The Korean Journal of Microbiology, Vol. 43, No. 2, June 2007, p. 142-146 Copyright 2007, The Microbiological Society of Korea Ginsenoside y ƒ w z ½ ³Á½» Á * w w w œw l (Panax ginseng) z w x š. ù, w š ù y w» t v w. z w probiotic œ w, w ginsenoside p y» w t. w Bacillus ³ ³ ginsenoside y. 2.5% (w/v), 1% (w/v) w z z ginsenoside y y w. w Bacillus ³ ³ 10 7 CFU/ml w š, Bacillus ginsenoside Rg 1, Rb 2, Rc, Rd ƒ ³ p ginsenoside y., ³ z w ³ ƒ w. Key words ý Bacillus, biotransformation, ginseng, ginsenoside, lactic acid bacteria w ƒ (Araliaceae), (Panax) w w wš. w w w C. A. Meyerƒ 1843 e w w Panax ginseng C. A. Meyer š w. yg š r 1990 22.9% 2002 9.6% s w ƒš (4). w,, 6 ƒ w t w w w ƒ š (5). w w zwš, wz w s (ginsenoside) yw w,» ƒ e š w. y š s (ginsenoside) Shibata (13, 14) ƒ x š, z ginsenoside w w z x š (8, 9). z w v p(probiotic) œ w ginsenoside ww ginsenoside yw w s šz s w. w ginsenoside y û (1, 2, 3, 10), Bacillus *To whom correspondence should be addressed. Tel: 82-31-670-4706, Fax: 82-31-675-0432 E-mail: cjcha@cau.ac.kr glucosidase z ƒ šƒ (6, 11, 12). Bacillus ³ w z g, z y w» w w w w. z z s w w ginsenoside y y w. ginsenoside y d w Bacillus ³ w ³, t 1 16S rrna w ƒ ƒ¾ t w (Table 1). ³ w w (Table 2). 4 w, w z» y w. ³ yww (2.5%, w/v, 1%, w/v) š, 121 o C, 1.5», 15 ³w. ³» w Bacillus ³ wš 10 CFU/ml w Bacillus 2 2.5% (w/v) (50 ml), ³ 1%(w/v) (50 ml) wš ƒƒ 37 C o 30 o C, 200 rpm w. w w z Bacillus ³ LB (Conda, Spain) š, ³ MRS (Difco, France) š wš, 24 w z g d w. 72 w z, saponin Shibata (14) w, saponin w ginsenoside ww. w ethyl ether 3z w wš, s 142
Vol. 43, No. 2 z 143 Table 1. Bacillus spp. used for ginseng fermentation and biotransformation Microorganism Bacillus sp. B1 Bacillus sp. B2 Bacillus sp. B3 Bacillus sp. B7 Bacillus sp. B8 Bacillus sp. B10 Bacillus sp. B11 Bacillus sp. B1-1 Bacillus sp. B1-2 Bacillus sp. B1-3 Bacillus sp. B1-4 Bacillus sp. B1-5 Bacillus sp. B2-1 Bacillus sp. B2-2 Bacillus sp. B2-3 Bacillus sp. B2-4 Bacillus sp. B2-5 Bacillus sp. B3-1 Bacillus sp. B3-2 Bacillus sp. B3-3 Bacillus sp. B3-5 Closest species based on 16S rrna similarity Bacillus olivae Fig. 1. Growth of Bacillus sp. in ginseng powder broth. Each curve represents the average of two independent experiments. Symbols: ù, Bacillus sp. B1;, Bacillus sp. B8;, Bacillus sp. B11; ÿ, Bacillus sp. B1-3; þ, Bacillus sp. B2-1; ý, Bacillus sp. B2-4; ü, Bacillus sp. B3-5 Table 2. Lactic acid bacteria used for ginseng fermentation and biotransformation Microorganism Lactococcus lactis Lactobacillus sakei Leuconostoc mensenteroides Lactobacillus plantarum Enterococcus faecium Leuconostoc citreum Leuconostoc citreum KM20 Weissella kimchii CHJ30 Pediococcus pentosaceus Leuconostoc kimchii Streptococcus pyogenes Leuconostoc gelidum y n-butanol 3z w z Butanol d Rotary evaporator (Model N-1000, EYELA, Japan) w w. saponin 40% acetonitrile ww HPLC v. w HPLC»» Fig. 2. Growth of lactic acid bacteria in ginseng powder broth. Each curve represents the average of two independent experiments. Symbols: ù, Lactococcus lactis; ø, Lactobacillus plantarum;, Leuconostoc gelidum ; ÿ, Streptococcus pyogenes ProStar 335 system (Varian, USA) š, column Atlantis dc18 (Waters, 4.6 250 mm, USA) w. Mobile phase HPLC acetonitrile (HPLC grade, J.T. Baker, USA) w, gradient 17% ACN w 30 40% ACN, 50 80% ACN, 55 17% ACN, 60 17% ACN š, 0.8 ml/min. UV 203 nm d w. x Bacillus 21 ³ 12 10 7 CFU/ml. 1 2 ù Bacillus 7 ³ 4 š. Bacillus»ƒ, 24 10 6 CFU/ml w š, z w y
144 Hee-Gyu Kim et al. Kor. J. Microbiol» (Fig. 1). ³ Bacillus w š ùkü 10 6 CFU/ml w 12 Bacillus (Fig. 2). ³ 2.5 % (w/v) w s ù w w ƒ. ³ Lactococcus lactis Lactobacillus plantarum ƒ ùkü. 72 ù z s y w» w Shibata (14) z HPLC ww. 3 3 Bacillus w ginsenoside y HPLC profile (Fig. 3). negative control ³ w š v w(37 o C, 200 rpm) k s w HPLC j m. Ginsenoside Rg 1, Rc, Rb 2, Rd 5ƒ w. s w w š, s w»., 5 s w s w p s (ratio) y w (Table 3). Bacillus 5ƒ yƒ w, ³ p y ƒ. Bacillus HPLC j m r, v Ginsenoside Rc w w y w š, Ginsenoside Rd v w ƒw y w. Ginsenoside Rc Rd y w, ginsenoside-α-arabinofuranase z ƒ y ƒ (15). 3 D profile, ginsenoside Rc Rb 1 w š Rd w ƒw. ginsenoside-αarabino-furanase w Rcƒ w β-glucosidase w Rb 1 w Rd y w. x Bacillus ³ w Table 3. Transformation of Ginsenoside Rg 1, Rc, Rb 2, and Rd by Bacillus spp. Strain Rg 1 Rb 1 Rc Rb 2 Rd B1 - + - + + B2 - + - - + B3 - + - + + B7 + + - - - B8 + - - - - B10 + - - - + B11 - + - - + B1-1 - + - + + B1-2 - + - - - B1-3 ND - - + + B1-4 + - - - ND B1-5 - + - + + B2-1 + - - - - B2-2 + - - + + B2-3 - - - + + B2-4 + - - + + B2-5 + - - - + B3-1 ND - - + + Fig. 3. HPLC profiles of transformation of ginsenoside Rg 1, Rc, Rb 2 and Rd by Bacillus strains. (A), negative control (B), Bacillus sp. B8 (C), Bacillus sp. B1-4 (D), Bacillus sp. B2-4, Ginsenosides; 1, Rg 1 ; 2 ; 3, Rc; 4, Rb 2 ; 5, Rd B3-2 ND - - + + B3-3 ND - - - + B3-5 ND + - ND + +: positive, -: negative, ND: not detected
Vol. 43, No. 2 z 145 Fig. 4. Proposed pathway of ginsenoside transformation by Bacillus spp. ginsenoside y 4 w (Fig. 4) ww w w z 21 Bacillus 12 ³, w probiotic w ƒ. Bacillus ³ ginsenoside yw y,,» d ¾ w. ³ probiotic ƒ w š, šw, ginsenoside w wy ³ yw w probiotic ƒeƒ q. 2006 w w ( ) w. š x 1. Bae, E.-A., N.-Y. Kim, M.J. Han, M.-K. Choo, and D.-H. Kim. 2003. Transformation of ginsenosides to compounds K (IH-901) by lactic acid bacteria of human intestine. J. Microbiol. Biotechnol. 13, 9-14. 2. Chi, H., D.-H. Kim, and G.-E. Ji. 2003. Transformation of ginsenosides Rb2 and Rc from Panax ginseng by food microorganisms. Biol. Pharm. Bull. 28, 2102-2105. 3. Chi, H. and G.-E. Ji. 2005. Transformation of ginsenosides Rb1 and Re from Panax ginseng by food microorganisms. Biotechnol. Lett. 27, 765-771. 4. Im, B.O., S.K. Ko, H.B. Jeong, and Y.D. Kim. 2005. Marketing strategy of ginseng product used tree analysis in domestic market. Food Marketing Research 22, 19-43. 5. Jeong, H.B., S.H. Park, S.K. Ko, S.H. Cho, and B.O. Im. 2005. Actual consumption conditions and consumer perception of ginseng in the major countries. J. Ginseng Res. 29, 152-158. 6. Kelly, C.T., F. O'Reilly, and W.M. Fogarty. 1983. Extracellular α- glucosidase of an alkalophilic microorganism, Bacillus sp. ATCC 21591. FEMS Microbiol. Lett. 20, 55-59. 7. Kim, M.K., J.W. Lee, K.Y. Lee, and D.-C. Yang. 2005. Microbial conversion of major ginsenoside Rb1 to pharmaceutically active minor ginsenoside Rd. J. Microbiol. 43, 456-462. 8. Kim, S.W., H.Y. Kwon, D.W. Chi, J.H. Shim, J.D. Park, Y.H. Lee, S.E. Pyo, and D.K. Rhee. 2002. Reversal of P-glycoprotein-mediated multidrug resistance by ginsenoside Rg3. Biochem. Pharmacol. 65, 75-82. 9. Liu, W.K., S.X. Xu, and C.T. Che. 2000. Anti-proliferative effect of ginseng saponins on human prostate cancer cell line. Life Sciences 67, 1297-1306. 10. Luan, H.W., X. Liu, X.H. Qi, Y. Hu, D.C. Hao, Y. Cui, and L. Yang, 2006. Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzing β-d-glucosidase from China white jade snail. Proc. Biochem. 41, 1974-1980 11. Pajni, S., N. Dhillon, D.V. Vadehra, and P. Sharma. 1989. Carboxymethyl cellulase, β-glucosidase and xylanase production by Bacillus isolates from soil. Int. Biodeterior. 25, 1-5. 12. Rowe, G.E. and A. Margaritis. 2004. Enzyme kinetic properties of α-1,4-glucosidase in Bacillus thuringiensis. Biochem. Engin. J. 17, 121-128. 13. Sanata, S., N. Kondo, J. Shoji, O. Tanaka, and S. Shibata. 1974. Studies on the saponins of ginseng. I. Structure of ginsenoside-r0, Rb1, Rb2, Rc and Rd. Chem. Pharm. Bull. 22, 421-428. 14. Shibata, S., T. Tanaka, T. Ando, M. Sado, S. Tsushima, and T. Ohsawa. 1966. Chemical studies on oriental plant drugs (XIV). Protopanaxadiol, a genuine sapogenin of ginseng saponins. Chem.
146 Hee-Gyu Kim et al. Kor. J. Microbiol Pharm. Bull. 14, 595-600. 15. Zhang, C., H. Yu, Y. Bao, L. An, and F. Jin. 2002. Purification and characterization of ginsenoside-α-arabinofuranase hydrolyzing ginsenoside Rc into Rd from the fresh root of Panax ginseng. Process Biochem. 37, 793-798. (Received June 7, 2007/Accepted June 13, 2007) ABSTRACT : Screening for Ginseng-Fermenting Microorganisms Capable of Biotransforming Ginsenosides Hee-Gyu Kim, Ki-Yeon Kim, and Chang-Jun Cha* (Department of Biotechnology, Chung- Ang University, Ansung 456-756, Republic of Korea) Panax ginseng has been drawing world-wide attention since it was used for medicinal purposes and its effects was discovered in scientific manners. However, it is necessary to develope new ginseng products as functional foods to compete with western ginseng. Fermented ginseng could be an excellent solution, where useful probiotics are provided and ginsenosides are specifically transformed to functional forms. In this study, we investigated the growth and ginsenoside biotransformation by 21 Bacillus strains isolated from Chongkukjang and 12 lactic acid bacteria. 2.5% (w/v) and 1% (w/v) of ginseng were used in culture media containing only ginseng powder as a sole nutrient source, and their biotransformation abilities were tested after the growths were checked. All used Bacillus strains and lactic acid bacteria were able to grow well in ginseng powder media at higher levels than 10 7 CFU/ml. Most of Bacillus strains displayed ginsenoside transformation in a strain-specific manner. Therefore, the results of this study demonstrated that the strains tested in this study could be used as potential starters for the ginseng fermentation.