Bifidobacterium longum ntuha2012 and application thereof in preparation of rare ginsenoside by fermentation
By combining fermentation and high-temperature steam treatment with Bacteroides fragilis TZF-004, the problem of low efficiency in the preparation of rare ginsenosides has been solved, achieving efficient and environmentally friendly preparation of rare ginsenosides, which is suitable for pharmaceuticals, health products, and cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JILIN AGRICULTURAL UNIV
- Filing Date
- 2026-07-01
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies are insufficient for the efficient preparation of rare ginsenosides. The content of rare ginsenosides in natural ginseng is low and the production cost is high. Traditional methods are inefficient and cause significant environmental pollution. There is a lack of efficient and environmentally friendly preparation methods.
Rare ginsenosides Rg6, F2, F4, 20(S)-Rh2, 20(R)-Rh2, 20(S)-Rh4 and 20(R)-Rh4 were prepared by fermentation transformation combined with high-temperature steam treatment using Bacteroides fragilis strain TZF-004.
The process achieves efficient conversion in a short time, shortens the preparation cycle, and produces rare ginsenosides with significant biological activity, making them suitable for the pharmaceutical, health product, and cosmetic fields.
Smart Images
Figure CN122484009A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of Bacteroides fragilis (… Bacteroides fragilis TZF-004 and its application in the fermentation and conversion of rare ginsenosides. Background Technology
[0002] Ginseng ( Panax ginseng CAMey. is a traditional and precious Chinese medicinal herb, whose main active ingredient is ginsenosides. Numerous studies have shown that ginsenosides possess good pharmacological activities in anti-tumor, anti-inflammatory, antioxidant, immunomodulatory, neuroprotective, and cardiovascular protective effects. Among them, rare ginsenosides such as Rg6, F2, F4, 20(S)-Rh2, 20(R)-Rh2, 20(S)-Rh4, and 20(R)-Rh4 typically exhibit higher biological activity and broader development and utilization value, and are in high demand in the pharmaceutical, health food, and functional product fields.
[0003] However, natural ginseng contains extremely low levels of rare ginsenosides, and direct extraction not only results in low yields but also in high production costs, making it difficult to meet the needs of industrial applications. Existing preparation methods mainly include heat treatment, acid hydrolysis, alkaline hydrolysis, enzymatic conversion, and microbial conversion. Compared with traditional chemical methods, microbial conversion has advantages such as mild reaction conditions, high specificity, less environmental pollution, and less damage to the target product structure, and therefore has gradually become an important technical route for the preparation of rare ginsenosides.
[0004] In recent years, functional bacterial strains derived from the gut microbiota have received increasing attention in the field of natural product biotransformation. Some anaerobic bacteria can produce glycoside hydrolases such as β-glucosidase, which can then directionally cleave the glycosyl groups in ginsenosides, promoting the conversion of primary saponins into rare saponins. If a ginsenoside-converting strain that can achieve efficient conversion within a short culture and fermentation cycle can be screened, and combined with appropriate physical treatment methods, it is hoped that a more efficient, shorter-cycle method suitable for further scale-up of rare ginsenosides can be established.
[0005] Bacteroides fragilis ( Bacteroides fragilis Bacteroides fragilis is an important bacterial species in the gut microbiota, possessing strong metabolic capabilities for carbohydrates and glycosides. To date, there are few reports on the application of Bacteroides fragilis in the fermentation and conversion of these rare ginsenosides. Therefore, providing a ginsenoside preparation strain and method that can be combined with high-temperature steam treatment to achieve efficient conversion in a short time is of significant research and application value. Summary of the Invention
[0006] The purpose of this invention is to provide a strain of Bacteroides fragilis (Bacteroides fragilis TZF-004 and its application in the fermentation and conversion of rare ginsenosides provide a new, efficient, and environmentally friendly route for the preparation of ginsenosides Rg6, F2, F4, 20(S)-Rh2, 20(R)-Rh2, 20(S)-Rh4, and 20(R)-Rh4.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0008] The present invention provides a strain of Bacteroides fragilis ( Bacteroides fragilis TZF-004 was deposited at the China Center for Type Culture Collection on April 22, 2026, with accession number CCTCC NO: M2026760.
[0009] The present invention also provides the aforementioned Bacteroides fragilis ( Bacteroides fragilis Application of TZF-004 in the fermentation and conversion of rare ginsenosides.
[0010] As one preferred embodiment, the application mainly includes the following steps:
[0011] Step 1: Preparation of Bacteroides fragilis ( Bacteroides fragilis TZF-004 bacterial suspension;
[0012] Step 2: Fermentation and transformation;
[0013] Step 3: High-temperature steam treatment and drying purification.
[0014] As one preferred embodiment, the specific operation process of step one is as follows: Bacteroides fragilis (… Bacteroides fragilis TZF-004 was inoculated into BHI liquid medium and anaerobically cultured at 37℃±0.5℃ for 24–48 h. Subsequently, it was centrifuged at 4℃ and 8000–12000 rpm for 5–10 min, and 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 fragilis ( Bacteroides fragilis TZF-004 bacterial suspension.
[0015] As one preferred embodiment, the specific operation process of step two is as follows: Prepare BHI liquid fermentation medium, add total ginsenosides at a ratio of 0.5 mg / mL to the sterilized BHI liquid fermentation medium, and filter it through a 0.22 μm microporous membrane for sterilization; Bacteroides fragilis (… Bacteroides fragilis TZF-004 is 1.0×10 9The inoculum was inoculated into BHI liquid fermentation medium supplemented with total ginsenosides at a concentration of CFU / mL, and anaerobic fermentation was carried out at 37℃±0.5℃ for 7 days.
[0016] In a preferred embodiment, the BHI 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, 5.0 g / L sodium chloride, 2.0 g / L glucose, and 2.5 g / L disodium hydrogen phosphate; pH 7.4 ± 0.2.
[0017] As one preferred embodiment, the specific operation process of step three is as follows: After fermentation, the obtained fermentation product is subjected to high-temperature steam treatment at 121°C three times, each time for 1 hour and 20 minutes; then extracted with water-saturated n-butanol, concentrated under reduced pressure at 55°C, and freeze-dried at -80°C and 5Pa. The residue is dissolved in methanol and centrifuged at 10,000 rpm and 4°C for 15 minutes. The supernatant is collected, the solvent is recovered under reduced pressure, and then freeze-dried under vacuum at -80°C and 5Pa to obtain a dried product containing rare ginsenosides such as Rg6, F2, F4, 20(S)-Rh2, 20(R)-Rh2, 20(S)-Rh4 and 20(R)-Rh4.
[0018] The beneficial effects of this invention are as follows:
[0019] This invention screened fecal samples from obese mice with fatty liver after oral ginseng intervention and obtained a probiotic strain TZF-004 that produces β-glucosidase and can efficiently convert ginseng total saponins. It was identified as Bacteroides fragilis (…). Bacteroides fragilis TZF-004 was deposited at the China Center for Type Culture Collection on April 22, 2026, with accession number CCTCC NO: M2026760.
[0020] This invention relates to a strain of Bacteroides fragilis that was independently isolated and identified. Bacteroides fragilis TZF-004 can be used for the bio-fermentation conversion of total ginsenosides, and can be further combined with high-temperature steam treatment to prepare a variety of rare ginsenosides. Compared with existing single heat treatment or single fermentation methods, this invention, through a combined process of "short-time pre-cultivation, short-cycle fermentation conversion, and high-temperature steam treatment," not only shortens the overall preparation cycle, but also produces a product containing a variety of highly active rare ginsenosides with significant bioactivity, showing broad application prospects in the fields of medicine, health products, and cosmetics. Attached Figure Description
[0021] Figure 1 Bacteroides fragilis ( Bacteroides fragilis ) Colorimetric pattern of TZF-004 on screening medium.
[0022] Figure 2 Bacteroides fragilis ( Bacteroides fragilis Biochemical colorimetric diagram of TZF-003 in HBI lactic acid bacteria biochemical identification strips.
[0023] Figure 3 Day 0 of ginseng total saponin fermentation, via Bacteroides fragilis ( Bacteroides fragilis HPLC chromatograms of TZF-004 fermentation and conversion after 7 days and samples after high-temperature steam treatment.
[0024] Figure 4 The HPLC chromatograms are of the standards for ginsenosides and secondary saponins. Detailed Implementation
[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Other implementation methods obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.
[0026] Implementation Case 1: Isolation, identification, and preservation information of Bacteroides fragilis TZF-004.
[0027] 1. Isolation of strains
[0028] In February 2026, fecal samples were collected from obese mice with fatty liver after oral ginseng intervention. 1 mL of the sample suspension was added to BHI medium for anaerobic enrichment culture for 24 h. The enriched bacterial culture was then streaked onto BHI agar plates and anaerobically cultured at 37°C for 24 h. Single colonies were picked for microscopic examination, and strains with good growth and stable colony morphology were selected. Repeated streak isolation and purification were performed until a pure culture with consistent colony morphology was obtained. Further inoculation was performed on aescin selection medium: used to isolate strains producing β-glucosidase (BHI agar medium with 1 g / L aescin and 0.5 g / L ferric citrate added, 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 strain was inoculated into BHI liquid 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 TZF-004.
[0029] 2. Identification of strains
[0030] (1) Bacteroides fragilis strainBacteroides fragilis The physiological and biochemical identification results of TZF-004 are as follows:
[0031] This strain grows well under anaerobic conditions, with an optimal growth temperature of 37℃ and a suitable pH of 7.0–8.0. It exhibits a uniform turbidity in BHI liquid medium. Figure 2 As shown, the strain exhibits the ability to utilize substrates such as aescin, cellobiose, maltose, salicin, sucrose, raffinose, inulin, lactose, hippuric acid, mannitol, and sorbitol, all of which are positive, suggesting that it has good glycoside hydrolysis-related metabolic characteristics and is suitable for the biotransformation of total ginsenosides.
[0032] (2) Molecular biological identification and results are as follows:
[0033] The target strain was inoculated into fresh BHI liquid medium and cultured for 24–48 h. Bacterial DNA was extracted using a bacterial genome extraction 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-004 showed a highly specific band at approximately 1500 bp, consistent with the expected result, and sequencing was performed. The sequenced sequence was compared with the 16S rDNA gene sequences of some strains already registered in the NCBI database. The results showed that strain TZF-004 is similar to *Bacteroides fragilis* (…). Bacteroides fragilis The homology between strain TZF-004 and NCTC 9343 (NR 074784.2) reached 99.93%. Based on the above results, strain TZF-004 was identified as belonging to Bacteroides fragilis (…). Bacteroides fragilis ).
[0034] The primer pair sequence information is as follows:
[0035] 27F: 5′-agagttgatcctggctcag-3′;
[0036] 1492R: 5′-ggttaccttgttacgactt-3′.
[0037] 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℃.
[0038] 3. Preservation of bacterial strains
[0039] The present invention is based on Bacteroides fragilis ( Bacteroides fragilisTZF-004 was deposited on April 22, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession number CCTCC NO: M2026760.
[0040] Implementation Case 2: Preparation of Rare Ginsenosides
[0041] (1) Preparation of Bacteroides fragilis ( Bacteroides fragilis TZF-004 bacterial suspension
[0042] Bacteroides fragilis ( Bacteroides fragilis TZF-004 was inoculated into BHI liquid medium and anaerobically cultured at 37°C for 24 h. The culture was then centrifuged at 12000 rpm for 10 min 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 fragilis ( Bacteroides fragilis TZF-004 bacterial suspension.
[0043] (2) Fermentation and transformation
[0044] To prepare the BHI liquid fermentation medium, ginseng total saponins (purchased from Chengdu Efa Biotechnology Co., Ltd., HPLC ≥ 80%) were first added to the sterilized BHI liquid fermentation medium at a ratio of 0.5 mg / mL, and then filtered through a 0.22 μm microporous membrane for sterilization. Bacteroides fragilis (… Bacteroides fragilis TZF-004 according to 1.0×10 9 The inoculum was inoculated into BHI liquid fermentation medium supplemented with total ginsenosides at a concentration of CFU / mL, and anaerobic fermentation was carried out at 37°C for 7 days. The BHI liquid fermentation medium contained 10.0 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5.0 g / L dehydrated calf heart extract, 5.0 g / L sodium chloride, 2.0 g / L glucose, and 2.5 g / L disodium hydrogen phosphate; pH 7.4 ± 0.2 (25°C), and was prepared by sterilization at 121°C for 20 min.
[0045] (3) High-temperature steam treatment and drying purification
[0046] High-temperature steam treatment: After fermentation, the obtained fermentation product was subjected to high-temperature steam treatment at 121℃ three times, each time for 1 hour and 20 minutes. Drying and purification: Extraction was performed with water-saturated n-butanol, followed by concentration under reduced pressure at 55℃, and freeze-drying at -80℃ and 5Pa. The residue was dissolved in methanol and centrifuged at 10,000 rpm and 4℃ for 15 minutes. The supernatant was then subjected to vacuum freeze-drying at -80℃ and 5Pa after solvent recovery to obtain a dried product rich in rare ginsenosides.
[0047] Implementation Case 3: Identification of Ginsenosides by High Performance Liquid Chromatography (HPLC)
[0048] 1. Determination of Ginsenoside Components by High Performance Liquid Chromatography (HPLC)
[0049] The dried product rich in rare ginsenosides and the total ginsenoside raw material obtained in Case 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: Shim-pack GWS 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 an aqueous phase (A) and an 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.
[0050] 2. The results of the identification.
[0051] HPLC chromatographic identification results as follows Figure 3 and Figure 4 As shown, by comparing the retention time with that of ginsenoside standards, it can be determined that *Bacteroides fragilis* (a type of bacteria) in this invention is indeed present. Bacteroides fragilis The rare ginsenosides prepared by fermentation and high-temperature steam treatment of TZF-004 are: rare ginsenosides Rg6, F2, F4, 20(S)-Rh2, 20(R)-Rh2, 20(S)-Rh4, and 20(R)-Rh4. This demonstrates that the target components in the obtained dried product rich in rare ginsenosides can be obtained through *Bacteroides fragilis* (…). Bacteroides fragilis TZF-004 was prepared by fermentation and conversion combined with high-temperature steam treatment of ginsenosides.
[0052] This invention discloses a strain of Bacteroides fragilis ( Bacteroides fragilis The application of TZF-004 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 within the scope of this invention. The products of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the products and methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
Claims
1. A type of Bacteroides fragilis ( Bacteroides fragilis TZF-004, characterized in that, The strain was deposited at the China Center for Type Culture Collection on April 22, 2026, with accession number CCTCC NO: M2026760.
2. The Bacteroides fragilis as described in claim 1 ( Bacteroides fragilis The application of TZF-004 in the fermentation and conversion preparation of rare ginsenosides Rg6, F2, F4, (S)-Rh2, (R)-Rh2, (S)-Rh4, and (R)-Rh4 is characterized by, Includes the following steps: Step 1: Preparation of Bacteroides fragilis ( Bacteroides fragilis TZF-004 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: Bacteroides fragilis (… Bacteroides fragilis TZF-004 was inoculated into BHI liquid medium and anaerobically cultured at 37-39℃ for 24-48 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, to obtain Bacteroides fragilis ( Bacteroides fragilis TZF-004 bacterial suspension.
4. The application according to claim 2, characterized in that, The specific operation procedure for step two is as follows: Prepare BHI liquid fermentation medium, add total ginsenosides to the sterilized BHI liquid fermentation medium at a ratio of 0.5 mg / mL, and sterilize by passing through a 0.22 μM microporous membrane; add Bacteroides fragilis (… Bacteroides fragilis TZF-004 bacterial suspension at 1.0×10 8 ~1.0×10 9 The inoculum was inoculated into BHI liquid fermentation medium supplemented with total ginsenosides at a concentration of CFU / mL, and anaerobic fermentation was carried out at 37℃±0.5℃ for 7 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 121℃ three times, each time for 1 hour and 20 minutes; Drying and purification: Water-saturated n-butanol is used for extraction, followed by vacuum concentration at 55℃, and freeze-drying at -80℃ and 5Pa. The residue is dissolved in methanol and centrifuged at 10,000 rpm and 4℃ for 15 minutes. The supernatant is taken to recover the solvent under reduced pressure and then freeze-dried at -80℃ and 5Pa to obtain a dried product containing ginsenosides Rg6, F2, F4, 20(S)-Rh2, 20(R)-Rh2, 20(S)-Rh4, and 20(R)-Rh4.