. J. Health Sci., /*, 01* 01- (,**.). A novel fermented food product prepared from soybean protein-calcium-gel with Monascus fungus : changes in -+ Santon, H.C., Mode of action of gamma amino butyric acid on the cardiovascular system. Arch. Int. Pharmacodyn., protein and nitrogen compounds during fermentation. Food Sci. Technol. Res., +/, -,/ --* (,**3). +.-, +3/,*. ( +30-). /* + -, pp. +*,**2 +/*.-2..+,**0 /+ Yasuda, M., Aoyama, M., Sakaguchi, M., Nakachi, K. and -- : Kobamoto, N., Purification and characterization of a /0 p. 0-,**3 soybean-milk-coagulating enzyme from Bacillus pumilus. -. Appl. Microbiol. Biotechnol., /+, ( +333). /, Aoyama, M., Yasuda, M., Nakachi, K., Kobamoto, N.,

10 190 /1 /,*+* / 10 Oku, H. and Kato, F., Soybean-milk-coagulating activity TYO- 01, by in vitro refolding of pro-enzyme fixed on a of Bacillus pumilus derives from a serine proteinase. solid surface. J. Biochem., +-0, /.3 //0 (,**.). Appl. Microbiol. Biotechnol., /-, -3* -3/ (,***). // /- Aoyama, M., Toma, C., Yasuda, M. and Iwanaga, M., Sequence of the gene encoding an alkaline serine pro- / -0.*,**, teinase of Bacillus pumilus. TYO- 01. Microbiol. Immunol., /0.., (,***). Bacillus pumilus /. Takahashi, M., Sekine, T., Kuba, N., Nakamori, S., Yasuda, -2 1+* 1+,,*** M. and Takagi, H., The production of recombinant APRP,,, - - an alkaline protease derived from Bacillus pumilus

13 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.3,.+./ (,**1) 401 * * Changes in the Color and Pigmentation of Fermented Benikoji-rice Drink Gok

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