A strain of *Duboscirella nymphoides* TZF-002 and its application in the fermentation and conversion of rare ginsenosides.

Abstract

A strain of Dubois nystomatitis ( Dubosiella newyorkensis TZF-002 and its application in the fermentation and conversion of rare ginsenosides belong to the field of microbiology. The *Duboscirella nymphaeus* mentioned herein (…) Dubosiella newyorkensis TZF-002 was deposited at the China Center for Type Culture Collection on December 15, 2025, with accession number CCTCC NO: M 20252890. This invention uses total ginsenosides as raw material, first utilizing the high-β-glucosidase-producing *Duboscirella nystatin* (…). Dubosiella newyorkensis The TZF-002 method employs a mild and controlled fermentation process to transform primary ginsenosides, followed by high-temperature steam treatment, ultimately yielding rare ginsenosides Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5 efficiently. This method, for the first time, combines the enzymatic transformation characteristics of intestinal symbiotic bacteria with physical treatment, offering advantages such as mild conditions, low cost, and significantly superior product variety and content compared to single processes. It effectively improves the yield, bioavailability, and physiological activity of rare saponins.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Duboscirella nystatin* (…). Dubosiella newyorkensis TZF-002 and its application in the fermentation and conversion of rare ginsenosides. Background Technology

[0002] Ginseng ( Panax ginseng CAMey., a traditional and precious Chinese medicinal herb, relies on ginsenosides as its core pharmacological value. It exhibits clear and significant application potential in areas such as anti-tumor activity, anti-inflammatory and antioxidant activity, immune regulation, cardiovascular protection, and improvement of nerve function. Among these, rare ginsenosides such as Rg6, F4, Rh4, 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5 possess stronger biological activity and are in urgent demand in disease prevention and treatment, as well as health product development. However, the content of rare ginsenosides in natural ginseng is extremely low, and traditional preparation techniques suffer from low conversion efficiency, limited product variety, complex processes, and severe pollution, hindering their industrial application. In recent years, many researchers have focused on converting primary ginsenosides into highly active rare ginsenosides through heating, acid hydrolysis, alkaline hydrolysis, and microbial and enzymatic transformation. Compared with heat treatment, acid hydrolysis, and alkaline hydrolysis, microbial and enzymatic biotransformation is more environmentally friendly and efficient. Probiotic-mediated biotransformation is an important technical pathway for the efficient preparation of natural active ingredients. Studies have confirmed that some probiotic strains can synthesize β-glucosidase, which can accurately recognize and hydrolyze glycosidic bonds at specific sites in ginsenoside molecules, promoting the transformation of primary ginsenosides into rarer ginsenosides with stronger biological activity.

[0003] New York Dubois ( Dubosiella newyorkensis *Duboscirella nystatus* is a novel potential probiotic discovered in the gut microbiota of mammals in recent years. Its core physiological functions have been confirmed to be closely related to the regulation of intestinal barrier integrity, the maintenance of host metabolic homeostasis, and the regulation of mucosal immune responses, thus becoming one of the research hotspots in the field of gut microbiota. To date, *Duboscirella nystatus* has exhibited probiotic properties in several aspects, but its application in the preparation of rare ginsenosides has not been reported. Therefore, screening for a *Duboscirella nystatus* strain capable of combining fermentation conversion and thermal pyrolysis to prepare rare ginsenosides is key to achieving the preparation of highly active and high-content rare ginsenosides. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Dubois nystomatitis ( ). Dubosiella newyorkensisTZF-002 and its application in the fermentation and conversion of rare ginsenosides provide a new, efficient, and environmentally friendly approach for the preparation of ginsenosides Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, Rg5, etc.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The present invention provides a strain of Dubois nystomatitis (New York) Dubosiella newyorkensis TZF-002 was deposited at the China Center for Type Culture Collection on December 15, 2025, with accession number CCTCC NO: M 20252890.

[0007] The present invention provides a strain of Dubois nystomatitis (New York) Dubosiella newyorkensis Application of TZF-002 in the fermentation and conversion of rare ginsenosides.

[0008] As one preferred embodiment, the application mainly includes the following steps:

[0009] Step 1: Preparation of Dubstein's New York ( Dubosiella newyorkensis TZF-002 bacterial suspension;

[0010] Step 2: Fermentation and transformation;

[0011] Step 3: High-temperature steam treatment and drying purification.

[0012] As one preferred embodiment, the specific operation process of step one is as follows: *Duboscirella nystatin* (… Dubosiella newyorkensis TZF-002 was inoculated into liquid CMC 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, obtained from Dubsteinella nystatin (CFU / mL) Dubosiella newyorkensis TZF-002 bacterial suspension.

[0013] As one preferred embodiment, the specific operation process of step two is as follows: Prepare a liquid fermentation culture medium, add total ginsenosides to the sterilized liquid fermentation culture medium at a ratio of 0.5 mg / mL, and filter through a 0.22 μM microporous membrane for sterilization; Add *Duboscirella nysalis* (… Dubosiella newyorkensis TZF-002 according to 2×10 8 The inoculum was inoculated into liquid fermentation medium supplemented with total ginsenosides at a concentration of CFU / mL and anaerobic fermented at 37°C for 7-14 days.

[0014] In a preferred embodiment, the liquid fermentation medium comprises: 30.0 g / L casein peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 5.0 g / L dipotassium hydrogen phosphate, 0.5 g / L L-cysteine ​​hydrochloride, 0.0005 g / L resazurin, 4.0 g / L glucose, 1.0 g / L cellobiose, 1.0 g / L maltose, and 1.0 g starch; pH 7.0 ± 0.2.

[0015] As one preferred embodiment, the specific operation process of step three is as follows:

[0016] After fermentation, the fermentation product was subjected to high-temperature steam treatment at 115℃-121℃ 4-6 times, each treatment lasting 1-3 hours. Then, it was extracted with water-saturated n-butanol, concentrated under reduced pressure at 55℃, and freeze-dried at -80℃ and 5 Pa. The residue was dissolved in methanol and centrifuged at 10000 rpm and 4℃ for 15 minutes. The supernatant was then subjected to vacuum freeze-drying at -80℃ and 5 Pa to obtain a dried product containing ginsenosides Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention screened a probiotic strain TZF-002 from fecal samples of healthy mice. This strain produced β-glucosidase and could efficiently convert ginseng total saponins. It was identified as *Duboscirella nystatin*. Dubosiella newyorkensis TZF-002 was deposited at the China Center for Type Culture Collection on December 15, 2025, with accession number CCTCC NO: M20252890.

[0019] This invention relates to a strain of *Duboscirella nystatin* that was independently isolated and identified. Dubosiella newyorkensis TZF-002 can be used for fermentation and conversion of total ginsenosides, followed by high-temperature steam treatment to obtain rare ginsenosides. This invention is the first to use *Duboscirella nystatin*, a high-yield β-glucanase bacterium, to prepare rare ginsenosides. The two-step method of this invention yields a different variety and content of rare ginsenosides compared to existing single-method methods, significantly increasing the content of rare ginsenosides in ginseng extract. The prepared product containing various highly active rare ginsenosides exhibits significant biological activity and has broad application prospects in medicine, health products, cosmetics, and other fields, which is of great significance for promoting the high-quality development of the ginseng industry. Attached Figure Description

[0020] Figure 1 Dubois nystomatitis, a bacterium that produces high levels of β-glucosidase ( Dubosiella newyorkensis ) Colorimetric pattern of TZF-002 on aescin screening medium.

[0021] Figure 2 Day 0 of ginseng total saponin fermentation and via Dubola ( Dubosiella newyorkensis HPLC chromatogram of ginseng total saponins after fermentation and conversion by TZF-002. Detailed Implementation

[0022] 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.

[0023] Case Study 1: Isolation, Identification, and Preservation Information of Dubstein's bacterium NY 1. Isolation of strains

[0024] In May 2025, fecal samples were collected from healthy mice. 1 mL of fecal sample suspension was added to CMC medium and anaerobically cultured for 48 h. The streak plate method was used, with streaks applied to CMC medium plates and anaerobically 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. Further inoculation was performed on aescin selection medium: used to isolate strains producing β-glucosidase (CMC agar solid medium with 1 g / L aescin and 0.5 g / L ferric citrate added, sterilized 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 CMC 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 of the probiotic strains was named *Duboscirella nysicae* (New York). Dubosiella newyorkensis )TZF-002. Identification of strains

[0025] (1) Strain: Dubois nystomatitis ( Dubosiella newyorkensis The physiological and biochemical identification results of TZF-002 are as follows:

[0026] In the biochemical identification tubes of 11 carbohydrates, esculin, cellobiose, maltose, salicin, sucrose, raffinose, inulin, lactose, and hippuric acid showed positive reactions; while mannitol and sorbitol showed negative reactions. The optimal growth temperature is 37℃; the suitable pH is 7-8; and it exhibits mild to moderate uniform turbidity in liquid CMC medium.

[0027] (2) Molecular biological identification and results are as follows:

[0028] The target strain was inoculated into fresh CMC 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 TZF-002 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 TZF-002 is related to *Duboscirella nysplasia* (…). Dubosiella newyorkensis The homology of NYU-BL-A4 (NR 156910.1) reached 99.79%. Based on the above results, strain TZF-002 was identified as belonging to *Duborcella nystatin* (NYU-BL-A4). Dubosiella newyorkensis ).

[0029] The primer pair sequence information is as follows:

[0030] 27F: 5′-agagttgatcctggctcag-3′;

[0031] 1492R: 5′-ggttaccttgttacgactt-3′.

[0032] 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℃. 3. Preservation of bacterial strains

[0033] The present invention is based on Dubois nystomatitis (New York Dubois). Dubosiella newyorkensis TZF-002 was deposited on December 15, 2025, 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 20252890.

[0034] Implementation Case 2: Preparation of Rare Ginsenosides

[0035] 1. Preparation of Dubois NY ( Dubosiella newyorkensis TZF-002 bacterial suspension

[0036] New York Dubstein's bacteria ( Dubosiella newyorkensis TZF-002 was inoculated into liquid CMC medium and incubated statically at 37°C for 48 hours. The culture was then centrifuged at 4°C and 12000 rpm for 10 minutes, and the bacterial pellet was collected. The pellet was then resuspended in sterile PBS, and the viable count was adjusted to 1.0 × 10⁻⁶ cells / day. 8 ~1.0×10 9 CFU / mL, obtained from Dubsteinella nystatin (CFU / mL) Dubosiella newyorkensis TZF-002 bacterial suspension.

[0037] 2. Fermentation and Conversion

[0038] To prepare the liquid fermentation medium (CMC), total ginsenosides (purchased from Chengdu Efa Biotechnology Co., Ltd., HPLC ≥ 80%) were first added to the sterilized liquid fermentation medium (CMC) at a ratio of 0.5 mg / mL, and then filtered through a 0.22 μM microporous membrane for sterilization. *Duboscirella nysalis* (…) was then added… Dubosiella newyorkensis TZF-002 according to 2×10 8 The inoculum was inoculated into liquid fermentation medium supplemented with total ginsenosides at a concentration of CFU / mL, and anaerobic fermentation was carried out at 37℃ for 7 days. The liquid fermentation medium CMC contained 30.0 g / L casein peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 5.0 g / L dipotassium hydrogen phosphate, 0.5 g / L L-cysteine ​​hydrochloride, 0.0005 g / L resazurin, 4.0 g / L glucose, 1.0 g / L cellobiose, 1.0 g / L maltose, and 1.0 g starch; pH 7.0±0.2, sterilized at 121℃ for 20 min at 25℃.

[0039] 3. High-temperature steam treatment and drying purification

[0040] High-temperature steam treatment: After fermentation, the obtained fermentation product is subjected to high-temperature steam treatment at 115℃-121℃ 4-6 times, each treatment lasting 1-2 hours; Drying and purification: Extracted with water-saturated n-butanol, concentrated under reduced pressure at 55℃ and freeze-dried at -80℃ and 5pa. The residue is dissolved in methanol and centrifuged at 10000rpm and 4℃ for 15 minutes. The supernatant is then subjected to vacuum freeze-drying at -80℃ and 5pa to obtain a dried product rich in rare ginsenosides.

[0041] Implementation Case 3: Identification of Ginsenosides by High Performance Liquid Chromatography (HPLC)

[0042] 1. Determination of Ginsenoside Components by High Performance Liquid Chromatography (HPLC)

[0043] The dried product containing rare ginsenosides and the total ginsenoside raw material obtained in Example 2 were dissolved in chromatographic methanol, filtered through a 0.22 μm microporous membrane, and then used for HPLC chromatographic analysis. The HPLC chromatographic analysis method was as follows: Agilent ZORBAX SB-C18 column, injection volume 20 μL, elution rate 1.0 mL / min, column temperature 30℃, 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.

[0044] 2. Evaluation Results

[0045] HPLC chromatographic identification results as follows Figure 2 As shown, by comparing the retention time with that of ginsenoside standard (purchased from Shanghai Yuanye Biotechnology Co., Ltd., HPLC ≥ 98%), it can be determined that *Duboscirella* in this invention (…) was identified. Dubosiella newyorkensis The rare ginsenosides prepared from ginseng total saponins by fermentation and conversion using TZF-002 are: rare saponins Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5. This proves that the rare ginsenosides Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5 in the dried product containing rare ginsenosides can be obtained through fermentation with DuBocci (…). Dubosiella newyorkensis TZF-002 is prepared by fermentation and conversion of ginsenosides.

[0046] This invention discloses a strain of Dubois nystomatitis (…). Dubosiella newyorkensis The TZF-002 and its application in the fermentation and conversion of rare ginsenosides can be understood by those skilled in the art, who can appropriately modify the process parameters to achieve the desired results. 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 can clearly make modifications or appropriate alterations and combinations to 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. Dubois nystocia (New York) Dubosiella newyorkensis TZF-002, characterized in that, It was deposited at the China Center for Type Culture Collection on December 15, 2025, with accession number CCTCC NO: M 20252890.

2. The *Duboscirella nymphaeus* of claim 1 (… Dubosiella newyorkensis The application of TZF-002 in the fermentation and conversion preparation of rare ginsenosides Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5 is characterized by, Includes the following steps: Step 1: Preparation of Dubstein's New York ( Dubosiella newyorkensis TZF-002 bacterial suspension; Step 2: Fermentation and transformation; Step 3: High-temperature steam treatment and drying purification.

3. The application according to claim 2, characterized in that, The specific operation procedure for step one is as follows: (The text abruptly ends here, likely due to an incomplete sentence or a missing section.) Dubosiella newyorkensis TZF-002 was inoculated into liquid CMC medium and anaerobically cultured at 37-39℃ for 48-72 h. The culture was then centrifuged at 4℃ and 8000-12000 rpm for 5-10 min. The bacterial pellet was collected, resuspended in sterile PBS, and the viable count was adjusted to 1.0 × 10⁻⁶. 8 ~1.0×10 9 CFU / mL, obtained from Dubsteinella nystatin (CFU / mL) Dubosiella newyorkensis TZF-002 bacterial suspension.

4. The application according to claim 2, characterized in that, The specific operation process of step two is as follows: Prepare the liquid fermentation culture medium, add total ginsenosides to the sterilized liquid fermentation culture medium at a ratio of 0.5 mg / mL, and filter it through a 0.22 μM microporous membrane for sterilization; add Dubois nystolonifera (… Dubosiella newyorkensis TZF-002 bacterial suspension was prepared at 2×10⁻⁶. 8 The inoculum was inoculated into liquid fermentation medium supplemented with total ginsenosides at a concentration of CFU / mL and anaerobic fermented at 37°C for 7-14 days.

5. The application according to claim 2, characterized in that, The specific operation process of step three is as follows: High-temperature steam treatment: After fermentation, the obtained fermentation product is subjected to high-temperature steam treatment at 115℃-121℃ 4-6 times, each treatment lasting 1-3 hours; Drying and purification: Extracted with water-saturated n-butanol, concentrated under reduced pressure at 55℃ and freeze-dried at -80℃ and 5pa. The residue is dissolved in methanol and centrifuged at 10000rpm and 4℃ for 15 minutes. The supernatant is then subjected to vacuum freeze-drying at -80℃ and 5pa to obtain a dried product containing ginsenosides Rg1, Rg6, F4, Rh4, S-Rg3, R-Rg3, Rk1, and Rg5.

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