Production of minor ginsenosides by combining Stereum hirsutum and cellulase

Abstract

Minor ginsenosides (MGs) (include ginsenoside F2, Compound K, PPT, etc), which are generally not produced by ginseng plants naturally, are obtained by deglycosylation of major ginsenosides. However, the conventional processes used to produce deglycosylated ginsenosides focus on the use of intestinal microorganisms for transformation. In this study, an edible and medicinal mushroom Stereum hirsutum JE0512 was screened from 161 β-glucosidase-producing soil microorganisms sourced from wild ginseng using the plate coloration method. Furthermore, JE0512 was used for the production of CK from ginseng extracts (GE) in solid-state fermentation (SSF) using 20 g corn bran as substrate, 4 g GE, and 20% inoculation volume, and the results showed that the highest CK content was 29.13 mg/g. After combining S. hirsutum JE0512 with cellulase (Aspergillus niger), the MGs (F2, CK, and PPT) content increased from 1.66 to 130.79 mg/g in the final products. Our results indicate that the Stereum genus has the potential to biotransform GE into CK and the combination of S. hirsutum JE0512 and cellulase could pave the way for the production of MGs from GE.

Fig 1. The chemical structural formula of ginsenosides.

Glc, β-D-glucopyranosyl; arap, α-L-arabinopyranosyl; xyl, β-D-xylopyranosyl; araf, α-L-arabinofuranosyl; rha, α-L-rhamncpyranosyl; Gyp, gypenoside; C, compound.

Fig 2. Screening of strains biotransformed GE into CK.

A: Geniposide was hydrolyzed to genipin by β-glucosidase; B: Colonies on the plate without glucosidase activity (a), colonies with a blue circle had a higher β-glucosidase activity (b); C: Comparison the ability of strains to biotransform GE into CK. Values are means ± SD of three replications; D: HPLC profiles of ginsenosides in ginsenosides standard, GE, and GE fermented with JE0512.

Fig 3. UPLC-Q-TOF-MS analysis of the fermented sample of JE0512.

A: Total ion current chromatograms of CK standard; B: Total ion current chromatograms of JE0512 fermentation sample; C: Mass spectrum of CK standard; D: Mass spectrogram of JE0512 fermentation sample; E: The chemical structural formula of CK.

Fig 4. Identification of strain JE0512.

A: Colony morphology of the isolate JE0512 grown at 25°C on PDA. B: Phylogenetic analysis of the related species of the strain JE0512 using the neighbor-joining approach. The scale bars represent 0.002 substitutions per site. The tree was constructed using a neighbor-joining method with bootstrap values of 1000 replications.

Fig 5. Optimization of main SSF process parameters for maximum CK content and β-glucosidase activity.

A: Effect of the solid-state fermentation substrate (20 g); B: Effect of inoculation volume (%); C: Effect of the amount of ginseng extracts (g); D: Effect of fermentation period (d). Values are shown as means ± SD of three replications. A one-way ANOVA was used to assess the statistical significance of the differences in expression levels. Different letters (a-e) indicate significant differences between each other (P < 0.05).