Use of Lactobacillus rossiae DC05 for Bioconversion of ... - Springer Link

Food Sci. Biotechnol. 23(5): 1561-1567 (2014) DOI 10.1007/s10068-014-0212-3

RESEARCH ARTICLE

Use of Lactobacillus rossiae DC05 for Bioconversion of the Major Ginsenosides Rb1 and Re into the Pharmacologically Active Ginsenosides C-K and Rg2 Md. Amdadul Huq, Yeon-Ju Kim, Jin-Woo Min, Kwi Sik Bae, and Deok-Chun Yang

Received January 13, 2014; revised April 21, 2014; accepted April 22, 2014; published online October 31, 2014 © KoSFoST and Springer 2014

Abstract Rb1 and Re are the major ginsenosides in protopanaxadiol and protopanaxatriol with contents of 38.89 and 13.34%, respectively. β-Glucosidase-producing food grade Lactobacillus rossiae DC05 was isolated from kimchi using esculin-MRS agar and an enzyme of L. rossiae DC05 was used for bioconversion of the major ginsenosides Rb1 and Re. Strain DC05 showed strong activity in converting ginsenosides Rb1 and Re into the minor ginsenosides compound-K and Rg2, respectively. Within 4 days, 100% of ginsenoside Rb1 was decomposed and converted into C-K, while 85% of Re was decomposed and converted into Rg2 after 6 days of incubation. The biosynthesis rate of ginsenoside C-K was 72.88%, and the biosynthesis rate of Rg2 was 53.94%. Strain DC05 hydrolyzed ginsenosides Rb1 and Re along the pathway Rb1→Rd→F2→CK and the pathway Re→Rg2, respectively. The optimum temperature and pH of the enzyme were 30oC and 7.0, respectively. Keywords: major ginsenosides, bioconversion, Lactobacillus rossiae

Introduction Ginseng (Panax ginseng C.A. Mayer) is a well-known medicinal plant that is used in traditional medicine in Md. Amdadul Huq, Deok-Chun Yang () Graduate School of Biotechnology and Ginseng Bank, College of Life Science, Kyung Hee University, Yongin, Gyeonggi 446-701, Korea Tel: +82-31-201-2100; Fax: +82-31-205-2688 E-mail: [email protected] Yeon-Ju Kim, Jin-Woo Min, Kwi Sik Bae Department of Oriental Medicinal Materials and Processing, College of Life Science, Kyung Hee University, Yongin, Gyeonggi 449-701, Korea

China, Korea, Japan, and other Asian countries (1). The major active ingredients of ginseng are triterpene glycosides, known as ginsenosides (2,3), which have biological activities of anti-cancer (4), anti-aging (5), and anti-tumor (6). Recently, there have been a number of reports regarding the activity of ginsenosides as tumor MDR (multi-drug resistance) reversal agents (7-9). In addition, they have shown other activities, such as protection from free-radical damage, maintenance of normal cholesterol and blood pressure, rescue of hippocampal neurons from lethal ischemic damage (10,11), stimulation of the central nervous system, improvement of learning and memory, immunoregulation, and anti-fatigue properties (12,13). Currently, more than 180 ginsenosides are known from ginseng (14) and are classified into the 3 groups of protopanaxadiol (PPD), protopanaxatriol (PPT), and oleanane types. Different sugars are linked to C-3 and C-20 in the aglycon PPD and to C-6 and C-20 in the aglycon PPT by glycosidic bonds (Fig. 1). The minor ginsenosides, which are generally absent in ginseng plant (F2, Rg3, Rh2, compound K, Rg2, F1, and Rh1) can be produced by hydrolyzing the sugar moieties of the major ginsenosides, which are available in ginseng plant (Rb1, Rb2, Rc, Rd, Re, and Rg1), which comprise more than 80% of ginsenosides (15). Many studies have reported that minor ginsenosides are more active than major ginsenosides. Ginsenoside compound-K, which is not present in the ginseng root, has been reported to show biological activities of anti-genotoxic, anti-allergic effects, and the prevention of tumor invasion and metastasis (16,17). In addition, compound-K shows potential hepatoprotective and antiinflammatory activities (18,19). Ginsenoside Rg2 is an enzymatic metabolite generated from ginsenoside Re that shows a protective effect in human umbilical cord vein endothelial cells (VEC-304) against H2O2-induced cell

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Separation of ginsenosides Rb1 and Re Ginsenosides Rb1 and Re were separated from crude ginseng saponin using both silica gel open column chromatography and a reverse phase flash chromatography system. First, pure Rb1 and a mixture of Re and Rd were separated using silica gel open column chromatography with the solvent CHCl3:CH3OH:H2O (7:3:0.5). The reverse phase flash chromatography system was then used to separate Re from Rd with the solvent CH3OH: H2O (6:4). The Kitagawa protocol (27) was used with modification for ginsenoside extraction and separation.

Fig. 1. Chemical structures of PPD and PPT ginsenosides. Glc, β-D-glucopyranosyl; Arap, α-L-arabinopyranosyl; Araf, α-Larabinofuranosyl; Rha, α-L-rhamnopyranosyl

apoptosis, and inhibition of glutamate-induced neurotoxicity in PC12 cells (20,21). Therefore, much research has attempted conversion of major ginsenosides into the more active minor ginsenosides (22,23). Lactobacillus rossiae is a food grade lactic acid bacterial species usually found within the autochthonous microbiota of sourdoughs (24), spelt flour, pineapple, and the gastrointestinal tracts of humans and animals. Some strains of L. rossiae have been investigated for antifungal activity (25), and used in sourdough biotechnology for improved glutamate production, and wheat germ fermentation (26). In this study, L. rossiae DC05 from kimchi, a Korean traditional fermented food, was isolated for production of the pharmacologically active minor ginsenosides compound K and Rg2 from the major ginsenosides Rb1 and Re using enzymes from L. rossiae DC05. This is the first report of bioconversion of ginsenoside Rb1 to ginsenoside C-K and ginsenoside Re to ginsenoside Rg2 using L. rossiae DC05.

Materials and Methods Materials The standard ginsenosides Rb1, Rd, Re, Rg2, F2, and compound K were obtained from the Ginseng Genetic Resource Bank (Kyung-Hee University, Yongin, Korea). Ginsenosides Rb1 and Re were extracted from crude saponin of Panax ginseng. MRS broth was purchased from Difco (Miller, Becton Dickinson and Co., MD, USA). Silica gel-60 used for TLC was purchased from Merck, Germany. All chemicals and solvents were of analytical or HPLC grade.

Screening of β-glucosidase producing lactic acid bacteria Esculin-MRS agar was used to isolate β-glucosidaseproducing lactic acid bacteria from kimchi. The growth medium contained 3 g/L of esculin and 0.2 g/L of ferric citrate in MRS agar (Becton Dickinson) and was autoclaved for 15 min at 121oC. The lactic acid bacteria that produced β-glucosidase, which hydrolyzes esculin, appeared on esculin-MRS agar as colonies surrounded by a reddishbrown to dark brown zone. Single colonies from these plates were purified via transfer to new plates using a sterile loop. Phylogenetic analysis The 16S rRNA gene was amplified from the chromosomal DNA of strain DC05 using the universal bacterial primer sets 27F, 518F, 800R, and 1492R. Purified PCR products were sequenced by Genotech in Daejeon, Korea (28,29). The 16S rRNA gene sequences of closely related strain were collected from gene bank and aligned by CLUSTAL X program (30). A phylogenetic tree was constructed using the neighbor-joining method (31) with the MEGA 4.1 program. A bootstrap analysis with 1,000 replicates was also conducted to obtain confidence levels for the branches (32). The closest strains were included in the phylogenetic tree. Preparation of a microbial crude enzyme L. rossiae DC05 was grown in MRS broth at a temperature of 37oC with shaking (160 rpm) for 12 h until the absorbance at 600 nm reached 1.0. After centrifugation of the culture broth (8,000×g for 10 min at 4oC) using a MICRO 17R microcentrifuge (Hanil Science Industrial, Incheon, Korea), 2 volumes of ethanol (EtOH) were added to the supernatant. The solution was mixed thoroughly and placed in an ice chamber for 1 h. The protein pellet was collected via centrifugation (MICRO 17R; Hanil) at 8,000×g for 10 min at 4oC, and was dissolved in 20 mM sodium phosphate buffer (pH 7.0) (33). Effects of pH and temperature on the enzyme activity To determine the optimal pH for the enzyme, the pNP-βD-glucopyranoside (5 mM final concentration) hydrolyzing

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Bioconversion of Ginsenosides Rb1 and Re to C-K and Rg2

activity at 30oC in different buffers at pH values from 4.09.0 was studied. Buffers (20 mM) included citric acidsodium citrate (pH 4.0-5.0), sodium phosphate (pH 6.07.0), and Tris-HCl (pH 8.0-9.0). The pH stability of the enzyme was determined by the release of pNP (4-nitrophenol) after 48 h of incubation. The amount of released pNP was measured using a microplate reader at 405 nm. To determine the optimal temperature for the enzyme, the β-glucosidase activity was studied at temperatures from 20-55oC in 20 mM sodium phosphate buffer (pH 7.0). The thermostability of the enzyme was examined after 48 h of incubation. Enzymatic hydrolysis of ginsenosides The biotransformation procedure was carried out in a 20 mL screwcapped tube. An amount of 1 mL of enzyme was mixed with an equal volume of ginsenosides Rb1 and Re at 0.6 mg/mL in 20 mM sodium phosphate buffer. The mixture was then incubated at 30oC with shaking (160 rpm). During the reaction period, a 200 µL aliquot was taken every 24 h. The ginsenosides from this aliquot was collected by adding water-saturated n-butanol and analyzed using TLC, HPLC, and LC/MS. TLC analysis of ginsenosides TLC analysis was carried out using silica gel plates (60F254; Merck, Darmstadt, Germany) and a solvent system of CHCl3:CH3OH:H2O (65:35:10, v/v/v) as the developing solvent. Spots on TLC plates were detected by spraying with 10% (v/v) H2SO4, followed by heating at 110oC for 10 min. HPLC and LC/MS analysis of the biotransformation of ginsenosides The reaction mixture was extracted using n-butanol saturated with H2O, evaporated in vacuo, and the residue was dissolved in CH3OH and applied to HPLC analysis using a C18 (250×4.6 mm, particle size 5 µm) column with acetonitrile (solvent A) and distilled water (solvent B) as the mobile phases with 85% B for 5 min, 79% B for 20 min, 42% B for 55 min, 10% B for 12 min, and 85% B for 18 min at a flow rate of 1 mL/min. The ginsenoside was detected using UV analysis at 203 nm. The biosynthesis rates of ginsenoside C-K and Rg2 were calculated as: Ginsenoside C-K biosynthesis rate (%) Weighht of C-K ⁄ MW of C-K = ------------------------------------------------------------------- × 100 Weighht of Rb1 ⁄ MW of Rb1 Ginsenoside Rg2 biosynthesis rate (%) Weighht of Rg2 ⁄ MW of Rg2 = ------------------------------------------------------------------- × 100 Weighht of Re ⁄ MW of Re where the molecular weight (MW) of Rb1 was 1109, the MW of C-K was 623, the MW of Re was 947, and the MW of Rg2 was 785.

LC/MS analysis for ginsenosides was performed using an Agilent QQQ/MS (Santa Clara, CA, USA) with positive polarity and an ion trap analyzer. The ion spray was operated at 5 L/min of N2, at 3.5 Kv and 25 psi, at 300oC.

Results and Discussion Phylogenetic study The 16S rRNA gene sequence of the DC05 strain was aligned with other neighboring type strains, and taxonomic relationships were confirmed. The phylogenetic tree is shown in Fig. 2. Strain DC05 was identified as belonging to Lactobacillus, with the highest degree of 16S rRNA gene sequence similarity found with the L. rossiae DSM 15814 (99.1%), L. siliginis M1-212 (98.2%), and L. vaccinostercus strain LMG 9215 (92.9%). Hence, based on the phylogenetic tree and homology analysis, the DC05 strain was identified as L. rossiae DC05. Effects of temperature and pH on the enzyme activity This study focused on determining the enzyme optimum temperature via performance at temperatures of 20, 30, 37, 45, and 55oC. Results, shown in Fig. 3A, revealed that the reactions of the enzyme and pNP-β-D-glucopyranoside were greatly influenced by temperature. The maximum activity observed at 30oC was 89.93% degradation of pNP-β-Dglucopyranoside (pNPG) into pNP after 48 h of incubation in 20 mM sodium phosphate buffer at pH 7. The enzyme did not catalyze reactions at temperatures lower than the optimum value due to insufficient energy release. At high temperatures, enzymes lose activity due to denaturation. The pH stability of the enzyme was determined based on the hydrolyzing activity of pNPG to pNP at different pH values ranging from 4-9 at 30oC. The maximum activity of 89% degradation of pNPG to pNP was observed at pH 7.0 after 48 h of incubation (Fig. 3B). A total of 3 replicates were tested in the assays, and error bars are included in Fig. 3. TLC profile of the bioconversion of ginsenosides Rb1 and Re Ginsenoside Rb1 was converted into Rd via hydrolysis of a glucose unit at the C-20 position of the ginsenoside aglycone. Ginsenoside F2 was produced from Rd by additional hydrolysis of a single glucose moiety from the C-3 position. The concentrations of ginsenoside Rb1 and the decomposition products Rd and F2 exhibited regular changes with the reaction time (Fig. 4A). Within 3 days, Rb1 was fully hydrolyzed and converted into ginsenoside Rd, a small amount of F2, and C-K. The remaining Rd and F2 were fully converted into C-K within 4 days, indicating that ginsenosides Rd and F2 were intermediate metabolites and that C-K was the final product. Ginsenoside Re was transformed into Rg2 as the sole

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Fig. 2. Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain DC05 and related Lactobacillus species.

Fig. 3. Effect of temperature (A) and pH (B) on the activity of the enzyme determined using pNP-β-D-glucopyranoside as a substrate.

metabolite after 6 days of incubation (Fig. 4B). Hydrolysis of ginsenoside Re was started after 3 days and was almost fully decomposed after 6 days. Analysis of ginsenosides using HPLC Conversion of ginsenosides Rb1 and Re by L. rossiae DC05 was confirmed using quantitative HPLC analysis (Fig. 5). Peaks with retention times of 8.75, 18.46, 20.68, 22.59, 26.27, and 31.63 min corresponded to ginsenosides Re, Rg2, Rb1, Rd, F2, and C-K, respectively (Fig. 5A). Fig. 5B shown the peak of ginsenoside Rb1 as control. The peak for ginsenoside Rb1 fully disappeared after 4 days and a new peak appeared (Fig. 5C). This new peak had a retention time consistent

with the time of C-K. Fig. 5D shown the peak of ginsenoside Re as control. Ginsenoside Re disappeared almost entirely, followed by the appearance of a new peak (Fig. 5E). The retention time was similar to the time of ginsenoside Rg2. The biosynthesis rates of ginsenosides C-K and Rg2 were 72.88 and 53.94%, respectively. Analysis of ginsenosides using LC/MS Metabolites 1 (from Rb1) and 2 (from Re) from the transformation were subjected to LC/MS to determine MW values. The MW value of metabolite 1 was determined on the basis of a protonated molecular ion peak [MW+formic acid] at m/z 668 in MS-ESI, corresponding to the elemental formula


Fig. 4. Time-course TLC analysis of metabolites of ginsenosides Rb1 and Re converted by strain DC05. Ginsenoside Rb1 was used as a substrate (A); ginsenoside Re was used as a substrate (B); C, control; S, S1 and S2, saponin standards.

C36H62O8 (MW=623). Thus, metabolite 1 was confirmed as compound K (Fig. 6A). The molecular formula of metabolite 2 was determined to be C42H72O13 based on a protonated molecular ion peak [MW+formic acid] at m/z 830 (MW, 785) in MS-ESI. This result confirmed that metabolite 2 was Rg2 (Fig. 6B). Rb1 and Re are the major ginsenosides in protopanaxadiol and protopanaxatriol, which are present in high amounts in ginseng root. For these reasons, Rb1 and Re were selected as target materials. Ginsenosides from fresh ginseng (main root and fibrous root) were extracted and analyzed. Of the total ginsenosides, the contents of ginsenosides Rb1 and Re were 38.89 and 13.34%, respectively. Ginsenosides Rb1 and Re were separated from crude ginseng saponin using 2 step protocol. First, pure Rb1 and a mixture of Re and Rd were separated using a silica gel open column chromatography system with solvent CHCl3:CH3OH:H2O (7:3:0.5, v/v/v). Second, a reverse phase flash chromatography system was used to separate Re from Rd using solvent CH3OH: H2O (6:4, v/v). The ginsenosides compound K and Rg2 are promising natural products that could be used for treatment of some human diseases. Compound-K shows an anti-genotoxic activity, anti-allergic effects, prevention of tumor invasion and metastasis (16,17), and hepatoprotective and anti-

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Fig. 5. HPLC profiles of metabolites of ginsenosides Rb1 and Re converted by strain DC05. Ginsenoside standards (A); ginsenoside Rb1 control (B); ginsenoside Rb1 metabolites (C); ginsenoside Re control (D); ginsenoside Re metabolite (E)

Fig. 6. Mass spectra of ginsenosides Rb1 and Re after hydrolysis by strain DC05. Mass spectrum of compound K, m/z 668=[MW+formic acid], MW 623 (A); mass spectrum of Rg2, m/z 830=[MW+formic acid], MW 785 (B)

inflammatory activities (18,19). Ginsenoside Rg2 shows a protective effect in human umbilical cord vein endothelial cells (VEC-304) against H2O2-induced cell apoptosis, and inhibition of glutamate-induced neurotoxicity in PC12 cells (20,21). Unfortunately, the methods currently available for commercial production of compound K and Rg2 are difficult, which limits availability and development of

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Fig. 7. Bioconversion pathways of ginsenoside Rb1 to compound-K (A) and ginsenoside Re to Rg2 (B) using the β-glucosidase of L. rossiae DC05.

these compounds. β-Glucosidase producing food grade L. rossiae DC05 was isolated from kimchi using esculin-MRS agar and analyzed for bioconversion of the major ginsenosides Rb1 and Re. There is no known report that L. rossiae has been isolated from kimchi. This study is the first report of L. rossiae isolated from kimchi. An enzyme of L. rossiae DC05 showed excellent activity to convert Rb1 and Re. It was previously reported that L. rossiae isolated from wheat germ showed a strong beta glucosidase activity (34). This is the first report regarding bioconversion of the major ginsenosides Rb1 and Re into the pharmacologically active ginsenosides C-K and Rg2, respectively, using L. rossiae DC05. Several microbial sources have already been reported to convert major ginsenosides, but most of these can only convert either the PPD-type or the PPT-type (33,35), not both. However, L. rossiae DC05, which is a food grade lactic acid bacteria, can convert both the PPD-type and the PPT-type ginsenosides. When the ginsenoside Rb1 was used as a substrate for strain DC05, the ginsenoside was converted into C-K via the pathway Rb1→Rd→F2→CK (Fig. 7A). Ginsenoside Rb1 was converted into Rd via hydrolysis of a glucose unit at the C-20 position of the ginsenoside aglycone. Ginsenoside F2 was produced from

Rd by additional hydrolysis of a single glucose moiety at the C-3 position. Finally, the last glucose moiety at the C3 position was hydrolyzed, producing C-K. The ginsenoside Re was transformed into Rg2 via the Re→Rg2 pathway (Fig. 7B) by hydrolysis of a single glucose moiety at the C20 position. Ginsenoside Re also contains one glucose molecule and one rhamnose molecule at the C-6 position, but the enzyme of L. rossiae DC05 did not show any rhamnosidase activity. The optimum pH of the L. rossiae DC05 enzyme was 7.0 in a 20 mM sodium phosphate buffer. The crude enzyme from L. pentosus DC101 had a similar optimum pH (36). The optimum temperature was 30oC, which is similar to the optimum temperature of a crude enzyme from Leuconostoc citreum LH1 (33). This study demonstrated that ginsenosides C-K and Rg2 were produced from ginsenosides Rb1 and Re using an enzyme from food grade L. rossiae DC05. Therefore, L. rossiae DC05 could be useful for practical preparation of ginsenosides C-K and Rg2 in the food industry. Further work must be undertaken to isolate, clone, and express the β-glucosidase gene from L. rossiae DC05 so that the production cycle can be shortened and the yield can be increased.


Acknowledgments This research was supported by a grant from the Korea Institute of Planning and Evaluations for the Technology of Food, Agriculture, Forestry, and Fisheries (#111035-03-1-SB010), and Next-Generation BioGreen 21 (SSAC, PJ00952903), Republic of Korea. Disclosure The authors declare no conflict of interest.

References 1. Ligor T, Ludwiczuk A, Wolski T, Buszewski B. Isolation and determination of ginsenosides in American ginseng leaves and root extracts by LC-MS. Anal. Bioanal. Chem. 383: 1098-1105 (2005) 2. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: Multiple constituents and multiple actions. Biochem. Pharmacol. 58: 16851693 (1999) 3. Yuan CS, Wu JA, Osinski J. Ginsenoside variability in American ginseng samples. Am. J. Clin. Nutr. 75: 600-601 (2002) 4. Chae S, Kang KA, Chang WY, Kim MJ, Lee SJ, Lee YS, Kim HS, Kim DH, Hyun JW. Effect of compound K, a metabolite of ginseng saponin, combined with gamma-ray radiation in human lung cancer cells in vitro and in vivo. J. Agr. Food Chem. 57: 5777-5782 (2009) 5. Cho WC, Chung WS, Lee SK, Leung AW, Cheng CH, Yue KK. Ginsenoside Re of Panax ginseng possesses significant antioxidant and anti-hyperlipidemic efficacies in streptozotocin induced diabetic rats. Eur. J. Pharmacol. 550: 173-179 (2006) 6. Wang W, Rayburn ER, Hao M, Zhao Y, Hill DL, Zhang R, Wang H. Experimental therapy of prostate cancer with novel natural product anti-cancer ginsenosides. Prostate 68: 809-819 (2008) 7. Zhao Y, Bu L, Yan H, Jia W. 20S-protopanaxadiol inhibits Pglycoprotein in multidrug resistant cancer cells. Planta Med. 75: 1124-1128 (2009) 8. Choi CH, Kang G, Min YD. Reversal of P-glycoprotein-mediated multidrug resistance by protopanaxatriol ginsenosides from Korean red ginseng. Planta Med. 69: 235-240 (2003) 9. Jin J, Shahi S, Kang HK, Veen HW, Fan TP. Metabolites of ginsenosides as novel BCRP inhibitors. Biochem. Bioph. Res. Co. 345: 1308-1314 (2006) 10. Lim JH, Wen TC, Matsuda S, Tanaka J, Maeda N, Peng H, Aburaya J, Ishihara K, Sakanaka M. Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root. Neurosci. Res. 28: 191-200 (1997) 11. Wang XY, Zhang JT. Effect of ginsenoside Rb1 on long term potentiation in the dentate gyrus of anaesthetized rats. J. Asian Nat. Prod. Res. 5: 1-4 (2003) 12. Zhang JT, Liu Y, Qu ZW, Zhang XL, Xiao HL. Influence of ginsenoside Rb1 and Rg1 on some central neurotransmitter receptors and protein biosynthesis in the mouse brain. Acta Pharm. Sin. 23: 12-16 (1988) 13. Zhang DS, Zhang JT. Effects of total ginsenoside on learning and memory impairment induced by beta amyloid peptide (25-35). Chin. Pharmacol. Bull. 16: 422-425 (2000) 14. Christensen LP. Ginsenosides, chemistry, biosynthesis, analysis and potential health effects. Adv. Food Nutr. Res. 55: 1-99 (2008) 15. Noh KH, Son JW, Kim HJ, Oh DK. Ginsenoside compound K production from ginseng root extract by a thermostable betaglycosidase from Sulfolobus solfataricus. Biosci. Biotech. Bioch. 73: 316-321 (2009) 16. Hasegawa H. Proof of the mysterious efficacy of ginseng: Basic and clinical trials: Metabolic activation of ginsenoside: Deglycosylation by intestinal bacteria and esterification with fatty acid. J. Pharmacol. Sci. 95: 153-157 (2004) 17. Lee SJ, Sung JH, Lee SJ, Moon CK, Lee BH. Antitumor activity of a novel ginseng saponin metabolite in human pulmonary adenocarcinoma cells resistant to cisplatin. Cancer Lett. 144: 39-43 (1999)

1567 18. Choi K, Kim M, Ryu J, Choi C. Ginsenosides compound K and Rh2 inhibit tumor necrosis factor-a-induced activation of the NF-kB and JNK pathways in human astroglial cells. Neurosci. Lett. 421: 37-41 (2007) 19. Lee HU, Bae EA, Han MJ, Kim NJ, Kim DH. Hepatoprotective effect of ginsenoside Rb1 and compound K on tertbutyl hydroperoxide-induced liver injury. Liver Int. 25: 1069-1073 (2005) 20. Li N, Liu B, Dluzen DE, Jin Y. Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells. J. Ethnopharmacol. 111: 458-463 (2007) 21. Xin X, Zhong J, Wei D, Liu J. Protection effect of 20(S)-ginsenoside Rg2 extracted from cultured Panax notoginseng cells on hydrogen peroxide-induced cytotoxity of human umbilical cord vein endothelial cells in vitro. Process Biochem. 40: 3202-3205 (2005) 22. Chen GT, Yang M, Song Y, Lu ZQ, Zhang JQ, Huang HL, Wu LJ, Guo DA. Microbial transformation of ginsenoside Rb1 by Acremonium strictum. Appl. Microbiol. Biotechnol. 77: 1345-1350 (2008) 23. Chi H, Kim DH, Ji GE. Transformation of ginsenosides Rb2 and Rc from Panax ginseng by food microorganisms. Biol. Pharm. Bull. 28: 2102-2105 (2005) 24. Corsetti A, Settanni L, Sinderen DV, Felis GE, Dellaglio F, Gobbetti M. Lactobacillus rossi sp. nov. isolated from wheat sourdough. Int. J. Syst. Evol. Microbiol. 55: 35-40 (2005) 25. Valerio F, Favilla M, Bellis PD, Sisto A, Candia SD, Lavermicocca P. Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products. Syst. Appl. Microbiol. 32: 438-448 (2009) 26. Rizzello CG, Nionelli L, Coda R, Angelis MD, Gobbetti M. Effect of sourdough fermentation on stabilisation, and chemical and nutritional characteristics of wheat germ. Food Chem. 119: 10791089 (2010) 27. Kitagawa I, Taniyama T, Yoshikawa M, Ikenishi Y, Nakagawa Y. Chemical studies on crude drug processing. VI. Chemical structures of malonyl-ginsenosides Rb1, Rb2, Rc, and Rd isolated from the root of Panax ginseng C. A. MEYER. Chem. Pharm. Bull. 37: 2961-2970 (1989) 28. Lane DJ. 16S/23S rRNA sequencing. pp. 115-175. In: Nucleic Acid Techniques in Bacterial Systematics, Stackebrandt E, Goodfellow M (eds). Wiley, New York, NY, USA (1991) 29. Kim MK, Im WT, Ohta H, Lee M, Lee ST. Sphingopyxis granuli sp. nov., a β-glucosidase-producing bacterium in the family Sphingomonadaceae in α-4 subclass of the Proteobacteria. J. Microbiol. 43: 152-157 (2005) 30. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882 (1997) 31. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425 (1987) 32. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791 (1985) 33. Quan LH, Piao JY, Min JW, Yang DU, Lee HN, Yang DC. Bioconversion of ginsenoside Rb1 into compound K by Leuconostoc citreum LH1 isolated from kimchi. Braz. J. Microbiol. 42: 1227-1237 (2011) 34. Rizzello CG, Mueller T, Coda R, Reipsch F, Nionelli L, Curiel JA, Gobbetti M. Synthesis of 2-methoxy benzoquinone and 2,6dimethoxybenzoquinone by selected lactic acid bacteria during sourdough fermentation of wheat germ. Microb. Cell Fact. 12: 1-9 (2013) 35. Senthil K, Veena V, Mahalakshmi M, Pulla R, Yang DC, Parvatham R. Microbial conversion of major ginsenoside Rb1 to minor ginsenoside Rd by Indian fermented food bacteria. Afr. J. Biotechnol. 8: 6961-6966 (2009) 36. Quan LH, Cheng LQ, Kim HB, Kim JH, Son NR, Kim SY, Jin HO, Yang DC. Bioconversion of ginsenoside Rd into compound K by Lactobacillus pentosus DC101 isolated from Kimchi. J. Ginseng Res. 34: 288-295 (2010)