Advances in the Production of Minor Ginsenosides Using Microorganisms and Their Enzymes

Minor ginsenodes are of great interest due to their diverse pharmacological activities such as their anti-cancer, anti-diabetic, neuroprotective, immunomodulator, and anti-inflammatory effects. The miniscule amount of minor ginsenosides in ginseng plants has driven the development of their mass production methods. Among the various production methods for minor ginsenosides, the utilization of microorganisms and their enzymes are considered as highly specific, safe, and environmentally friendly. In this review, various minor ginsenosides production strategies, namely utilizing microorganisms and recombinant microbial enzymes, for biotransforming major ginsenosides into minor ginsenoside, as well as constructing synthetic minor ginsenosides production pathways in yeast cell factories, are described and discussed. Furthermore, the present challenges and future research direction for producing minor ginsenosides using those approaches are discussed.

Exploration and Characterization of Novel Glycoside Hydrolases from the Whole Genome of Lactobacillus ginsenosidimutans and Enriched Production of Minor Ginsenoside Rg3(S) by a Recombinant Enzymatic Process

Background: Several studies have reported that ginsenoside Rg3(S) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(S), which may improve nutritional activity, human health, and quality of life. Method: Lactobacillus ginsenosidimutans EMML 3041T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(S) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(S), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. Results: The L. ginsenosidimutans EMML 3041T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (α,β-glucosidase and α,β-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1→Rd→Rg3(S). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(S) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(S) with 74.3% chromatography purity. Conclusion: Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(S) in the functional food and pharmaceutical industries.

Optimum Conversion of Major Ginsenoside Rb1 to Minor Ginsenoside Rg3(S) by Pulsed Electric Field-Assisted Acid Hydrolysis Treatment

Ginsenoside Rg3(S) is a primary bioactive component in ginseng, which has pharmacological effects and nutritional activities. In the present study, pulsed electric field (PEF)-assisted acid hydrolysis processing was used to convert major ginsenoside Rb1 to minor ginsenoside Rg3(S). The optimum parameters of PEF assisted acid hydrolysis were analyzed by response surface methodology (RSM). The optimum processing conditions were: electric field intensity, 20 kVcm−1; acid concentration, 0.25 mol/L; pulse number, 10. The conversion rate of ginsenoside Rg3(S) achieved 68.58%, in accordance to the predicted value. The structure of hydrolyzed product was confirmed by 13C-NMR. The results suggested that PEF-assisted acid hydrolysis significantly enhanced conversion rate of ginsenoside Rg3(S).