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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 41, No. 4, pp. 399~404 (2009) The Korean Society of Food Science and Technology Bacillus subtilis Lactobacillus plantarum yw t e w Á û * œ w tœw Effect of the Mixed Culture of Bacillus subtilis and Lactobacillus plantarum on the Quality of Cheonggukjang Kyung-Eun Ju and Nam-Soon Oh* Department of Food Science and Technology, Kongju National University Abstract The goal of this study was to improve the quality of cheonggukjang by the optimization of the inoculation methods of the Bacillus subtilis (B. subtilis) and Lactobacillus plantarum (L. plantarum) strains. In order to optimize the mixed cultivation of B. subtilis and L. plantarum, the B. subtilis strain was inoculated into steamed soybeans after cultivation of L. plantarum. Inoculation size of B. subtilis was changed to the simultaneous inoculation method in order to stimulate the growth of the L. plantarum in cheonggukjang. The viable cell count of L. plantarum increased from 2 10 CFU/g to CFU/g and B. subtilis grew to 9 10 CFU/g. These results showed that 2 strains were successfully able to grow in the steamed soybean for good quality of cheonggukjang by optimization of the inoculation methods. The sensory evaluation indicated that a favorable aroma and overall acceptance of cheonggukjang by the optimized mixed cultivation of B. subtilis and L. plantarum, which was relatively higher than those of cheonggukjang by single strain inoculation of B. subtilis. Key words: cheonggukjang, Bacillus subtilis, Lactobacillus plantarum, mixed culture, product quality w g Bacillus subtilis(b. subtilis) g ù š m t» w w,, v t w š t. x wz (1,2), šx z (3), g l (4), w (5), w (6)» ƒ š ù, y y y y w š. p, z p w w wù, w wš t w» w,, e, y ƒw w (,) w w (9)wš w. w t w ³ wš w. ³ w w ½e, z t t w z ww. w *Corresponding author: Nam-Soon Oh, Department of Food Science and Technology, Kongju National University, Yesan, Chungnam , Korea Tel: Fax: nsoh@kongju.ac.kr Received May 13, 2009; revised June 29, 2009; accepted June 29, 2009 ³ w ü y(10), ³ (11), g l w(12), w (13) z ƒ š. t t B. subtilis Rhizopus oryzae koji yw ³ w (14) Pediococcus halophilus yw³ y w t (15) w. p, ³ y w B. subtilis Lactobacilli y w h w (16)ƒ š ù, w» w ƒ wš, ³ w t w». ³ Lactobacilli ³ z t w z³., ù t z t ½e z ³ Lactobacillus plantarum(l. plantarum) ³ t ³ y wš w., B. subtilis ³ L. plantarum ³ yw w, w ù ³ s y yw, t e w w. ³ ³ x w B. subtilis CKB ³ ( w B. subtilis) w, nutrient broth (Difco Laboratories, Detroit, MI, USA) 30 o C, 24 w ³ w. ³ L. plantarum ATCC 399

2 400 w t wz 41 «4y (2009) 014 ³ ( w L. plantarum) w, 0.05% L-cysteine(Sigma, St. Louis, MO, USA) ƒ MRS (Difco Laboratories) 3 o C, 24 w ³ w. w z 4 o C 24 ew. z 250 ml ƒv j 50 g š autoclave(da-ac-60, Dong-A Scientific Corp., Shiheung, Korea) w 121 o C 30 w. clean bench 50 o C þƒ k z ³ B. subtilis ³ CFU/g wš 3 o C 4 k (Shaking incubator, SI-600R, Jeio Tech Co., Ltd. Daejeon, Korea)w. ³ L. plantarum ³ x w. ³ d w B. subtilis ³ L. plantarum x³ ³ d 1 g ³ (0.% NaCl) w d w. B. subtilis ³ ³ plate count agar w z 30 o C 24 w, L. plantarum ³ TOS propionate agar medium (Yakult Pharmaceutical Ind. Co., Ltd., Tokyo, Japan) w z 3 o C 2 w. z ùkù colony w ³ (CFU/g) ùkü. Protease y Azocasein w protease Prestidge (1) d w., 5g yww z w š 25 mlƒ w. 10,000 rpm 20 (Centrifuge, Mega-1R, Hanil Science Industrial, Daejeon, Korea)w w ml 1 M MES-NaOH Buffer(pH 6.5) 0.05 ml, 0.05 ml, azocasein(2%(w/v)) 0.25 ml x yww z 3 o C w» 30 k z % perchloric acid 1 ml ƒw g. z 10,000 rpm 10 w z 1mL 10 N NaOH 0.15 ml ƒw z 430 nm Ÿ d w. Protease 1 unit 1 z y Ÿ ùkü. γ-gtp(r-glutamyltranspeptidase) γ-gtp d jp(am-15, Asan Pharmaceutical Co., Ltd. Seoul, Korea) γ-gtp y d w (Protocol sheet , Asan Pharmaceutical Co., Ltd.). 5g 5 w z k z w. w 4 o C, 10,000 rpm 10 w š, z z w. 3 o C 5» 1 ml z 0.02 ml ƒw z 3 o C w» 20 g. óù 3mL ƒw z yww 10 g ml š» g. 10 z yww 60 ü 635 nm Ÿ (Spectrophotometer, V-50, Jasco, Tokyo, Japan) d w, w γ-gtp y (U/g) ùkü. ph ph 1g 10 ml xk g yw w z ph meter(915pdc, Istek, Seoul, Korea) d w. k k k Formol (1) d w. 5g w 250 ml xk k z 10 ml 100 ml ƒv j wš, 0.1% phenolphthalein 2-3 ƒw z 0.1 N NaOH t y ¾ w. s (35-40%) 5.4 ml ƒw w y ¾ 0.1 N NaOH t k w w. k w œ x (19) k y d w. 5g yw kw z w. 10,000 rpm 10 w z 20 ml wš, w. 4% NaOH 0. ml ƒw z v j ¼. v j y 0.3 g š 150 ml z w. 100 ml» 0.05 N y 25 ml š 0 ml z 100 ml. w 100 ml 500 ml ƒ v j»š p - j yw 3-4 z (ph 4.) ùkú ¾ 0.05 N NaOH l k w. 5g w yw kw z w ü z 50 mlƒ. w 0.1% phenolphthalein 2-3 z w 0.1 N NaOH d w. m tœw w 15 w k z w. w w w,, p w,»y 5 w. ƒ x 1 ù, 2 ù, 3 m, 4, 5 sƒw. m Student s two tail t-test w. š ³ ph y B. subtilis ³ L. plantarum ³ w w. B. subtilis ³ CFU/g 5, L. plantarum 5 10 CFU/g w w. B. subtilis ³ w ù(fig. 1), B. subtilis ³ L. plantarum ³ (Fig. 2)w x ww. B. subtilis ³ w, B. subtilis ³» CFU/g 12 z CFU/g, ph» 6.5 w 24 z ƒw z 96 ph. (Fig. 1). B. subtilis ³ L. plantarum w yw w

3 L. plantarum ³ yw w 401 Fig. 1. Growth (ù) and ph ( ) during cultivation of B. subtilis strain in cooked soybean. Fig. 2. Growth of B. subtilis (ù) and L. plantarum (ø) and the ph ( ) in cooked soybean by mixed culture. Inoculum size of B. subtilis was CFU/g and L. plantarum was 5 10 CFU/g. (Fig. 2) B. subtilis ³ B. subtilis ³ y w w, B. subtilis j w e. L. plantarum» 5 10 CFU/g yw w 4 z CFU/g, CFU/g 6 ³ ƒ w. B. subtilis w» w xk v w w wš ü B. subtilisƒ w». B. subtilis L. plantarum (11,20) j. ù, ph y B. subtilis w B. subtilis L. plantarum ³ yw w» phƒ ph.2¾ w ƒ ³ w w. yw ³ p B. subtilis L. plantarum yw w L. plantarum ƒ k w» w L. plantarum w wš, ù B. subtilis w xw (Fig. 3). L. plantarum 2 10 CFU/g w wš 4,, 12 w z B. subtilis CFU/ Fig. 3. Growth of B. subtilis (ù) and L. plantarum (ø) and the ph ( ) in cooked soybean by various inoculation time. (A) Inoculation of B. subtilis after 4 h cultivation of L. plantarum, (B) Inoculation of B. subtilis after h cultivation of L. plantarum, (C) Inoculation of B. subtilis after 12 h cultivation of L. plantarum. Inoculum size of B. subtilis was CFU/g and that of L. plantarum was 2 10 CFU/g. g w w. L. plantarum w w z 4 B. subtilis w (Fig. 3(A), (B)) B. subtilis» CFU/g CFU/g 9 ƒw ù, 12 B. subtilis w (Fig. 3(C)) B. subtilis w» 1/ CFU/g 4 w. ³ w, hydrogen peroxide bacteriocins k (11,20)w» ƒ., L. plantarum w ³ w» B. subtilis. wr, L. plantarum» 2 10 CFU/g 4 z CFU/g ƒw ù, B. subtilis (Fig. 3(A))w 4 z š w w. L. plantarum (Fig. 3(B))z ³ CFU/g ƒw, B. subtilis w z L. plantarum ³ w š ƒw w CFU/g¾ ƒw. L. plantarum

4 402 w t wz 41 «4y (2009) 12 (Fig. 3(C))w L. plantarum ³ 2 10 CFU/ g ƒw w w ƒ w, B. subtilis w z Fig. 3(B) ùkù L. plantarum yw CFU/ g¾ ƒw. yw B. subtilis y ph y ùkû, L. plantarum w z 4 B. subtilis w (Fig. 3(A))» phƒ 5. ƒw 96 ph.2 w. L. plantarum w z B. subtilis w (Fig. 3(B))» ph ¾ w w z ƒw 96 ph 6.. ù, L. plantarum w z 12 B. subtilis w (Fig. 3(C))» ph 5. w, B. subtilis w z ph w 96 phƒ 4.9¾ w. B. subtilis ph yƒ ³ w (Fig. 3(C)) k w. yw t w» w L. plantarum w z B. subtilis w p t w ƒ. yw³ p L. plantarum B. subtilis ³ yw L. plantarum ƒ j» w B. subtilis ³ û w., B. subtilis CFU/g CFU/g û w L. plantarum w yw w (Fig. 4). L. plantarum 2 10 CFU/g w w. B. subtilis CFU/g w z 3 L. plantarum w (Fig. 4(B)), B. subtilis 24 z» ³ CFU/g CFU/g ƒw, CFU/g 9. L. plantarum» 2 10 CFU/g 24 z CFU/g ƒw ù, z w CFU/g w.» ph ƒw w ù, B. subtilis w ph(ph.6) û ùkû. B. subtilis CFU/g jš 2 L. plantarum w (Fig. 4(B)) B. subtilis» ³ CFU/g 24 z CFU/g ƒw CFU/g w ww x 3 w. ù L. plantarum» 2 10 CFU/g w 4 z 2 10 CFU/g 2 ¾ ƒw. 96 z CFU/ g ³ ƒ w. ph» ¾ ƒw ù, z ƒw ¾ ƒw. w û ph B. subtilis w L. plantarum yw». B. subtilis CFU/g û w L. plantarum (2 10 CFU/g) w yw w ³ w t k ƒ q. š (21) B. subtilis w 10 3 Fig. 4. Effect of inoculum size on the growth of B. subtilis (ù), L. plantarum (ø) and ph ( ). (A) Inoculum size of B. subtilis was CFU/g, (B) Inoculum size of B. subtilis was CFU/g. Inoculum size of L. plantarum was 2 10 CFU/g. CFU/g zƒ w š w, yw ù w w v ƒ ƒ. sƒ B. subtilis ³ L. plantarum ³ yw w yw p w. w 4 w ³ w yw w t p Table 1 ùkü. B. subtilis ³ w protease y 1 U/g In () ùkû, L. plantarum w z B. subtilis w yw w protease y 136 U/g, B. subtilis CFU/g w L. plantarum yw w protease y 110 U/g ³ yw w B. subtilis ³ w û ùkû. L. plantarum yw w B. subtilis ³» protease y w. γ-gtp(γ-glutamyltranspeptidase) x w z wù (22). ³ w γ-gtp y 1.11 U/g ³ yw y ùkü. B. subtilis ³ w y w ³ w ƒ ùkù» ƒ. k w w ùkü t, tœ 0.2% w. B. subtilis ³ w k w 0.44%, L. plantarum yw w k w ƒ ƒ 0.4% 0.46% ³ yw k w j w e. k

5 L. plantarum ³ yw w 403 Table 1. Quality characteristics of cheonggukjang by mixed culture of B. subtilis and L. plantarum 1) 2) 3) Inoculation Method Protease activity (U/g) γ-gtp activity (U/g) Amino type nitrogen (%) Ammonia type nitrogen (mg%) Titratable acidity (ml) Single strain inoculation 1) 1.0± ± ± ± ±0. Two stage inoculation 2) 136.4± ± ± ± ±0.5 Simultaneous inoculation 3) 110.2± ± ± ± ±0.5 Single strain inoculation of B. subtilis. Two stage inoculation of B. subtilis after h cultivation of L. plantarum. Simultaneous inoculation of B. subtilis and L. plantarum. Table 2. Sensory evaluation of cheonggukjang by mixed culture of B. subtilis and L. plantarum Inoculation Method Sweet smell Ammonia smell Yoghurt smell Overall acceptance Single strain inoculation 1) 3.0± ± ± ±0.54 Two stage Inoculation 2) 3.2± ± ± ±0.54 Simultaneous inoculation 3) 3.4± ± ±1.00 *3.5±0.50* *p<0.05, compared with the control group (by Student s two-tailed t-test) 1),2),3) See Table 1.»vw h w (23), 5.6 mg% yw mg% ùkû. ³ w ƒ L. plantarum yw w w ùkû. ³ yw w w» w ùkü. B. subtilis w L. plantarum yw w w»y w sƒ w (Table 2). sƒ L. plantarum yw w ƒ ³ w w t, p B. subtilis ³ w L. plantarum yw w»y. w w L. plantarum yw wš m w, t» w ƒ. B. subtilis ³ L. plantarum ³ yw w w ùkù ƒ t w wš sƒw. B. subtilis ³ L. plantarum yw w L. plantarum. yw L. plantarum ƒ j» w L. plantarum wš, z B. subtilis w L. plantarum ³ ƒ 2 10 CFU/g 4 z 6 10 CFU/g¾ ƒw. yw L. plantarum j B. subtilis» CFU/g, L. plantarum 2 10 CFU/g w w 24 z B. subtilis ³ 9 10 CFU/g¾ ƒw L. plantarum ³ 2 10 CFU/g 6 10 CFU/g ƒw., y mw B. subtilis ³ L. plantarum ³ œ w. l L. plantarum ³ ƒ ƒ k w ³ w w w, h ƒ, t»y. w k L. plantarum ³ yw w» t w» w ƒ. x 1. Lee JO, Ha SD, Kim AJ, Yuh CS, Bang IS, Park SH. Industrial application and physiological functions of cheonggukjang. Food Sci. Ind. 3: 69- (2005) 2. Kim WK, Choi KH, Kim YT, Park HH, Choi JY, Lee YS, Oh HI, Kwon IB, Lee SY. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from cheonggukjang. Appl. Environ. Microb. 62: (1996) 3. Heo S, Lee SK, Joo HK. Isolation and identification of fibrinolytic bacteria from Korean traditional cheonggukjang. J. Korean Soc. Agric. Chem. Biotechnol. 41: (199) 4. Cho YJ, Cha WS, Bok SK, Kim MU, Chun SS, Choi UK. Production and separation of anti-hypertensive peptide during cheonggukjang fermentation with Bacillus subtilis CH J. Korean Soc. Appl. Biol. Chem. 43: (2000) 5. Park HG, Lee MH, Yoon SH. Effect of cheonggukjang on lipid contents in rats fed high cholesterol diet. J. Korean Soc. Hyg. Sci. 12: 1-6 (2006) 6. Kim JI, Kang MJ, Kwon TW. Antidiabetic effect of soybean and cheonggukjang. Korea Soybean Digest 20: (2003). Kim JH, Kim SI, Kim JK, Im DK, Park JH, Lee JU, Byun MW. Effect of green tea powder on the improvement of sensorial quality of cheonggukjang. J. Korean Soc. Food Sci. Nutr. 35: (2006). In JP, Lee SK, Ahn BK, Chung IM, Jang CH. Flavor improvement of cheonggukjang by addition of Yucca (Yucca shidigera) extract. Korean J. Food Sci. Technol. 34: 5-64 (2002) 9. Park WJ, Park HY, Yoo JH, Rhee MS. Effect of Artmisia asiatica Nakaki extract on the flavor of cheonggukjang. Food Eng. Prog. 5: (2001) 10. Rhee YH, Kang MS. Physico-chemical characteristics and - galactosidase activity of Lactobacillus plantarum from kimchi. Agr. Chem. Biotechnol. 39: (1996) 11. Ha CG, Cho JK, Chai YG, Heo KC. Isolation and identification of lactic acid bacteria containing superior activity of bile salts deconjugation. Korean J. Food Sci. Anim. Res. 24: (2004) 12. Rhim KH, Kim JG, Han JH. Effect of fermented milk on rats fed by hypercholesterolemic diet. Kor. J. Env. Hlth. Soc. 19: -9 (1993) 13. Yoon TJ, Yoo YC, Kang TB, Lee KH, Kwak JH, Baek YJ, Huh

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