Pseudoxanthomonas wennieri strain TY3-10 and application thereof in preparation of rare ginsenoside

Abstract

The invention provides a pseudoxanthomonas wenibertii strain TY3-10 and application thereof in preparation of rare ginsenoside, and belongs to the technical field of biological medicine. The preservation number of the pseudoxanthomonas wenibertii strain TY3-10 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.35238. The pseudoxanthomonas wenibertii The strain TY3-10 has the characteristic of producing beta-glucosidase and beta-xylosidase, and can be used for converting various ginsenosides and notoginsenoside into rare ginsenosides F2, Rd2, Fd, C-Mc1 and Rh1. The invention further provides a method for preparing the rare ginsenoside, and ginsenoside is subjected to a conversion reaction under the action of the pseudoxanthomonas wenibertii strain TY3-10 to obtain the rare ginsenoside. The invention provides a new process for preparing the rare ginsenoside, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, specifically relating to a strain of *Xanthomonas winnipes* (…). Pseudoxanthomonas winnipegensis ) strain TY3-10 and its application in the preparation of rare ginsenosides. Background Technology

[0002] Ginseng is the plant ginseng (Panax ginseng) of the Araliaceae family. Panax ginseng The dried roots and rhizomes of *C. A. Mey.* contain ginsenosides as their main active ingredient. Ginsenosides possess biological activities such as relieving fatigue, inhibiting tumor proliferation, regulating glucose metabolism, and modulating inflammatory responses. Studies have found that secondary metabolites of ginsenosides, after transformation, exhibit even stronger biological activity. These secondary metabolites are named "rare ginsenosides" due to their scarcity in natural ginseng. In contrast, ginsenosides extracted directly from plants in the Araliaceae family are called "proto-ginsenosides."

[0003] Rare ginsenosides exhibit superior transmembrane absorption efficiency and targeted efficacy compared to native ginsenosides, thus possessing greater medicinal development value and broader application potential. However, the abundance of rare ginsenosides in natural plants is less than 0.1%, severely restricting large-scale production and application. Traditional preparation methods for rare ginsenosides include physical methods, chemical methods, and biotransformation methods (microorganisms, enzymes). Physicochemical methods (acid-base catalysis, thermal pyrolysis, etc.) suffer from drawbacks such as violent reactions and complex byproducts, easily leading to non-specific glycosidic bond breakage and changes in active conformation. Although enzymatic transformation offers the advantage of glycosidic bond selectivity, its industrialization has been limited by poor enzyme stability and high immobilization costs. While gut microbiota can achieve the biotransformation of native ginsenosides, the influence of individual microbial composition results in limitations such as difficulty in controlling transformation efficiency and product stability. Summary of the Invention

[0004] In view of this, the present invention provides a strain of *Pseudomonas winnipes* TY3-10, which can convert total ginsenosides or monomers thereof from ginseng root into a variety of rare ginsenosides.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain of *Xanthomonas winnipes* (…). Pseudoxanthomonas winnipegensis The strain TY3-10 has the accession number CGMCC No.35238.

[0006] The present invention provides a bacterial agent containing the aforementioned *Xanthomonas winnipes* strain TY3-10.

[0007] This invention provides the application of the *Xanthomonas winnipes* strain TY3-10 or the bacterial agent in the conversion of ginsenosides to prepare rare ginsenosides.

[0008] Preferably, the ginsenosides include total ginsenosides from ginseng root or ginsenoside monomers.

[0009] Preferably, the ginsenoside monomers include at least one of the following: ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc.

[0010] Preferably, the rare ginsenosides include at least one of the following: rare ginsenoside F2, rare ginsenoside Rd2, rare ginsenoside Fd, rare ginsenoside C-Mc1, and rare ginsenoside Rh1.

[0011] This invention provides a method for preparing rare ginsenosides, wherein ginsenosides are subjected to a transformation reaction under the action of the *Xanthomonas yunnanensis* strain TY3-10 or the bacterial agent to obtain rare ginsenosides.

[0012] Preferably, the ginsenosides include at least one of the following: total ginsenosides from ginseng root, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc. The rare ginsenosides include at least one of the following: rare ginsenoside F2, rare ginsenoside Rd2, rare ginsenoside Fd, rare ginsenoside C-Mc1, and rare ginsenoside Rh1.

[0013] Preferably, the solution used for the conversion reaction is a fermentation medium; the fermentation medium contains the following components: sucrose 0.7~1.3 g / L, tryptone 7~13 g / L, yeast extract 3~7 g / L and sodium chloride 0.7~1.3 g / L.

[0014] Preferably, the conversion reaction is aerobic fermentation; the fermentation temperature is 26~34℃.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a strain of *Xanthomonas winnipes*, TY3-10, with the accession number CGMCC No. 35238. The strain TY3-10 exhibits the characteristics of producing β-glucosidase and β-xylosidase, capable of degrading the glucosyl and xylose groups in the chemical structure of ginsenosides, specifically the outer sugars at C-3, C-6, and C-20, converting total ginsenosides from ginseng root into rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1.

[0016] This invention provides a method for preparing rare ginsenosides. Ginsenosides are transformed under the action of *Pseudomonas winnipes* strain TY3-10 or the aforementioned bacterial agent to obtain rare ginsenosides. Currently, there are no research reports on the preparation of rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1 by *Pseudomonas winnipes* through fermentation of total or monomeric ginsenosides from ginseng roots. Compared to traditional biotransformation technologies, this invention utilizes the β-glucosidase and β-xylosidase production characteristics of strain TY3-10, using total ginsenosides from ginseng roots or their monomers as substrates, to completely transform them into rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1 under mild conditions. This achieves high-efficiency transformation while also possessing environmental compatibility advantages, effectively avoiding high energy consumption and pollution problems, and is suitable for large-scale industrial production. The rare ginsenosides Rd2, Fd, and C-Mc1 in the transformation products are rare ginsenosides that can only be obtained through transformation by human intestinal bacteria. This invention develops a novel process for the in vitro preparation of rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1, which has broad application prospects. Attached Figure Description

[0017] Figure 1 The colorimetric pattern of the Winnipeg pseudoxanthomonas strain TY3-10, which produces β-glucosidase and β-xylosidase, on the selection medium. Figure 2 The figures show HPLC chromatograms of total ginseng root saponins fermented on day 0 and day 3; where a is the HPLC chromatogram of total ginseng root saponins fermented on day 0, b is the HPLC chromatogram of total ginseng root saponins fermented on day 3 by *Pseudomonas winnipes* strain TY3-10, and c is the HPLC chromatogram of total ginseng root saponins fermented on day 3 by *Pseudomonas winnipes* strain TY3-10. Pseudoxanthomonas beigongshangi HPLC chromatogram after 3 days of fermentation; Figure 3 HPLC chromatograms of ginsenoside Rf fermentation after 0 and 3 days. Figure 4 HPLC chromatograms of ginsenoside Rb1 fermented for 0 days and 3 days; Figure 5 HPLC chromatograms of ginsenoside Rb2 fermentation on days 0 and 3. Figure 6 HPLC chromatograms of ginsenoside Rb3 fermented for 0 days and 3 days; Figure 7 HPLC chromatograms of ginsenoside Rc fermentation after 0 and 3 days; Figure 8 Biotransformation synthesis route for preparing rare ginsenosides F2, Rd2, Fd, C-Mc1 and Rh1 from total ginsenosides of ginseng root by fermentation of Winnipeg pseudoxanthomonas strain TY3-10.

[0018] Information on the preservation of biological materials Winnipeg Pseudomonas ( Pseudoxanthomonas winnipegensis It was deposited on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35238 and strain number TY3-10. Detailed Implementation

[0019] This invention provides a strain of *Xanthomonas winnipes* TY3-10, with accession number CGMCC No. 35238.

[0020] In this invention, the *Xanthomonas winnipes* strain TY3-10 was screened from rhizosphere soil samples of ginseng under forest cover in Tonghua County, Jilin Province, and possesses the characteristics of producing β-glucosidase and β-xylosidase. The *Xanthomonas winnipes* strain TY3-10, when grown on a esculin selection medium, forms colonies that are dark brown to black. Figure 1Pseudoxanthomonas winnipegensis The homology between the strain and the bacteria reached 100%, therefore, strain TY3-10 was identified as *Pseudomonas winnipes* (…). Pseudoxanthomonas winnipegensis ).

[0021] The present invention provides a bacterial agent containing the aforementioned *Xanthomonas winnipes* strain TY3-10.

[0022] This invention provides the application of the *Xanthomonas winnipes* strain TY3-10 or the bacterial agent in the conversion of ginsenosides to prepare rare ginsenosides.

[0023] In this invention, the ginsenosides preferably include total ginsenosides from ginseng root or ginsenoside monomers; the ginsenoside monomers include at least one of the following: ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc. The rare ginsenosides include at least one of the following: rare ginsenoside F2, rare ginsenoside Rd2, rare ginsenoside Fd, rare ginsenoside C-Mc1, and rare ginsenoside Rh1. The conversion of ginsenosides to prepare rare ginsenosides preferably includes at least one of the following: conversion of ginsenoside Rf to rare ginsenoside Rh1, conversion of ginsenoside Rb1 to rare ginsenoside F2, conversion of ginsenoside Rb2 to rare ginsenoside Rd2, conversion of ginsenoside Rb3 to rare ginsenoside F2 and / or rare ginsenoside Fd, and conversion of ginsenoside Rc to rare ginsenoside C-Mc1.

[0024] In this embodiment of the invention, total ginseng root saponins and ginseng saponin monomers (ginsenosides Rf, Rb1, Rb2, Rb3, and Rc) were used as raw materials, and fermentation products were obtained by fermentation using the strain TY3-10. The fermentation products were identified by HPLC chromatography. The results showed that the retention times of the total ginseng root saponin conversion products were consistent with those of the rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1 standards; the retention time of the ginsenoside Rb1 fermentation conversion product was consistent with that of the rare ginsenoside F2 standard; the retention time of the ginsenoside Rb3 fermentation conversion product was consistent with that of the rare ginsenosides F2 and Fd standards; the retention time of the ginsenoside Rf fermentation conversion product was consistent with that of the rare ginsenoside Rh1 standard; the retention time of the ginsenoside Rb2 fermentation conversion product was consistent with that of the rare ginsenoside Rd2 standard; and the retention time of the ginsenoside Rc fermentation conversion product was consistent with that of the rare ginsenoside C-Mc1 standard. This demonstrates that the *Xanthomonas winnipes* strain TY3-10 can be used to convert ginsenosides into rare ginsenosides. It also shows that the *Xanthomonas winnipes* strain TY3-10 can remove glucose or xylose from the C-3, C-6, and C-20 positions during the fermentation and conversion of ginsenosides.

[0025] This invention provides a method for preparing rare ginsenosides, wherein ginsenosides are subjected to a transformation reaction under the action of the *Xanthomonas yunnanensis* strain TY3-10 or the bacterial agent to obtain rare ginsenosides.

[0026] In this invention, the ginsenosides are preferably the same as those used in the above applications, and will not be repeated here. The concentration of the ginsenosides is preferably 0.5~2 mg / L, more preferably 0.7~1.5 mg / L, even more preferably 0.9~1.2 mg / L, and most preferably 1 mg / L. The inoculum size of the *Pseudomonas winnipes* strain TY3-10 is preferably 1×10⁻⁶. 7 ~3×10 7 CFU / mL, more preferably 1.5 × 10⁻⁶ 7 ~2.5×10 7 CFU / mL, the optimal value is 2×10⁻⁶. 7 CFU / mL. The *Xanthomonas winnipes* strain TY3-10 is preferably inoculated in the form of a bacterial suspension. The preferred method for preparing the bacterial suspension is to inoculate *Xanthomonas winnipes* strain TY3-10 into liquid LB medium and culture to obtain a bacterial suspension of *Xanthomonas winnipes* strain TY3-10, isolate the bacterial cells, and suspend the bacterial cells in PBS to obtain the bacterial suspension. The culture time is preferably 18-24 h, more preferably 20-22 h, and most preferably 21 h. The culture temperature is preferably 26-34 °C, more preferably 28-32 °C, more preferably 29-31 °C, and most preferably 30 °C. The culture rotation speed is preferably 150-220 rpm, more preferably 175-210 rpm, and most preferably 200 rpm.

[0027] In this invention, the solution used for the conversion reaction is a fermentation medium; the fermentation medium preferably contains the following components in the following amounts: sucrose 0.7-1.3 g / L, tryptone 7-13 g / L, yeast extract 3-7 g / L, and sodium chloride 0.7-1.3 g / L; more preferably, it contains the following components in the following amounts: 8-12 g / L, yeast extract 4-6 g / L, and sodium chloride 0.8-1.2 g / L; and most preferably, it contains the following components in the following amounts: sucrose 1 g / L, tryptone 10 g / L, yeast extract 5 g / L, and sodium chloride 10 g / L. The pH value of the fermentation medium is preferably 6.0-7.0, more preferably 6.2-6.8, and most preferably 6.5. In this invention, the fermentation medium is conducive to the growth of strain TY3-10 and induces the production of β-glucosidase.

[0028] In this invention, the conversion reaction is preferably aerobic fermentation. The rotation speed is preferably 150-220 rpm, more preferably 175-210 rpm, and most preferably 200 rpm. The fermentation temperature is preferably 26-34℃, more preferably 28-32℃, more preferably 29-31℃, and most preferably 30℃. The fermentation time is preferably 24 hours or more, more preferably 36-72 hours, more preferably 48-60 hours, and most preferably 54 hours. This fermentation method facilitates the conversion of major ginsenosides into rare ginsenosides.

[0029] Following fermentation, a fermentation product is obtained. The fermentation product is preferably extracted, concentrated, and dried using an organic reagent to obtain rare ginsenosides. The organic reagent preferably includes n-butanol and / or methanol. The rare ginsenosides are preferably the same as those described above and will not be repeated here.

[0030] In this invention, the TY3-10 strain can degrade the glucose and xylose groups in the chemical structure of ginsenosides, with the linkage positions being the outer sugars at C-3, C-6, and C-20. This allows it to convert total ginsenosides from ginseng roots into rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1. The biotransformation synthesis route for these rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1 is as follows: Under the action of the *Pseudomonas winnipes* strain TY3-10, ginsenoside Rb1 in total ginsenosides from ginseng roots loses the outer glucose at the C-20 and C-3 positions to generate rare ginsenoside F2; ginsenoside Rb2 in total ginsenosides from ginseng roots loses the outer glucose at the C-3 position to generate rare ginsenoside Rd2; and so on. Ginsenoside Rb3 in ginsenosides loses glucose at the C-3 position to form rare ginsenoside Fd, while a small amount of rare ginsenoside Fd loses xylose at the C-20 position to form rare ginsenoside F2; ginsenoside Rc in total ginsenosides of ginseng root loses glucose at the C-3 position to form rare ginsenoside C-Mc1; ginsenoside Rf in total ginsenosides of ginseng root loses glucose at the C-6 position to form rare ginsenoside Rh1.

[0031] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Source of materials Aescin screening medium: Add 1 g / L aescin and 0.5 g / L ferric citrate to R2A agar solid medium and autoclave at 121℃ for 20 min. Fermentation medium: 1 g / L sucrose, 10 g / L tryptone, 5 g / L yeast extract and 1 g / L sodium chloride, pH 6.5, prepared by sterilization at 121℃ for 20 min; Total saponins from ginseng root: Total saponins from ginseng root (HPLC purity ≥60%) were purchased from Ji'an Jiayuan Biotechnology Co., Ltd.

[0033] Primary ginsenoside monomers: ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc (HPLC purity ≥98%) were purchased from Chengdu Manster Biotechnology Co., Ltd.

[0034] Example 1 Isolation, identification and preservation of bacterial strains 1. Isolation of strains Soil samples from the rhizosphere of ginseng under forest cover were collected in September 2024 from Tonghua County, Jilin Province. 1g of fresh soil sample was weighed, added to 99ml of sterile water, and incubated for 24h. After serial dilution, the samples were plated on LB agar plates and incubated at 30℃ for 24h. Single colonies were picked and streaked onto LB agar plates using the streak method. After incubation at 30℃ for 24h, single colonies were picked for microscopic examination. Streak inoculation and purification were continued until a purified product with a uniform colony morphology was obtained. The purified product was further inoculated onto aesculin selection medium to isolate strains producing β-glucosidase. Strains with a dark brown to black ring around their positive morphology were considered positive (e.g.,...). Figure 1 (As shown in the image) This indicates that the strain possesses the ability to produce β-glucosidase. The obtained pure culture was inoculated into liquid LB 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 *Xanthomonas winnipegii* was named TY3-10.

[0035] 2. Identification of strains (1) Physiological and biochemical identification results of strain TY3-10: Gram-negative, capable of utilizing glucose, maltose, sucrose, galactose, xylose, melibiose, amygdalin and esculin, and positive; negative for lactose, rhamnose, arabinose, mannitol, sorbitol and inositol. The optimal growth temperature is 30℃; the suitable pH is 6.0~7.0; tolerant of 2% NaCl; grows uniformly turbid in liquid LB medium, and the cells form a pale yellow precipitate after prolonged standing.

[0036] (2) Molecular biological identification: The target strain was inoculated into fresh LB liquid medium and cultured for 24 h. Bacterial DNA was extracted using a kit from Tiangen Biotech Co., Ltd., and its 16S rDNA sequence was amplified. The primers used were a species-universal primer set consisting of 1492R (5-ggttaccttgttacgactt-3, SEQ ID NO: 2) and 27F (5-agagtttgatcctggctcag-3, SEQ ID NO: 3), and PCR amplification was performed. The PCR amplification conditions were as follows: pre-denaturation: 94℃ for 5 min; denaturation: 94℃ for 30 s, annealing: 55℃ for 30 s, extension: 72℃ for 90 s, for a total of 35 cycles; final extension: 72℃ for 5 min, and stored at 4℃.

[0037] Electrophoresis results of 16S rDNA PCR products of strain TY3-10 showed a highly specific band at a molecular weight of approximately 1500 bp, consistent with the expected result. Sequencing revealed that the 16S rDNA gene sequence of strain TY3-10 is 1483 bp in length, as shown in SEQ ID NO: 1.

[0038] The sequencing 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 TY3-10 is similar to *Xanthomonas winnipes* (…). Pseudoxanthomonas winnipegensis The homology between the strain TY3-10 and *Xanthomonas winnipes* reached 100%. Based on the above results, strain TY3-10 was identified as belonging to *Xanthomonas winnipes*. Pseudoxanthomonas winnipegensis ).

[0039] 3. Preservation of bacterial strains The strain TY3-10 of *Xanthomonas winnipes* was deposited on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35238.

[0040] 4. Preparation of suspension of *Xanthomonas winnipes* strain TY3-10 The strain TY3-10 of *Pseudomonas winnipegii* was inoculated into liquid LB medium and cultured at 30°C for 24 h. The culture was then centrifuged at 8000 rpm for 10 min at 4°C, and the bacterial pellet was collected. The pellet was resuspended in sterile 0.85% physiological saline solution, and the viable count was adjusted to 1.0 × 10⁻⁶ cells / mL. 9 CFU / mL was used to obtain a suspension of *Xanthomonas winnipegii* strain TY3-10.

[0041] Example 2 Total saponins from ginseng root were converted by fermentation using *Pseudomonas wani* strain TY3-10. 1. Preparation of rare ginsenosides from the fermentation and conversion of total ginsenosides from ginseng root In the liquid fermentation medium, the addition amount of total saponins from ginseng root was 1 mg / mL, sterilized by filtration through a 0.22 μM microporous membrane, and then adjusted according to 2 × 10⁻⁶. 7 The *Xanthomonas yunnanensis* strain TY3-10 from Example 1 was inoculated at a CFU / mL inoculum and fermented aerobically at 30°C for 3 days. After fermentation, the fermentation product was extracted with water-saturated n-butanol, then concentrated under reduced pressure at 50°C and freeze-dried at -80°C and 5 Pa. The residue was dissolved in methanol and centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant was then subjected to reduced pressure to recover the solvent and freeze-dried under vacuum at -80°C and 5 Pa to obtain the dried fermentation product.

[0042] 2. Identification of rare ginsenosides The dried fermentation product was weighed and redissolved in chromatographic methanol. Rare ginsenosides F2, Rd2, Fd, C-Mc1 and Rh1 were detected by high performance liquid chromatography (HPLC). The total ginsenosides from ginseng root and the dried product obtained above were dissolved in chromatographic methanol, filtered through a 0.22 μm microporous membrane and then used for HPLC chromatographic analysis.

[0043] The HPLC chromatographic analysis method was as follows: Agilent Eclipse XDB-C18 column, injection volume 10 μL, elution rate 1.0 mL / min, column temperature 30℃, and detection wavelength 203 nm. The mobile phase consisted of water (A) and acetonitrile (B), and gradient elution was performed as follows: 0 min, 79% A, 21% B; 5 min, 74% A, 26% B; 9 min, 68% A, 32% B; 39 min, 66.5% A, 33.5% B; 42 min, 64% A, 36% B; 44 min, 62% A, 38% B; 59 min, 35% A, 65% B; 64 min, 35% A, 65% B; 69 min, 15% A, 85% B; 75 min, 15% A, 85% B; 80 min, 79% A, 21% B.

[0044] HPLC chromatographic identification results as follows Figure 2As shown in Figures a and b, qualitative analysis of the ginsenoside components in the products before and after fermentation revealed that the retention times of the fermentation products containing total ginsenosides from ginseng root were consistent with those of the rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1 standards. This demonstrates that total ginsenosides from ginseng root can be fermented using the *Pseudomonas winnipes* strain TY3-10, which produces β-glucosidase and β-xylosidase exonucleases, and the resulting fermentation products are rich in rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1. Furthermore, as shown in Figures a and b, the ginsenosides exhibit the same retention times as the rare ginsenosides F2, Rd2, Fd, C-Mc1, and Rh1. Figure 2 As shown, the biotransformation synthesis route for preparing rare ginsenosides using *Pseudomonas wanens* strain TY3-10 via fermentation is as follows: Primary ginsenoside Rb1 is converted to rare ginsenoside F2 by removing glucose from the C-20 and C-3 positions; primary ginsenoside Rb2 is converted to rare ginsenoside Rd2 by removing glucose from the C-3 position; primary ginsenoside Rb3 is converted to rare ginsenoside Fd by removing glucose from the C-3 position, and a small amount of rare ginsenoside Fd is further converted to rare ginsenoside F2 by removing xylose from the C-20 position; primary ginsenoside Rc is converted to rare ginsenoside C-Mc1 by removing glucose from the C-3 position; and primary ginsenoside Rf is converted to rare ginsenoside Rh1 by removing glucose from the C-6 position.

[0045] Comparative Example 1 Total saponins from ginseng root were converted by fermentation using commercially available Xanthomonas pseudoepiploicum strains. 1. Preparation of commercially available Xanthomonas pseudoepiploicella bacterial suspension Pseudoxanthomonas strains Pseudoxanthomonas beigongshangi (Accession number: JCM 33961) Purchased from Beijing Bio-Biobio Biotechnology Co., Ltd. *Xanthomonas pseudoepiploiculatus* strain Pseudoxanthomonas beigongshangi Inoculate into liquid LB medium, incubate at 30°C for 24 h, centrifuge at 8000 rpm for 10 min at 4°C, and collect the bacterial pellet; resuspend in sterile 0.85% physiological saline solution and adjust the viable count to 1.0 × 10⁻⁶. 9 CFU / mL, obtained Xanthomonas pseudoxanthomonas strain Pseudoxanthomonas beigongshangi bacterial suspension.

[0046] 2. Strains Pseudoxanthomonas beigongshangi Fermentation and transformation of total saponins from ginseng root In the liquid fermentation medium, the addition amount of total saponins from ginseng root was 1 mg / mL, sterilized by filtration through a 0.22 μM microporous membrane, and then adjusted according to 2 × 10⁻⁶. 7 Inoculate with *Xanthomonas pseudoepiploicum* strain at a dose of CFU / mL. Pseudoxanthomonas beigongshangiThe bacterial suspension was aerobically fermented at 30℃ for 3 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 10,000 rpm for 15 min at 4℃. The supernatant was then subjected to reduced pressure to recover the solvent and freeze-dried under vacuum at -80℃ and 5pa to obtain the dried fermentation product.

[0047] 3. The identification of the total saponin conversion products from ginseng root was the same as in Example 2.

[0048] HPLC chromatographic qualitative analysis results are as follows Figure 2 As shown in Figure c, a comparison with the results of TY3-10 in the transformation of ginseng root total saponins shows that when using the same strain TY3-10 for fermentation and transformation to prepare rare ginseng saponins, the initial saponins Rc, Rb2, Rb3, and Rd in the total ginseng root saponins are almost completely eliminated, and the amount of rare ginseng saponins C-Mc1, Rd2, Fd, and F2 generated is also greater. Therefore, the transformation efficiency and the yield of the target product of strain TY3-10 are significantly better than those of strain C-Mc1. Pseudoxanthomonas beigongshangi It has outstanding advantages in industrial applications.

[0049] Example 3 Ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc were transformed by fermentation using the Winnipeg pseudoxanthomonas strain TY3-10. 1. Preparation of rare ginsenosides by fermentation and conversion of primary ginsenoside monomers The method for preparing rare ginsenosides by fermentation and conversion of total ginsenosides from ginseng root in Example 2 is the same, except that the total ginsenosides from ginseng root are replaced with each primary ginsenoside monomer.

[0050] 2. Identification of rare ginsenosides The fermentation products of each ginsenoside monomer were filtered through a 0.22 μm microporous membrane, extracted with water-saturated n-butanol, followed by low-temperature reduced pressure concentration and vacuum freeze-drying (-80℃, 5 Pa). The residue was dissolved in methanol and centrifuged at 10,000 rpm for 15 min at 4℃. The supernatant was subjected to vacuum freeze-drying after solvent recovery under reduced pressure to obtain the dried product. Rare ginsenosides Rh1, F2, Fd, Rd2, and C-Mc1 were detected by high-performance liquid chromatography (HPLC): the ginsenoside monomers and the dried product obtained above were dissolved in chromatographic methanol, filtered through a 0.22 μm microporous membrane, and then used for HPLC analysis.

[0051] The HPLC chromatographic analysis method was as follows: Agilent Eclipse XDB-C18 column, injection volume 10 μL, elution rate 1.0 mL / min, column temperature 30℃, and detection wavelength 203 nm. The mobile phase consisted of water (A) and acetonitrile (B), and gradient elution was performed as follows: 0 min, 79% A, 21% B; 5 min, 74% A, 26% B; 9 min, 68% A, 32% B; 39 min, 66.5% A, 33.5% B; 42 min, 64% A, 36% B; 44 min, 62% A, 38% B; 59 min, 35% A, 65% B; 64 min, 35% A, 65% B; 69 min, 15% A, 85% B; 75 min, 15% A, 85% B; 80 min, 79% A, 21% B.

[0052] HPLC chromatographic identification results as follows Figures 3 to 7 As shown, qualitative analysis of ginsenoside components in the products before and after fermentation revealed that the retention time of ginsenoside Rb1 fermentation product was consistent with that of rare ginsenoside F2 standard; the retention time of ginsenoside Rb3 fermentation product was consistent with that of rare ginsenosides F2 and Fd standards; the retention time of ginsenoside Rf fermentation product was consistent with that of rare ginsenoside Rh1 standard; the retention time of ginsenoside Rb2 fermentation product was consistent with that of rare ginsenoside Rd2 standard; and the retention time of ginsenoside Rc fermentation product was consistent with that of rare ginsenoside C-Mc1 standard. This indicates that when using *Pseudomonas winnipes* strain TY3-10 for fermentation of ginsenosides, the glucose or xylose at positions C-3, C-6, and C-20 can be removed. Figure 8 ).

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative intent, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Pseudomonas winnipegii* ( Pseudoxanthomonas winnipegensis ) strain TY3-10, characterized in that, The accession number is: CGMCC No.35238.

2. A microbial agent, characterized in that, Contains the Winnipeg pseudoxanthomonas strain TY3-10 as described in claim 1.

3. The application of the Winnipeg pseudoxanthomonas strain TY3-10 of claim 1 or the bacterial agent of claim 2 in the conversion of ginsenosides to prepare rare ginsenosides.

4. The application according to claim 3, characterized in that, The ginsenosides include total ginsenosides from ginseng root or ginsenoside monomers.

5. The application according to claim 3, characterized in that, The ginsenoside monomers include at least one of the following: ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc.

6. The application according to any one of claims 3 to 5, characterized in that, The rare ginsenosides include at least one of the following: rare ginsenoside F2, rare ginsenoside Rd2, rare ginsenoside Fd, rare ginsenoside C-Mc1, and rare ginsenoside Rh1.

7. A method for preparing rare ginsenosides, characterized in that, Rare ginsenosides are obtained by transformation reaction of ginsenosides under the action of the *Xanthomonas yunnanensis* strain TY3-10 of claim 1 or the bacterial agent of claim 2.

8. The method according to claim 7, characterized in that, The ginsenosides include at least one of the following: total ginsenosides from ginseng root, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, and ginsenoside Rc. The rare ginsenosides include at least one of the following: rare ginsenoside F2, rare ginsenoside Rd2, rare ginsenoside Fd, rare ginsenoside C-Mc1, and rare ginsenoside Rh1.

9. The method according to claim 7, characterized in that, The solution used for the conversion reaction is a fermentation medium; the fermentation medium contains the following components: sucrose 0.7~1.3 g / L, tryptone 7~13 g / L, yeast extract 3~7 g / L and sodium chloride 0.7~1.3 g / L.

10. The method according to any one of claims 7 to 9, characterized in that, The conversion reaction is aerobic fermentation; the fermentation temperature is 26~34℃.

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