Bifidobacterium longum SJY-001 and application thereof in fermentative preparation of ginsenoside

Abstract

A strain of Bacteroides monomorpha SJY-001 producing β-glucosidase and its application in the fermentation and conversion of ginsenosides belong to the field of probiotic fermentation. This strain was deposited at the China Center for Type Culture Collection on March 19, 2026, with accession number CCTCC NO: M2026473. This technology uses ginsenoside Rb1 as raw material and utilizes a high-β-glucosidase-producing strain for fermentation transformation to first obtain primary ginsenoside Rd, which is then further directionally transformed into highly active rare ginsenosides (S)-Rg3 and Rg5, achieving multi-stage continuous directional biotransformation. This method abandons the traditional high-pressure processes such as strong acid and strong alkali hydrolysis, high temperature and high pressure, and has the advantages of mild reaction conditions, environmental friendliness, low preparation cost, high product purity and structural stability. It provides a safe, efficient, green and low-cost new preparation route for ginsenoside Rd and rare ginsenosides (S)-Rg3 and Rg5, and has outstanding innovation and broad prospects for promotion in the industrial production and high-value application of natural drug active ingredients.

Description

Technical Field

[0001] This paper discusses a strain of Bacteroides monomorpha SJY-001 that produces β-glucosidase and its application in the fermentation and conversion of ginsenosides. It falls under the field of probiotic fermentation technology, specifically involving a strain of Bacteroides monomorpha (…). Bacteroides uniformis Application of SJY-001 in the preparation of ginsenosides from ginsenoside Rb1 through fermentation conversion. Background Technology

[0002] Ginseng ( Panax ginseng Ginsenosides (CA Mey.) are a traditional and precious Chinese medicinal herb belonging to the genus Panax in the family Araliaceae. Their core active ingredient is ginsenosides. Modern pharmacological studies have shown that ginsenosides possess various important physiological activities, including anti-tumor, anti-inflammatory, immunomodulatory, and cardiovascular protective effects, and are widely used in the pharmaceutical and health fields. Compared to the naturally abundant primary ginsenoside Rb1, rare ginsenosides (S)-Rg3 and Rg5, obtained through deglycosylation or structural modification, exhibit stronger pharmacological activity, higher lipid solubility, and greater bioavailability, showing great application potential in innovative drug development and high-end functional food development. Furthermore, ginsenoside Rd, as a key active monomer, not only possesses multiple pharmacological activities such as anti-inflammatory effects, improved microcirculation, nerve cell protection, and enhanced stress tolerance, but also plays a core intermediate role in the biotransformation pathway of highly active rare ginsenosides, serving as a crucial link between primary saponins and high-value-added rare ginsenosides. However, rare ginsenosides are present in extremely low amounts in natural ginseng, making large-scale supply difficult through extraction and separation alone, severely restricting the development and application of related products. Furthermore, current ginsenoside Rd preparation technologies generally suffer from key bottlenecks such as weak transformation specificity, easily compromised product activity, and poor process compatibility, severely limiting their large-scale production and industrial application. This invention discloses a method using Bacteroides monomorpha (… Bacteroides uniformis A biotransformation method for preparing ginsenoside Rd and rare ginsenosides (S)-Rg3 and Rg5 using strain SJY-001. This strain possesses excellent characteristics of high β-glucosidase production, enabling targeted hydrolysis of the characteristic glycosidic bonds of ginsenoside Rb1, thereby achieving efficient and complete conversion of Rd and the target rare ginsenosides. This method requires no external enzyme preparations or chemical reagents, and the fermentation process is closely integrated with downstream purification, with simple operation, significantly reducing production costs. It provides stable and feasible technical support for the large-scale, green, and efficient preparation of ginsenoside Rd and rare ginsenosides (S)-Rg3 and Rg5.

[0003] Bacteroides monomorpha (Bacteroides uniformis)This is a novel potential probiotic discovered in the mammalian gut microbiota in recent years. Its core physiological functions have been confirmed to be closely related to the maintenance of intestinal barrier integrity, regulation of host metabolic homeostasis, and regulation of mucosal immune response. Numerous studies have shown that this strain can optimize the gut microbiota structure, inhibit pathogenic bacterial colonization, and upregulate the expression of tight junction proteins in intestinal epithelial cells by secreting short-chain fatty acids, demonstrating promising application prospects in the prevention and intervention of gut-related diseases. However, it is noteworthy that while *Bacteroides monomorpha* exhibits probiotic properties in multiple aspects, its application in the biotransformation of ginsenosides has not yet been reported. Therefore, this invention is the first to utilize *Bacteroides monomorpha* (… Bacteroides uniformis The SJY-001 strain, using ginsenoside Rb1 as a substrate, successfully achieved the biotransformation of ginsenoside Rd, rare ginsenosides (S)-Rg3, and Rg5 through directed fermentation after 7 days. This method requires no exogenous enzymes or chemical reagents, features mild reaction conditions, high conversion efficiency, and effectively maintains the natural active structure of the products. The overall process is green, simple, and easy to scale up, demonstrating promising prospects for industrial application. The obtained ginsenosides Rd, rare ginsenosides (S)-Rg3, and Rg5 have a wide range of applications, showing broad potential and market prospects in disease intervention and functional food development. Summary of the Invention

[0004] The purpose of this invention is to provide a monomorphic Bacteroides strain ( Bacteroides uniformis The application of SJY-001 in the fermentation and transformation of ginsenoside Rb1 to prepare ginsenoside Rd, rare ginsenoside (S)-Rg3 and Rg5 provides a new and efficient route for the preparation of ginsenoside Rd, rare ginsenoside (S)-Rg3 and Rg5.

[0005] The technical solution adopted by this invention to solve the technical problem is as follows:

[0006] The monomorphic Bacteroides provided by this invention ( Bacteroides uniformis SJY-001 was deposited at the China Center for Type Culture Collection on March 19, 2026, with accession number CCTCC M 2026473, located in Wuhan, China.

[0007] The present invention provides a strain of β-glucosidase-producing Bacteroides monomorpha ( Bacteroides uniformis The application of SJY-001 fermentation and transformation of ginsenoside Rb1 to prepare ginsenoside Rd, and rare ginsenosides (S)-Rg3 and Rg5.

[0008] In a preferred embodiment, the ginsenosides include ginsenoside Rd, rare ginsenosides (S)-Rg3 and Rg5.

[0009] As a preferred embodiment, the present invention provides a monomorphic Bacteroides strain ( Bacteroides uniformis The fermentation and conversion of SJY-001 ginsenoside Rb1 to prepare ginsenoside Rd, and its application in rare ginsenosides (S)-Rg3 and Rg5, specifically includes the following steps:

[0010] Ginsenoside Rb1 was added to sterilized brain and heart extract broth BHI liquid fermentation medium at a ratio of 0.5 mg / mL, and then filtered through a 0.22 μm microporous membrane for sterilization. The resulting Bacteroides monomorpha (…) Bacteroides uniformis SJY-001 was used to prepare a bacterial suspension, and the bacterial suspension was prepared at a ratio of 2×10⁻⁶. 8 The inoculum was inoculated into the above-mentioned liquid fermentation medium supplemented with ginsenoside Rb1 at a CFU / mL inoculum and anaerobic fermented at 37±0.5℃ for 7 days. After fermentation, the fermentation product was extracted with water-saturated n-butanol, then concentrated under reduced pressure at 50℃ and freeze-dried at -80℃ and 5Pa. The residue was dissolved in methanol and centrifuged at 10000rpm and 4℃ for 15min. The supernatant was then freeze-dried under vacuum at -80℃ and 5Pa to obtain the dried product, which contained ginsenoside Rd and rare ginsenosides (S)-Rg3 and Rg5.

[0011] In a preferred embodiment, the liquid fermentation medium comprises: 10.0 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5.0 g / L dehydrated calf heart extract, 2.5 g / L disodium hydrogen phosphate, 5.0 g / L sodium chloride, and 2.0 g / L glucose; pH 7.4 ± 0.2.

[0012] As a preferred embodiment, Bacteroides monomorpha ( Bacteroides uniformis The specific process for preparing SJY-001 into a bacterial suspension is as follows:

[0013] Bacteroides monomorphum ( Bacteroides uniformis SJY-001 was inoculated into liquid BHI medium and anaerobically cultured at 37°C for 48–72 h. After centrifugation at 4°C and 8000–12000 rpm for 5–10 min, the bacterial pellet was collected and resuspended in sterile PBS to adjust the viable count to 1.0 × 10⁻⁶. 8 ~1.0×10 9 CFU / mL, to obtain Bacteroides monomorphosum ( Bacteroides uniformis ) SJY-001 bacterial suspension.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention screened a probiotic strain SJY-001 from fecal samples of healthy mice. This strain produces β-glucosidase and can completely ferment ginsenoside Rb1 into ginsenoside Rd, and contains rare ginsenosides (S)-Rg3 and Rg5. The strain SJY-001 was identified as a monotypic bacterium. Bacteroides uniformis The *Bacteroides monomorpha* was deposited on March 19, 2026, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2026473. This invention identifies *Bacteroides monomorpha* (…). Bacteroides uniformis A novel fermentation process, SJY-001, was used to convert ginsenoside Rb1 into ginsenoside Rd, rare ginsenosides (S)-Rg3, and Rg5. HPLC analysis was used to identify the converted ginsenosides Rd, (S)-Rg3, and Rg5. This process utilizes Bacteroides monomorpha (…). Bacteroides uniformis SJY-001 possesses the characteristic of producing β-glucosidase. Using ginsenoside Rb1 as a raw material, it utilizes Bacteroides monomorpha (… Bacteroides uniformis The SJY-001 fermentation method was used to prepare ginsenoside Rd, rare ginsenoside (S)-Rg3, and Rg5. HPLC analysis was used to identify the converted ginsenosides Rd, (S)-Rg3, and Rg5. The results showed that the retention time of the ginsenoside Rb1 conversion product was consistent with that of the ginsenoside Rd, (S)-Rg3, and (Rg5) standards. Compared with traditional biotransformation technologies, this process has significant technical advantages and application value. This system can efficiently achieve the complete and directional conversion of ginsenoside Rb1 under mild, controllable, and non-stress conditions. It not only has high conversion efficiency, stable conversion rate, and excellent product purity, but also maximizes the preservation of the natural configuration and bioactivity of rare saponins. Furthermore, this process does not require the addition of exogenous enzymes, the fermentation process is stable and controllable, simple to operate, and has good reproducibility, possessing strong potential for industrial scale-up and suitable for large-scale industrial production. The ginsenosides Rd, (S)-Rg3, and Rg5 prepared by this method are all high-value-added active ingredients with clear pharmacological effects, high bioavailability, and broad application prospects. They can be widely used in various fields such as innovative drug development, functional health foods, special dietary foods, and high-end cosmetics. This technology provides a new technical route for the efficient utilization, in-depth development, and high-value transformation of ginseng resources, and has important scientific value and practical significance for enhancing the added value of the ginseng industry chain and promoting the modernization of traditional Chinese medicine and the high-quality development of the bio-manufacturing industry. Attached Figure Description

[0016] Figure 1 A monomorphobacterium that produces high levels of β-glucosidase ( Bacteroides uniformis ) SJY-001 colorimetric pattern on aescin screening medium.

[0017] Figure 2 The HPLC chromatograms of ginsenoside Rb1 on days 0 and 7 of fermentation are shown.

[0018] Figure 3 Bacteroides monomorpha ( Bacteroides uniformis The biotransformation and synthesis route of SJY-001 for the fermentation and conversion of ginsenoside Rb1 to ginsenoside Rd, and rare ginsenosides (S)-Rg3 and Rg5. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] I. Isolation of bacterial strains

[0021] Fecal samples were collected from mice with chronic alcoholic liver injury after oral ginseng intervention in June 2025. 1 mL of fecal sample suspension was added to BHI medium for anaerobic enrichment culture for 48 h. The streak plate method was used, with streaks applied to BHI medium plates and anaerobic cultured at 37°C for 48 h. Single colonies were examined under a microscope, and Gram-negative strains were selected and cultured at 37°C for 48 h. This streaking and purification process continued until a purified product with a uniform colony morphology was obtained. The culture was then further inoculated onto aescin selection medium (used to isolate strains producing β-glucosidase; 1 g / L aescin and 0.5 g / L ferric citrate were added to BHI agar solid medium, and autoclaved at 121°C for 20 min). Strains with a brownish-red to dark brown ring around them were considered positive strains (e.g., ...). Figure 1 As shown in the figure, this indicates that the strain possesses the ability to produce β-glucosidase. The obtained pure culture was inoculated into liquid BHI medium for further cultivation. 0.8 mL of the bacterial culture was mixed with 0.2 mL of glycerol and stored at -80°C. One strain of *Bacteroides monomorpha* (…) was… Bacteroides uniformis It was named SJY-001.

[0022] II. Identification of the strain

[0023] 1. The physiological and biochemical identification results of strain SJY-001 are as follows:

[0024] Gram-negative, it is positive for aescin in 11 carbohydrate patterns, and negative for cellobiose, maltose, salicin, sucrose, raffinose, inulin, lactose, hippuric acid, mannitol, and sorbitol. The optimal growth temperature is 37°C; the suitable pH is 7–8; and it exhibits mild to moderate homogeneous turbidity in liquid BHI medium.

[0025] 2. Molecular biological identification and results are as follows:

[0026] The target strain was inoculated into fresh BHI liquid medium and cultured for 48 h. Bacterial DNA was extracted using a kit from Tiangen Biotech Co., Ltd., and its 16S rDNA sequence was amplified. A species-universal primer set consisting of 1492R and 27F was used for amplification. Electrophoresis of the 16S rDNA PCR product of strain SJY-001 showed a highly specific band at approximately 1500 bp, consistent with the expected result. Sequencing was performed, and the sequence is shown in SEQ ID NO.1 of the sequence listing. The sequenced sequence was compared with the 16S rDNA gene sequences of some strains already registered on the website http: / / www.ncbi.nlm.nih.gov. The results showed that strain SJY-001 is similar to Bacteroides monomorpha (…). Bacteroides uniformis The homology of strain SJY-001 with JCM 5828 (NR 040866.1) reached 99.78%. Based on the above results, strain SJY-001 was identified as belonging to Bacteroides monomorpha (…). Bacteroides uniformis ).

[0027] The primer pair sequence information is as follows:

[0028] 27F: 5′-agagttgatcctggctcag-3′;

[0029] 1492R: 5′-ggttaccttgttacgactt-3′.

[0030] The PCR amplification conditions used were as follows: pre-denaturation: 94℃ for 3 min; denaturation: 94℃ for 40 s, annealing: 56℃ for 40 s, extension: 72℃ for 1 min, for a total of 35 cycles; final extension: 72℃ for 5 min, and storage at 4℃.

[0031] III. Preservation of bacterial strains

[0032] The monomorphic Bacteroides of the present invention ( Bacteroides uniformisSJY-001 was deposited on March 19, 2026, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province (Wuhan University Collection Center), with accession number CCTCC NO: M 2026473.

[0033] IV. Bacteroides monomorpha ( Bacteroides uniformis Preparation of SJY-001 bacterial suspension

[0034] The obtained monomorphic Bacteroides ( Bacteroides uniformis SJY-001 was inoculated into liquid BHI medium and incubated statically at 37°C for 48 hours. The culture was then centrifuged at 12000 rpm for 10 minutes at 4°C, and the bacterial pellet was collected. The pellet was resuspended in sterile PBS, and the viable count was adjusted to 1.0 × 10⁻⁶ cells / mL. 8 ~1.0×10 9 CFU / mL, to obtain Bacteroides monomorphosum ( Bacteroides uniformis ) SJY-001 bacterial suspension.

[0035] V. Bacteroides monomorpha ( Bacteroides uniformis SJY-001 fermentation conversion of ginsenoside Rb1 to prepare ginsenoside Rd, rare ginsenosides (S)-Rg3 and Rg5

[0036] 1. Preparation of liquid fermentation medium: 10.0 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5.0 g / L dehydrated calf heart extract, 2.5 g / L disodium hydrogen phosphate, 5.0 g / L sodium chloride, and 2.0 g / L glucose; pH 7.4 ± 0.2, sterilized at 121℃ for 20 min.

[0037] 2. Fermentation and transformation: Ginsenoside Rb1 (purchased from Chengdu Efa Biotechnology Co., Ltd., HPLC ≥ 98%) was added to the sterilized liquid fermentation medium at a ratio of 0.5 mg / mL, and then filtered through a 0.22 μM microporous membrane for sterilization. The Bacteroides monomorpha (… Bacteroides uniformis SJY-001 was used to prepare a bacterial suspension, and the bacterial suspension was prepared at a ratio of 2×10⁻⁶. 8 The inoculum was inoculated into liquid fermentation medium supplemented with ginsenoside Rb1 at a CFU / mL level and anaerobic fermented at 37℃ for 7 days. After fermentation, the fermentation product was extracted three times with water-saturated n-butanol. The fermentation product was concentrated under reduced pressure at 50℃ and freeze-dried at -80℃ and 5Pa. The residue was dissolved in methanol and centrifuged at 10000 rpm and 4℃ for 15 min. The supernatant was depressurized to recover the solvent and then freeze-dried under vacuum at -80℃ and 5Pa to obtain the dried product. The dried product contained ginsenoside Rd and rare ginsenosides (S)-Rg3 and Rg5.

[0038] VI. Identification of Ginsenosides by High Performance Liquid Chromatography (HPLC)

[0039] Ginsenoside Rd, rare ginsenoside (S)-Rg3, and Rg5 were detected by high-performance liquid chromatography (HPLC). Ginsenoside Rb1 and the dried products obtained above were dissolved in chromatographic methanol, filtered through a 0.22 μm microporous membrane, and then used for HPLC analysis. The HPLC chromatographic method was as follows: Agilent ZORBAX SB-C18 column, injection volume 20 μL, elution rate 1.0 mL / min, column temperature 30 °C, and detection wavelength 203 nm. The mobile phase consisted of aqueous phase (A) and acetonitrile phase (B), and gradient elution was performed as follows: 0 min, 81.50% A, 18.50% B; 20 min, 79.50% A, 20.50% B; 30 min, 70% A, 30% B; 45 min, 65% A, 35% B; 60 min, 55% A, 45% B; 70 min, 40% A, 60% B; 80 min, 30% A, 70% B; 90 min, 20% A, 80% B; 91 min, 81.50% A, 18.50% B; 95 min, 81.50% A, 18.50% B.

[0040] HPLC chromatographic identification results as follows Figure 2 As shown, qualitative and quantitative analysis of ginsenoside components in the products before and after fermentation revealed that the retention times of the ginsenoside Rb1 conversion product were consistent with those of ginsenoside Rd, rare ginsenoside (S)-Rg3, and Rg5 standards. This demonstrates that ginsenoside Rb1, after fermentation by β-glucosidase-producing Bacteroides monomorpha (…),… Bacteroides uniformis SJY-001 is fermented into ginsenoside Rd, and rare ginsenosides (S)-Rg3 and Rg5.

[0041] In utilizing Bacteroides monomorphosum ( Bacteroides uniformis The biotransformation and synthesis routes for preparing ginsenoside Rd, rare ginsenosides (S)-Rg3, and Rg5 from ginsenoside Rb1 via fermentation of SJY-001 bacterial suspension are as follows: Figure 3 As shown: The ginsenoside Rb1 in Bacteroides monomorpha ( Bacteroides uniformis SJY-001 hydrolyzes the glucose at the C-20 position of ginsenoside Rb1 to generate primary ginsenoside Rd, then removes one glucose molecule to generate rare ginsenoside (S)-Rg3, and then dehydrates to generate rare ginsenoside Rg5.

[0042] This invention discloses a monomorphic Bacteroides strain ( Bacteroides uniformisThe fermentation and conversion of ginsenoside Rb1 into ginsenoside Rd, and its application in rare ginsenosides (S)-Rg3 and Rg5, as described in SJY-001, can be used as a reference for those skilled in the art to appropriately modify process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The products of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the products described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. Bacteroides monomorpha ( Bacteroides uniformis SJY-001, characterized in that, The monomorphobacteria ( Bacteroides uniformis It was deposited at the China Center for Type Culture Collection on March 19, 2026, with accession number: CCTCC NO: M 2026473.

2. The monomorphic Bacteroides as described in claim 1 ( Bacteroides uniformis The application of SJY-001 in the fermentation and transformation of ginsenoside Rb1 to prepare ginsenosides, wherein the ginsenosides include ginsenoside Rd, rare ginsenosides (S)-Rg3 and Rg5.

3. The application according to claim 2, characterized in that, The biotransformation synthesis route of ginsenoside Rd is as follows: ginsenoside Rb1 is synthesized in Bacteroides monomorpha (… Bacteroides uniformis Under the action of SJY-001, a glucose residue at the C-20 position of ginsenoside Rb1 is hydrolyzed to generate ginsenoside Rd, which then loses a glucose residue to generate rare ginsenoside (S)-Rg3, and is subsequently dehydrated to generate rare ginsenoside Rg5.

4. The application according to claim 2, characterized in that, comprising the following steps: Ginsenoside Rb1 was added to sterilized BHI liquid culture medium at a ratio of 0.5 mg / mL, and then filtered through a 0.22 μm microporous membrane for sterilization. (The remaining text appears to be unrelated and possibly machine-translated gibberellins.) Bacteroides uniformis SJY-001 was used to prepare a bacterial suspension, and the bacterial suspension was prepared at a ratio of 2×10⁻⁶. 8 The inoculum was inoculated into BHI liquid medium supplemented with ginsenoside Rb1 at a CFU / mL level and anaerobic fermented at 37±0.5℃ for 7 days. After fermentation, the fermentation product was extracted with water-saturated n-butanol, then concentrated under reduced pressure at 50℃ and freeze-dried at -80℃ and 5Pa. The residue was dissolved in methanol and centrifuged at 10000rpm and 4℃ for 15min. The supernatant was then subjected to vacuum freeze-drying at -80℃ and 5Pa after solvent recovery under reduced pressure to obtain the dried product. The dried product contained ginsenoside Rd and rare ginsenosides (S)-Rg3 and Rg5.

5. The application according to claim 4, characterized in that, Bacteroides monomorphum ( Bacteroides uniformis The specific process for preparing SJY-001 into a bacterial suspension is as follows: Bacteroides monomorpha (… Bacteroides uniformis SJY-001 was inoculated into liquid BHI medium and anaerobically cultured at 37°C for 48 hours. After centrifugation at 8000-12000 rpm for 5-10 minutes at 4°C, the bacterial pellet was collected and resuspended in sterile phosphate-buffered saline (PBS) to adjust the viable count to 1.0 × 10⁻⁶. 8 ~1.0×10 9 CFU / mL, to obtain Bacteroides monomorphosum ( Bacteroides uniformis ) SJY-001 bacterial suspension.

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