Biotransformation of Major Ginsenoside Rb1 to Rd by Dekkera anomala YAE-1 from Mongolian Fermented Milk (Airag)
1Department of Animal Biosystem Science, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 34134, Republic of Korea
2Department of Pharmacology Science, College of Pharmacology, Chungnam National University, Daejeon 34134, Republic of Korea
3Agricultural Research Station, Fort Valley State University, Fort Valley, GA 31030, USA and and Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA
J. Microbiol. Biotechnol. 2020; 30(10): 1536-1542
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
In Korea, ginseng products are widely used as health functional foods and generally enjoy brisk sales. In 2018, health functional food sales totaled US$2.1 billion and red ginseng products accounted for 44%, or US$927 million in sales . Biologically functional components found in most ginseng species include ginsenosides, polysaccharides, peptides, polyacetylenic alcohols, and fatty acids . Ginsenoside Rd is the main hydrolyzed product of ginsenosides Rb1, Rb2, and Rc, which are components in more than 80% of ginseng roots [3, 4]. Among ginsenosides, the minor ginsenoside Rd has been reported to have important biological activities including immunosuppressive  and anti-inflammatory properties . Ginsenosides Rk1 and Rg5, products of ginsenoside Rd, prevent wrinkle formation and the loss of collagen from epithelial cells . Moreover, the anti-inflammatory properties of ginsenoside Rd inhibit iNOS and COX-2, which are known to play pivotal roles in the pathogenesis of acute and chronic inflammation. Ginsenoside Rd has therapeutic effects on inflammation and may therefore have applications in the treatment of inflammatory diseases . Moreover, ginsenoside Rd prevents Pb-induced decrease in NSC proliferation by inhibiting microgliosis .
Airag is the local name for fermented mare’s milk in Mongolia. The traditional beverage is fermented by a co-culture of yeasts and lactic acid bacteria and is produced by churning mare’s milk in a leather sack with a wooden masher.
Airag does not use an inoculated starter and is instead fermented by mixing the lactic acid bacteria and yeast in mare’s milk. Therefore, the diversity of yeast and lactic acid bacteria is very high.
The conversion of ginsenosides by LAB enzymes has great potential importance for human health due to biological, immunological and medicinal functions. We have hypothesized that the β-glucosidase activity of the
Materials and Methods
Collection of Airag Samples
Airag was prepared according to the method . Airag samples were collected from four different mare milk farms located in close proximity to Ulaanbaatar, Mongolia.
Isolation of Strains with β-Glucosidase Activity from Airag
The experimental strains were isolated from airag, a Mongolian fermented milk product. Samples were plated according to the method [12-14]. Esculin-positive colonies were inoculated in pH 4.0 Yeast Malt broth (HiMedia Laboratories, USA), and sample preparation and β-glucosidase activity were determined by the procedure . Conversion of ginsenosides was checked by TLC analysis .
18S Ribosomal RNA Gene Sequencing of the Strain YAE-1 with β-Glucosidase Activity
18S rRNA gene sequencing of selected strains was conducted according to the method . PCR sequences were compared with those in the NCBI database using BLAST. The primers used were ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'.
Effect of Temperature and pH on Growth of
D. anomala YAE-1
To determine the optimum pH (3.0-7.0) and temperature (20-40°C) for growth of strain YAE-1, 3% (v/v) of the yeast strain was inoculated in YM broth for 72 h in an incubator, and the specimen was checked for viable cell count.
Enzyme Activity by API ZYM Kit
Enzyme activity was detected using the API ZYM Kit (BioMérieux, Mercy I’Etoile, France) in accordance with the manufacturer's instructions. Selected colonies from the surface agar plates were suspended in medium (0.085% NaCl, Ref. 20070, BioMérieux,) to a turbidity adjusted to a McFarland No. 5.0-6.0 standard (BioMérieux). Each microcupule of the API ZYM gallery containing 19 dehydrated chromogenic enzyme substrates was inoculated with 50 μl of the suspension, and the strip was incubated 4 h at 37°C. (BioMérieux).
Analysis of Carbohydrate Utilization
Carbohydrate fermentation tests were performed according to the method . Selected colonies from the surface agar plates were suspended in medium (0.085% NaCl) to a turbidity adjusting to a McFarland No. 2.0 standard (BioMérieux) and inoculated into API C medium. Each microcupule of the API 20C AUX kit containing 19 carbohydrates with pH detector enzyme substrates was inoculated with 100 μl of the suspension. The first microcupules were used as a negative control. The strips were incubated 48 and 72 h at 29 ± 2°C.
β-Glucosidase Activity of
D. anomala YAE-1 Supernatant
β-Glucosidase activity of the supernatant was determined by the rate of hydrolysis of 5 mM p-nitrophenyl-β-D-glucopyranoside (PNPG, Sigma-Aldrich, Germany) at 40°C and pH 7.0 (50 mM potassium phosphate buffer). The optimum pH and temperature on β-glucosidase activity were measured over a range of pH from 3.0 to 9.0 and temperature from 25°C to 70°C, respectively. β-Glucosidase activity of the supernatant was determined according to the method .
Conversion of Ginsenoside Rb1 by Supernatant of
D. anomala YAE-1
Selected colonies were inoculated in YM broth (pH 4.0) (HiMedia Laboratories) for 3 days at 30°C. Supernatant activities were tested for hydrolysis of the major ginsenoside Rb1 under optimum condition (40°C, pH 5.0) of β-glucosidase. The reaction condition was cultured at 40°C for 48 hours. Conversion of ginsenoside Rb1 was checked by TLC and HPLC analysis .
Fermentation of Ginseng Root by
D. anomala YAE-1
Ginseng roots were sliced and sterilized at 75°C for 10 min. A 20% (w/v) ginseng root solution was incubated with 3% (v/v) of yeast strain YAE1 at 30°C for 7 days. The fermentation characteristics of ginseng root by the strain YAE-1 were analyzed by TLC and HPLC, and viable cell counts were performed according to the method .
Analysis of Ginsenoside Hydrolysates
Hydrolyzed ginsenosides obtained from filtrate samples showing β-glucosidase activity were analyzed using the reversed-phase HPLC system Shimadzu LC-6AD (Shimadzu, Japan) and an ACE-5-C18 column (4.6 × 250 mm) equilibrated with solvent A (H2O). HPLC analysis was conducted according to the method .
All analyses were repeated at least 3 times and are expressed as means ± standard deviation (SD) .
Results and Discussion
Isolation and Screening of the Strain Exhibiting β-Glucosidase Activity
Samples of the Mongolian traditional fermented dairy product airag were collected from 5 different farms. The respective isolated strains were: Airag A (strain YAA1-YAA7), Airag B (strain YAB1-YAB8), Airag C (strain YAC1-YAC6), Airag D (strain YAD1-YAD8), Airag E (strain YAE1-YAE8), and they were examined for β-glucosidase activity. Fifteen strains showed esculin-positive reaction. The YAE-1 strain was found to be the strongest esculin positive. When the major ginsenosides Rb1, Rb2, Rd, Re, and Rg1 were hydrolyzed by the strains, strain YAE-1 can hydrolyze ginsenoside Rb1 converted to Rd in minor quantity, while no conversions occurred for ginsenosides Rb2, Rd, Re and Rg1 (Fig. 1). β-Glucosidase is mainly a hydrolyzing enzyme for conversion of protopanaxadiol ginsenosides in ginseng. Many types of microorganisms with β-glucosidase activity have been used to hydrolyze ginsenosides Rb1, Rb2, Rc, and Rd to minor ginsenoside F2 and compound K [15-18].
TLC analysis of ginsenoside Rb1, Rb2, Rd, Re and Rg1 fermented byTLC was performed on Silica gel 60 F254 plates. A solvent mixture of chloroform: methanol: water (65:35:10, v/v/v, lower phase) was used as the developing solvent. D. anomalaYAE-1 at 30°C for 48 h.
Strain YAE-1 was selected for DNA analysis. The PCR produced 18S rRNA sequences of the strain YAE-1. The sequence of the strain was compared using the NCBI database, and it was found to be 99.9% homologous with
Phylogenetic tree based on 18S rRNA sequences showing the position of yeast strain YAE-1.Scale length is 0.01.
Optimum Temperature and pH for Growth of
D. anomala YAE-1
The effect of temperature and pH on growth of
Figure 3. Effect of temperature and pH on growth of
D. anomalaYAE-1. A: Temperature, B: pH.
By changing the pH of the medium,
Enzymatic Activity and Carbohydrate Utilization of
D. anomala YAE-1
Enzymatic activities of
Optimum Temperature and pH of β-Glucosidase Activity in Supernatant
The β-glucosidase of the supernatant exhibited maximum enzyme activity at 40°C (Fig. 4), but gradually decreased activity at 45 to 65°C, and finally showed significantly decreased activity above 65°C. The optimum pH for the activity occurred around 4.0 to 5.0 at 30°C. The enzyme activity was rapidly decreased above pH 5. Enzyme maximal activity was at pH 5.0 (Fig. 4). On the other hand, the optimum pH and temperature of β-glucosidase isolated from
Figure 4. Optimum temperature and pH of β-glucosidase activity in supernatant of
Biotransformation of Ginsenoside Rb1 to Rd by
D. anomala YAE-1
D. anomala YAE-1 exhibiting β-glucosidase activity was incubated in YM broth (pH 4.0) for 48 h at optimum condition of 30°C. Hydrolysis of the major ginsenoside Rb1 was tested under optimum condition for β-glucosidase (40°C, pH 5.0). After 48 h, 3.095 mg/ml of ginsenoside Rb1 was fully hydrolyzed by supernatant with β-glucosidase activity, and released 3.0 mg/ml of ginsenoside Rd (Fig. 5), (Table 1A). Our results showed that Rd was the main ginsenoside in the final fermentation product of ginsenoside Rb1. Enzymatic hydrolysis made it possible to produce minor ginsenosides. The biological activities of ginsenosides increase according to their molecular mass. . Many reports have been conducted on the conversion of the ginsenoside Rb1 into Rd during fermentation [13, 20, 21]. Our results on the biotransformation of ginsenoside Rb1 by
Biotransformation of ginsenoside Rb1 hydrolyzed by supernatant ofA: HPLC analysis, 0 h, B-4 h, C-8 h, D-16 h, E-24 h, F-48 h; TLC analysis. D. anomalaYAE-1 for 48 h.
Characteristics of Ginseng Root Fermented by
D. anomala YAE-1
D. anomala YAE-1 hydrolyzed ginsenoside Rb1 to ginsenoside Rd in 20% fermented ginseng root. The peak size of ginsenoside Rb1 was small and that of ginsenoside Rd was significantly large after 7 days, as revealed in HPLC chromatograms (Fig. 6). Furthermore, the quantity of ginsenoside Rd was elevated from 0 to 1.404 mg/ml at the same period (Table 1B). In fermented ginseng roots, ginsenoside Rb1 was converted to Rd, reducing ginsenoside Rb1 and increasing Rd. The factor that absolutely affects ginsenoside conversion is fermentation time. Rd was increased as the main ginsenoside in the final fermentation product of ginseng roots, confirming that Rb1 was decreased continuously during the 7-day fermentation. The quantity of minor ginsenoside Rd was elevated from 0 to 0.060 ± 0.011 mg/ml at 7 days in
Biotransformation of ginsenoside of fermented ginseng root byA: HPLC analysis, A-0 day, B-1 days, C-3 days, D-7 days. B: TLC analysis. D. anomalaYAE-1 during 0, 1, 3, 7 days.
This study was supported by a grant (NRF-K2A4A1036939) from the National Research Foundation of the Republic of Korea.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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