A strain of Pediococcus acidilactici QM70-3 and its application in the fermentation conversion for preparing rare ginsenosides

Abstract

A Pediococcus acidilactici QM70-3 and its application in the fermentative conversion for preparing rare ginsenosides belong to the field of probiotic fermentation. The Pediococcus acidilactici QM70-3 was deposited at the China Center for Type Culture Collection on February 25, 2025, with the deposit number of CCTCC NO: M 2025298. In the present invention, ginsenoside Re is used as the raw material, and the rare ginsenosides Rg2, Rg6, F4 and Rh1 are prepared by fermentative conversion using Pediococcus acidilactici QM70-3 with high β-glucosidase activity. After fermentation, the ginsenoside monomer Re can be completely converted into Rg2, Rg6, F4 and Rh1. This technology does not rely on processes such as alkali hydrolysis, high temperature and high pressure, and steam treatment, and has the characteristics of mild conditions, environmental friendliness and outstanding economy. The present invention provides a new way for the preparation of rare ginsenosides Rg2, Rg6, F4 and Rh1, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic fermentation, and particularly relates to a strain of Pediococcus acidilactici QM70-3 and application thereof in preparing rare ginsenosides by fermentation and conversion. Background Art

[0002] Ginseng (Panax ginseng CAMey.) is a treasure among traditional Chinese herbal medicines in my country. Its key active ingredients, ginsenosides, have biological activities such as relieving physical fatigue, inhibiting tumor proliferation, regulating sugar metabolism and regulating inflammatory response. Studies have shown that rare ginsenosides have superior transmembrane absorption efficiency and targeting efficacy compared with primary ginsenosides. However, the abundance of rare saponins in natural plants is less than 0.1%, which seriously restricts large-scale production. Traditional physical and chemical methods (acid-base catalysis, thermal cracking, etc.) have defects such as violent reactions and complex by-products, which can easily cause nonspecific cleavage of glycosidic bonds and changes in active conformations. Although enzymatic conversion has the advantage of regional selectivity, it is limited by industrial barriers such as poor stability of enzyme preparations and high immobilization costs. Although intestinal flora can achieve the bioconversion of primary saponins, it is affected by differences in individual microbial composition, and there are limitations such as difficulty in controlling conversion efficiency and product stability. Based on this, in vitro bioconversion technology has become a key direction to break through the bottleneck. The biotransformation system with microbial fermentation and enzyme catalysis as the core can efficiently prepare rare ginsenosides by simulating the intestinal metabolic process and directed removal of sugar groups under mild conditions. Among them, the probiotic fermentation method, with its environmental friendliness, strong substrate specificity, and high product purity, can not only avoid the violent reaction of the chemical method, but also solve the problem of high cost of the direct enzymatic method, providing an innovative path for improving the bioavailability of ginseng products. The in-depth development of this technology is of strategic significance to the construction of the value system of the entire ginseng industry chain.

[0003] Lactic acid bacteria are a widely recognized type of probiotics that play an important role in improving intestinal health and immune function and are widely used in industries such as pharmaceutical manufacturing and fermented foods. Some lactic acid bacteria can produce β-glucosidase, such as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus, which can convert primary ginsenosides into a series of rare ginsenosides with low natural content, such as Rg6, Rh2, F2, etc. These transformation products have significant value in the medical field. Pediococcus acidilactici, as a probiotic, has multiple functions, including regulating the intestinal flora, improving intestinal function, enhancing immunity, and increasing food flavor. In addition, there is no research report on the preparation of rare ginsenosides Rg2, Rg6, F4, and Rh1 by fermenting ginsenoside Re monomer with Pediococcus acidilactici. This invention patent uses Pediococcus acidilactici QM70-3 with high-yield β-glucosidase, takes ginsenoside Re monomer as the substrate, and completely converts ginsenoside Re monomer into rare ginsenosides Rg2, Rg6, F4, and Rh1 after 14 days of fermentation. This process is suitable for large-scale industrial production, and the prepared rare ginsenosides Rg2, Rg6, F4, and Rh1 have a wide range of uses and broad application prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Pediococcus acidilactici QM70-3 and its application in the fermentation conversion for the preparation of rare ginsenosides, so as to provide a new way for the preparation of rare ginsenosides Rg2, Rg6, F4, and Rh1.

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

[0006] The Pediococcus acidilactici QM70-3 provided by the present invention was deposited at the China Center for Type Culture Collection on February 25, 2025, and the deposit number is: CCTCC NO: M 2025298.

[0007] A strain of Pediococcus acidilactici QM70-3 provided by the present invention and its application in the fermentation conversion for the preparation of rare ginsenosides.

[0008] As a preferred embodiment, the Pediococcus acidilactici has the characteristic of producing β-glucosidase.

[0009] As a preferred embodiment, the rare ginsenosides include Rg2, Rg6, F4, and Rh1.

[0010] As a preferred embodiment, the biotransformation synthesis route of rare ginsenosides Rg2, Rg6, F4 and Rh1 is as follows: Ginsenoside Re is hydrolyzed at the C-20 glucose position by Pediococcus acidilactici QM70-3 to generate rare ginsenoside Rg2, which dehydrates to generate rare ginsenoside F4 and Rg6. At the same time, rare ginsenoside Rg2 removes a rhamnose at the C-6 position to generate rare ginsenoside Rh1.

[0011] As a preferred embodiment, the present invention provides a strain of Pediococcus acidilactici QM70-3 and its application in the fermentation conversion for preparing rare ginsenosides, which specifically includes the following steps:

[0012] Dissolve ginsenoside Re in 2 mL of dimethyl sulfoxide, water bath at 50 °C for 10 min, add it to the sterilized liquid fermentation medium according to the addition amount of 0.5 g / L, and then sterilize it through a microporous filter membrane. Prepare a cell suspension of Pediococcus acidilactici QM70-3, and add the cell suspension according to an inoculation amount of 2×10 7 CFU / ml to the liquid fermentation medium added with ginsenoside Re, and anaerobically ferment at 37 °C for 7-14 days; after the fermentation is completed, extract the obtained fermentation product with water-saturated n-butanol, then carry out vacuum concentration at 50 °C and freeze-dry at -80 °C under 5 Pa conditions. The residue is dissolved in methanol and centrifuged at 10000 rpm for 15 min at 4 °C. The supernatant is depressurized to recover the solvent and then vacuum freeze-dried at -80 °C under 5 Pa to obtain a dried product, and this dried product contains dried products of rare ginsenosides Rg2, Rg6, F4 and Rh1.

[0013] As a preferred embodiment, the liquid fermentation medium includes: 1 g / L of glucose, 1 g / L of tryptone, 0.5 g / L of yeast extract powder and 1 g / L of sodium chloride; pH 6.0-7.0.

[0014] As a preferred embodiment, the specific process of preparing a cell suspension of Pediococcus acidilactici QM70-3 is as follows:

[0015] Inoculate Pediococcus acidilactici QM70-3 into liquid MRS medium, anaerobically culture at 37 °C for 24-48 h, centrifuge at 4 °C and 8000-12000 rpm for 5-10 min to collect the cell precipitate, and suspend it with sterile PBS to adjust the viable cell count to 1.0×10 7 ~1.0×108 CFU / ml to obtain a suspension of Pediococcus acidilactici QM70-3.

[0016] The beneficial effects of the present invention are as follows:

[0017] In the present invention, a strain of lactic acid bacterium QM70-3 that produces β-glucosidase and can completely ferment and convert ginsenoside Re into rare ginsenosides Rg2, Rg6, F4, and Rh1 was screened from a pickled platycodon sample homemade by farmers in Yanbian Prefecture, Jilin Province. At the same time, the strain QM70-3 was identified and the result was Pediococcus acidilactici. It was deposited in the China Center for Type Culture Collection on February 25, 2025, and the deposit number is: CCTCC NO: M 2025298. The present invention determined a new process for fermenting and converting rare ginsenosides Rg2, Rg6, F4, and Rh1 by Pediococcus acidilactici QM70-3, and at the same time, the transformed rare ginsenosides Rg2, Rg6, F4, and Rh1 were identified by HPLC chromatographic analysis. This process utilizes the property of Pediococcus acidilactici QM70-3 to produce β-glucosidase, uses ginsenoside Re as the raw material, and ferments and converts it by Pediococcus acidilactici QM70-3 to prepare rare ginsenosides Rg2, Rg6, F4, and Rh1. At the same time, the transformed rare ginsenosides Rg2, Rg6, F4, and Rh1 were identified by HPLC chromatographic analysis. The results showed that the retention times of the transformation products of ginsenoside Re were consistent with those of the standard products of rare ginsenosides Rg2, Rg6, F4, and Rh1. Compared with traditional biotransformation technologies, this process can achieve efficient transformation under mild conditions, and at the same time has the advantage of environmental compatibility, effectively avoiding high energy consumption and pollution problems, being suitable for large-scale industrial production, and the prepared rare ginsenosides Rg2, Rg6, F4, and Rh1 have a wide range of uses and broad application prospects. Description of the Drawings

[0018] Figure 1 It is a chromogenic diagram of Pediococcus acidilactici QM70-3 with high β-glucosidase production on the screening medium.

[0019] Figure 2 They are HPLC chromatograms of ginsenoside Re on the 0th day and 14th day of fermentation. Among them, a is the HPLC chromatogram of ginsenoside Re on the 0th day of fermentation, and b is the HPLC chromatogram of ginsenoside Re on the 14th day of fermentation.

[0020] Figure 3 This is the biotransformation synthesis route for Pediococcus acidilactici QM70-3 to ferment and transform ginsenoside Re into rare ginsenosides Rg2, Rg6, F4, and Rh1. Specific Embodiments

[0021] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0022] I. Isolation of Strains

[0023] In September 2024, a sample of pickled platycodon root made by farmers in Yanbian Prefecture, Jilin Province, was selected. 1 ml of the original pickled vegetable solution was taken and added to MRS medium for enrichment culture for 24 h. Using the plate streaking method, it was streaked on an MRS medium plate containing bromocresol purple and cultured at 37 °C for 24 h. Single colonies that could cause bromocresol purple to turn yellow were picked for microscopic examination, and among them, strains with a positive Gram stain were selected and cultured at 37 °C for 24 h. Then, it was continuously streaked and inoculated for isolation and purification until a pure culture with a single colony morphology was obtained. Further inoculated into aesculin screening medium (used to isolate strains producing β-glucosidase, adding 1 g / L of aesculin and 0.5 g / L of ferric citrate to MRS agar solid medium, autoclaved at 121 °C for 20 min), a strain with a brownish-red to dark brown circle around it was a positive strain (as Figure 1 shown), indicating that the strain had the ability to produce β-glucosidase. The obtained pure cultured strain was inoculated into liquid MRS medium for continued culture. 0.8 ml of the bacterial liquid was taken and mixed with 0.2 ml of glycerol, and then placed in a -80 °C refrigerator for storage. One of the lactic acid bacteria strains was named QM70-3.

[0024] II. Identification of Strains

[0025] 1. The physiological and biochemical identification results of strain QM70-3 are as follows:

[0026] Gram stain positive. Among 11 carbohydrate patterns, aesculin, cellobiose, salicin, and 1% sodium hippurate were positive. While maltose, sucrose, mannitol, sorbitol, raffinose, inulin, and lactose were negative reactions. The optimal growth temperature was 37 °C; the suitable pH was 6.0 - 7.0; it was tolerant to 6.5% NaCl; it grew uniformly turbid in liquid MRS medium, and the bacteria formed a white precipitate after standing for a long time.

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

[0028] The target strain was inoculated into fresh MRS liquid medium and cultured for 24 h. The genomic DNA of the strain was extracted using the kit from Tiangen Biochemical Technology Co., Ltd., and the 16S rDNA sequence was amplified. The primers used for amplification were the genus-specific primers composed of 1492R and 27F. The electrophoresis result of the 16S rDNA PCR product of strain QM70-3 showed a specific band at about 1500 bp in molecular weight, which was consistent with the expected result. Then it was sequenced, and the sequence was shown as SEQ ID NO.1 in the sequence listing. The sequenced sequence was aligned with the 16S rDNA gene sequences of some strains registered on the website http: / / www.ncbi.nlm.nih.gov. The result showed that the homology between strain QM70-3 and Pediococcus acidilactici NGRI 0510Q (NR 041640.1) reached 100%. Based on the above results, strain QM70-3 was identified as Pediococcus acidilactici.

[0029] Among them, the sequence information of the primer pair is specifically as follows:

[0030] 27sF: 5′-agagttgatcctggctcag-3′; 1492R: 5′-ggttaccttgttacgactt-3′. The PCR amplification conditions used were as follows: pre-denaturation: 94°C for 2 min; denaturation: 94°C for 30 s, annealing: 55°C for 30 s, extension: 72°C for 30 s, for a total of 30 cycles; final extension: 72°C for 2 min, and stored at 4°C.

[0031] III. Preservation of the Strain

[0032] Pediococcus acidilactici QM70-3 of the present invention was deposited at the China Center for Type Culture Collection (CCTCC for short) on February 25, 2025. The address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (the preservation center of Wuhan University), and the deposit number is: CCTCC NO: M 2025298.

[0033] IV. Preparation of Pediococcus acidilactici QM70-3 Bacterial Suspension

[0034] The obtained Pediococcus acidilactici QM70-3 was inoculated into a liquid MRS medium and cultured statically at 37 °C for 24 h, then centrifuged at 4 °C and 8000 rpm for 10 min to collect the cell precipitate; it was suspended with sterile PBS, and the viable cell count was adjusted to 1.0×10 9 CFU / mL to obtain a Pediococcus acidilactici QM70-3 bacterial suspension.

[0035] V. Preparation of rare ginsenosides by fermentation and transformation of ginsenoside Re with Pediococcus acidilactici QM70-3

[0036] 1. Preparation of liquid fermentation medium: The liquid fermentation medium contains 1 g / L of glucose, 1 g / L of tryptone, 0.5 g / L of yeast extract powder and 1 g / L of sodium chloride; the pH is 6.0 - 7.0, and it is obtained by sterilization at 121 °C for 20 min.

[0037] 2. Fermentation and transformation: First, dissolve ginsenoside Re in 2 mL of dimethyl sulfoxide, add it to the liquid fermentation medium after a 10-min water bath at 50 °C. The addition amount of ginsenoside Re is 0.5 g / L, and it is filtered and sterilized with a 0.22-μm microporous filter membrane, and then inoculate the Pediococcus acidilactici QM70-3 bacterial suspension at an inoculum size of 2×10 7 CFU / mL and ferment anaerobically at 37 °C for 14 days. After the fermentation is completed, the obtained fermentation product is extracted three times with n-butanol saturated with water, then concentrated under reduced pressure at 50 °C and freeze-dried at -80 °C and 5 Pa. Its product is extracted with methanol, centrifuged at 10000 rpm for 15 min at 4 °C. After the supernatant is recovered from the solvent under reduced pressure, it is vacuum freeze-dried at -80 °C and 5 Pa to obtain a dry product, and this dry product contains rare ginsenosides. The dry product contains rare ginsenosides Rg2, Rg6, F4 and Rh1.

[0038] VI. Identification of rare ginsenosides by high performance liquid chromatography (HPLC)

[0039] Detect rare ginsenosides Rg2, Rg6, F4 and Rh1 by high performance liquid chromatography (HPLC):

[0040] Dissolve ginsenoside Re and the above-obtained dried product in chromatographic methanol respectively. After filtration through a 0.22 μm microporous filter membrane, it is used for HPLC chromatographic analysis. The HPLC chromatographic analysis method is as follows: The chromatographic column is an Agilent pursuit5 SB C18 chromatographic column, the injection volume is 20 μL, the elution rate is 1.0 mL / min, the column temperature is 30 °C, and the detection wavelength is 203 nm. The mobile phase is water (A) and acetonitrile (C), and gradient elution is carried out as follows: 0 min, 81.50% A, 18.50% C; 20 min, 79.50% A, 20.50% C; 30 min, 70% A, 30% C; 45 min, 65% A, 35% C; 60 min, 55% A, 45% C; 70 min, 40% A, 60% C; 80 min, 30% A, 70% C; 90 min, 20% A, 80% C; 91 min, 81.50% A, 18.50% C; 95 min, 81.50% A, 18.50% C.

[0041] The HPLC chromatographic identification results are as Figure 2 shown. Through the qualitative and quantitative analysis of ginsenoside components in the products before and after fermentation, it can be known that the retention times of the conversion products of ginsenoside Re are consistent with those of the rare ginsenoside standards Rg2, Rg6, F4, and Rh1. This proves that ginsenoside Re can be fermented and converted into rare ginsenosides Rg2, Rg6, F4, and Rh1 by Pediococcus acidilactici QM70-3 producing β-glucosidase.

[0042] In the process of using the Pediococcus acidilactici QM70-3 bacterial suspension to ferment and convert to prepare rare ginsenosides Rg2, Rg6, F4, and Rh1, its biotransformation synthesis route is as Figure 3 shown: Ginsenoside Re is hydrolyzed at the C-20 position of glucose under the action of Pediococcus acidilactici QM70-3 to generate rare ginsenoside Rg2, which dehydrates to generate rare ginsenoside F4 and Rg6. At the same time, rare ginsenoside Rg2 loses a rhamnose at the C-6 position to generate rare ginsenoside Rh1.

[0043] The present invention discloses a strain of Pediococcus acidilactici QM70-3 and its application in the fermentative conversion for preparing rare ginsenosides. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The products of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to the products described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. A strain of Pediococcus acidilactici QM70-3, characterized in that, The Pediococcus acidilactici was deposited at the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025298.

2. Use of a strain of Pediococcus acidilactici QM70-3 as described in claim 1 in the fermentation conversion of ginsenoside Re to prepare rare ginsenosides, wherein the rare ginsenosides are selected from Rg2, Rg6, F4 and Rh1.

3. The application according to claim 2, characterized in that, The biotransformation synthesis routes of the rare ginsenosides Rg2, Rg6, F4 and Rh1 are as follows: Ginsenoside Re is hydrolyzed at the C-20 position of glucose under the action of Pediococcus acidilactici QM70-3 to generate the rare ginsenoside Rg2. Further, the rare ginsenoside Rg2 is dehydrated to generate the rare ginsenosides F4 and Rg6, and at the same time, the rare ginsenoside Rg2 removes a rhamnose at the C-6 position to generate the rare ginsenoside Rh1.

4. The application according to claim 2, characterized in that, It includes the following steps: Dissolve ginsenoside Re in 2 mL of dimethyl sulfoxide, add it to the liquid fermentation medium after a 10-minute water bath at 50 °C. The addition amount of ginsenoside Re is 0.5 g / L, filter and sterilize it with a 0.22-μm microporous membrane. Prepare Pediococcus acidilactici QM70-3 into a bacterial suspension, and inoculate the bacterial suspension into the liquid fermentation medium added with ginsenoside Re at an inoculum size of 2×10 7 CFU / ml, and carry out anaerobic fermentation at 37±0.5 °C for 7 to 14 days; after the fermentation is completed, extract the obtained fermentation product with n-butanol saturated with water, then carry out reduced pressure concentration at 50 °C and freeze-dry it at -80 °C under 5 Pa. Dissolve the residue in methanol, centrifuge it at 10,000 rpm for 15 minutes at 4 °C. After the supernatant is recovered by reduced pressure to remove the solvent, carry out vacuum freeze-drying at -80 °C under 5 Pa to obtain a dried product, and the dried product contains rare ginsenosides Rg2, Rg6, F4 and Rh1.

5. The application according to claim 4, characterized in that The specific process of preparing Pediococcus acidilactici QM70-3 into a bacterial suspension is as follows: Inoculate Pediococcus acidilactici QM70-3 into liquid MRS medium, anaerobically culture at 37 °C for 24 - 48 h, centrifuge at 4 °C and 8000 - 12000 rpm for 5 - 10 min, collect the bacterial cell precipitate, suspend it with sterile PBS, and adjust the viable cell count to 1.0×10 7 ~1.0×10 8 CFU / ml to obtain the Pediococcus acidilactici QM70-3 bacterial suspension.

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