454 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /,, No.+*,./..0+ (,**/) 20 Bacillus subtilis KFP 2.- * * * * Soft Natto Manufactured with Star

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1 454 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /,, No.+*,./..0+ (,**/) 20 Bacillus subtilis KFP 2.- * ** *** * ** *** Soft Natto Manufactured with Starter of Bacillus subtilis KFP 2.- Isolated from Douchi Saori Takahashi, Rie Katsumata, Kumi Yoshizawa, Momoko Yamaki, Miyuki Osako, Mariko Murakami, Sayaka Mouri, Mizuki Sato, Hiroko Meguro, Hiroko Kubokura*, Kanae Hirose, Tadayoshi Tanaka**, Sugio Watanabe*** and Kan Kiuchi Faculty of Home Economics, Kyoritsu Womens University,,,+ Hitotsubashi, Chiyoda-ku, Tokyo +*+2.-1 * Presen address : Department of the Science of Living, Kyoritsu Womens College,,,+ Hitotsubashi, Chiyoda-ku, Tokyo +*+2.-1 ** Department of the Science of Living, Kyoritsu Womens College,,,+ Hitotsubashi, Chiyoda-ku, Tokyo +*+2.-1 *** Suzuyo Kogyo Co.,,33 Ooyamahigashicyo, Itabashi-ku, Tokyo +1-**+. We collected non-salted Douchi from Yunnan Province, China, and isolated the dominant bacteria for the production of Natto. We identified the most predominant bacteria as Bacillus subtilis, calleditkfp2.-. The toughness of Natto made by us with this strain was about.* of commercial Natto. Formol nitrogen content and relative viscosity of Natto made by us with B. subtilis KFP 2.- was about -* and /*, respectively, of those of the commercial Natto. Organoleptic assessment revealed that the stickiness of Natto made by us was weaker than commercial Natto, but that the former was softer. The appearance, clack of soybean and toughness were considered good. The softer Natto will be useful for individuals who experience di$culty swallowing or in mastication. (Received Jan.,2,,**/ ; Accepted Jul. 2,,**/) +*+2.-1,,+ * +*+2.-1,,+ ** +*+2.-1,,+ *** +1-**+.,33 Corresponding author +

21 : 455 + KFP 2.- Bergey s Manual of Systematic Bacteriology Vol.,, Bergey s Manual, Marble../ mm /.1 mm,**+ - NBP NaCl *./, Beef extract +, Phytone Peptone + NBP.. NBP / / SY-No.

2 21 : KFP 2.- Bergey s Manual of Systematic Bacteriology Vol.,, Bergey s Manual, Marble../ mm /.1 mm,**+ - NBP NaCl *./, Beef extract +, Phytone Peptone + NBP.. NBP / / SY-No.,* 2 / 0 03 A a g /, ;, ; - ;. ; / ; 0 ; 1 SPSS +* Scheffé,* , + Fig. + / KFP 2.-, Table + KFP 2.- *.2 mm + Bacillus 1 Nutrient broth V-P broth ph 0 1 D L D D D ph 0 2 Nutrient broth Bergey s Manual +* /* Bergey s Manual d * /* KFP 2.- Bergey s Manual Bacillus subtilis KFP 2.- B. subtilis KFP 2.- Fig. + Douchi collected at Wayao in China - / B. subtilis KFP Fig., B. subtilis KFP * 2.- +*+-.2 +/,*.-,*,.

3 456 /, +*,**/ +* 22 Table + The characteristics of KFP 2.- Characteristics KFP 2.- Bacillus subtilis* Gram s stain Spores heat-resistance test Cell diameter Spores round Sporangium swollen Catalase test Anaerobic growth Voges-Proskauer test *42 *42 ph in V-P broth0 1 d Acid from D-Glucose L-Arabinose D-Xylose D-Mannitol Gas from Glucose Hydrolysis of Casein Gelatin Starch Utilization of Citrate Propionate Degradation of tyrosine Deamination of phenylalanine Egg-yolk lecithinase Nitrate reduced to nitrite Formation of Indole Dihydroxyacetone ND Growth at ph 042, nutrient broth /41, nutrient broth Growth in NaCl / 1 +* ND Growth at / +* -* -1.* /* // 0/ d d Growth with lysozyme present d *The characteristics of Bacillus subtilis are those described in Bergey s Manual of Systematic Bacteriology Vol.,. Symbols are as follows : (), 3* or more strains are negative ; (), 3* or,more strains are positive ; (d), ++23 of strains are positive ; (ND), No data available. / KFP KFP Fig * /,* / /. Fig..,/* g 0** g -3-1*.- g.- /* g -** g g.1 +** g./* g,2+1*.- g.-.- / Table, *.3. KFP *.-2 KFP *.,2 B. subtilis KFP 2.- Table -,.*0 KFP *, KFP ,+ KFP GC-MS

4 23 : 457 Fig., Temperature and humidity programs for manufacturing natto with Bacillus subtilis KFP 2.-, Temperature program.- (initial temperature is.-), Temperature program.1 (initial temperature is.1), Humidity program +* ++,-, -,,/,-/, -, KFP Fig. / KFP *,- -, - KFP KFP Table. B. subtilis KFP 2.- KFP KFP Fig. 0 KFP2.-.-, p*.*/ KFP2.-.1 p*.*/ KFP2.-.1, p*.*/ KFP KFP 2.- Fig. - Oxygen concentration during manufacturing Natto with B. subtilis KFP 2.- (A) Natto manufactured by using the temperature Program.- shown in Fig., (B) Natto manufactured by using the temperature Program.1 shown in Fig.,, O, concentration in the chamber, O, concentration in the natto package

5 458 /, +*,**/ +* 24 Fig.. Toughness of three kinds of Natto The three markings of poles show the using temperature programs for natto manufactured and commercial natto as follows :, Temperature program.- ;, Temperature program.1 ;, Commercial natto. Table, Formol nitrogen concentrations in Natto Table - Relative viscosity of Natto Natto Formol nitrogen () Natto Relative viscosity Natto + Natto, Commercial natto *4-2 *4,2 *43. Natto + Natto, Commercial natto +4+* +4,+,4*0 Natto + and, were made with B. subtilis KFP 2.- using the temperature programs.- and.1, respectively, shown in Fig.,. Natto + and, were made with B4 subtilis KFP 2.- using the temperature programs.- and.1, respectively, shown in Fig.,..1 - KFP 2.- B. subtilis B. subtilis natto 1 +- B. subtilis KFP 2.- Bacillus subtilis natto B. subtilis natto

6 25 : 459 Fig. / Chromatogram of volatile components in Natto made with B. subtilis KFP 2.- The components with peak numbers in the chromatogram are as follows : No. +/, ethanol ; No. 0*, pyrazine ; No. 1-,,-methylpyrazine ; No. 1/, --hydroxy-,-butanone (acetoin) ; No. 10,,,/-dimethylpyrazine ; No. 22,,,-,/- trimethylpyrazine ; No. +*+,,-methylpropanoic acid (isobutyric acid) ; A (No.,+),,,--butanedione (diacetyl) ; B (No. +*1), --methylbutanoic acid (isovaleric acid) ; C (No. +*2),,-methylbutanoic acid. Table. Organoleptic assessment of Natto made with B. subtilis KEP 2.- using temperature programs.- and.1 Commercial Manufactured natto natto Program.- Program.1 Appearance.4**4* b -42*4. b 04**4* a Lysis of bacilli.4**4* b 04*+4. a /42+4+ ab Color of soybean.4**4* a,4.+4+ b -4**4* ab Clack of soybean.4**4* b 040*43 a b Stickiness.4**4* a +4**4* b +4**4* b Smell.4**4* -4.+4/,42*4. Di#erent odor from natto.4**4*.4*+43-4*,41 Toughness.4**4* b 14**4* a.40+4/ b Umami.4**4*.4,+40-4*+4, Taste of soybean.4**4* a /4*+4, a +42*4. b Bitter taste.4**4*,42,4. -42,4* Overall estimation.4**4* ,+4+ Fancy.4**4* -4* Data are presented as means.d. values (n++). Assessment was scored on a scale of + to 1 (+far inferior,,inferior, -slightly inferior,.equal, /slightly better, 0better, 1far better) compared to commercial natto. a,b Mean values with di#erent superscript letters in the same row were significantly di#erent at p*4*/ (Sche#é post hoc test).,* PSP KFP KFP KFP , B. subtilis KFP 2.- +, 0 +, B. subtilis KFP 2.-, - B. subtilis KFP 2.-

7 460 /, +*,**/ +* 26 Fig. 0 Photographs of Natto made with B. subtilis KFP 2.- and commercial Natto (A), Before agitation of natto we made ; (B), Before agitation of marketed natto ; (C), After agitation of natto we made ; (D), After agitation of marketed natto. B. subtilis KFP 2.- KFP 2.-. B. subtilis KFP 2.- B. subtilis KFP 2.- KFP 2.- KFP 2.- Bacillus subtilis, B. subtilis KFP 2.-, KFP **.* - *.3. *.,2 *.-2,.*0 +.+* +.,+. p*.*/ p*.*/ +

8 27 : ,+0,,* +330, Claus, D. and Berkley, R.C.W., Genus Bacillus Cohn +21,, +1.AL. Bergey s Manual of Systematic Bacteriology, Vol.,, Eds. by Sneath, P.H.A., Mair, N., S., Sharpe, M., E. and holt, J.G. William & Wilkins Co. (Baltimore, U.S.A.). pp. ++*/ Sulistyo, J., Taya, N., Funane, K. and Kiuchi, K., Production of natto starter. Nippon Shokuhin Kogyo Gakkaishi, -/,,12, , /1/ /2, +33/ /.2,11,20,**+ 0 pp. +, * 1 p.,, +33* 2 p. /2 +33* 3 p * +* SPSS Inc., SPSS Base +*.* J for Windows user s guide, p Tanaka, T., Muramatsu, K., Kim, H-R., Watanabe, T., Takeyasu, M., Kanai, Y. and Kiuchi, K., Comparison of volatile components from Chungkuk-jang and Itohikinatto, Biosci. Biotechnol. Biochem.,0,, +..* , /*,12,2/,**- +- Kim, H., Muramatsu, K., Kanai, Y., Tanaka, T., Takeyasu, M. and Kiuchi, K., Manufacture of chungkukjang with elastase activity. Food Sci. Technol., -,,/+,/ * ,

406 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.3,.*0.++ (,**1) 18 KFP. * * * * Sporulation of Bacillus subtilis (natto) KFP. Isolated

406 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.3,.*0.++ (,**1) 18 KFP. * ** *** * ** *** Sporulation of Bacillus subtilis (natto) KFP. Isolated from Takahashi natto Starter Derived from Food Extracts