Pediococcus acidilactici QM70-3 and application thereof in preparation of rare ginsenoside through fermentation conversion

Abstract

The invention discloses pediococcus acidilactici QM70-3 and application thereof in preparation of rare ginsenoside through fermentation conversion, and belongs to the field of probiotic fermentation, the pediococcus acidilactici QM70-3 is preserved in China Center for Type Culture Collection on February 25, 2025, and the preservation number is CCTCC NO: M 2025298. According to the invention, ginsenoside Re is used as a raw material, and the rare ginsenosides Rg2, Rg6, F4 and Rh1 are prepared by fermentation and conversion of pediococcus acidilactici QM70-3 capable of producing beta-glucosidase at high yield. After fermentation, the ginsenoside monomer Re can be completely converted into Rg2, Rg6, F4 and Rh1, and the technology does not need to depend on processes such as alkaline hydrolysis, high temperature and high pressure, steam treatment and the like, and has the characteristics of mild conditions, environmental friendliness, outstanding economical efficiency and the like. The invention provides a new way for the preparation of rare ginsenosides Rg2, Rg6, F4 and Rh1, and has a better application prospect.

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 probiotic that improves intestinal health and immune function, and occupies an important position in the pharmaceutical manufacturing and fermented food industries. 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 rare natural content, such as Rg6, Rh2, F2, etc. These conversion products have significant value in the medical field. As a probiotic, Pediococcus acidilactici has multiple functions, including regulating intestinal flora, improving intestinal function, enhancing immunity, and increasing food flavor. In addition, there are no reports on the preparation of rare ginsenosides Rg2, Rg6, F4 and Rh1 by fermenting ginsenoside Re monomers with Pediococcus acidilactici. The present invention adopts the high-yield β-glucosidase-producing Pediococcus acidilactici QM70-3, and uses the ginsenoside monomer Re as the substrate. After 14 days of fermentation, the ginsenoside monomer Re is completely converted into rare ginsenosides Rg2, Rg6, F4 and Rh1. The 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 and conversion 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 problem is as follows:

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

[0007] The present invention provides a strain of Pediococcus acidilactici QM70-3 and application thereof in preparing rare ginsenosides by fermentation and conversion.

[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 the rare ginsenosides Rg2, Rg6, F4 and Rh1 is as follows: ginsenoside Re is hydrolyzed with glucose at the C-20 position under the action of Pediococcus acidilactici QM70-3 to generate rare ginsenoside Rg2, which is then dehydrated to generate rare ginsenosides 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 use in fermentation and conversion to prepare rare ginsenosides, which specifically includes the following steps:

[0012] Ginsenoside Re was dissolved in 2 mL of dimethyl sulfoxide, and then added to the sterilized liquid fermentation medium at a rate of 0.5 g / L. After sterilization through a microporous filter membrane, Pediococcus acidilactici QM70-3 was prepared into a bacterial suspension, and the bacterial suspension was heated to 2 × 10 7 The inoculum amount of CFU / ml is inoculated into a liquid fermentation medium added with ginsenoside Re, and anaerobically fermented at 37°C for 7 to 14 days; after the fermentation is completed, the fermentation product is extracted with water-saturated n-butanol, then concentrated under reduced pressure at 50°C and freeze-dried at -80°C, 5PA, the residue is dissolved in methanol and centrifuged at 10000rpm, 15min, 4°C, the supernatant is decompressed and the solvent is recovered, and then vacuum freeze-dried at -80°C, 5PA to obtain a dry product, which contains rare ginsenosides Rg2, Rg6, F4 and Rh1.

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

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

[0015] Pediococcus acidilactici QM70-3 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24-48 h. The bacterial precipitate was collected after centrifugation at 4°C and 8000-12000 rpm for 5-10 min and suspended in sterile PBS to adjust the viable cell count to 1.0×10 7 ~1.0×108 CFU / ml, and obtain Pediococcus acidilactici QM70-3 bacterial suspension.

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

[0017] The present invention screened a 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 from a homemade pickled cabbage platycodon sample made by a farmer in Yanbian Prefecture, Jilin Province. At the same time, the strain QM70-3 was identified as Pediococcus acidilactici, and was deposited in the China Center for Type Culture Collection on February 25, 2025, with a deposit number of CCTCC NO: M 2025298. The present invention determined a new process for preparing rare ginsenosides Rg2, Rg6, F4 and Rh1 by fermentation and conversion of Pediococcus acidilactici QM70-3, and HPLC chromatographic analysis was used to identify the converted rare ginsenosides Rg2, Rg6, F4 and Rh1. The process utilizes the characteristics of Pediococcus acidilactici QM70-3 in producing β-glucosidase, takes ginsenoside Re as raw material, and uses Pediococcus acidilactici QM70-3 to ferment and transform rare ginsenosides Rg2, Rg6, F4 and Rh1. At the same time, HPLC chromatography analysis is used to identify the transformed rare ginsenosides Rg2, Rg6, F4 and Rh1. The results show that the retention time of the conversion product of ginsenoside Re is consistent with that of the standards of rare ginsenosides Rg2, Rg6, F4 and Rh1. Compared with traditional biotransformation technology, this process can achieve efficient transformation under mild conditions, and at the same time has the advantages of environmental compatibility, effectively avoiding high energy consumption and pollution problems, and is suitable for large-scale industrial production. The prepared rare ginsenosides Rg2, Rg6, F4 and Rh1 have a wide range of uses and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a color diagram of Pediococcus acidilactici QM70-3, which produces high β-glucosidase, on the screening culture medium.

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

[0020] Figure 3 The present invention provides a biotransformation synthesis route for the fermentation and transformation of ginsenoside Re by Pediococcus acidilactici QM70-3 to produce rare ginsenosides Rg2, Rg6, F4 and Rh1. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] 1. Isolation of strains

[0023] Samples of homemade kimchi platycodon from farmers in Yanbian Prefecture, Jilin Province were selected in September 2024. 1 ml of kimchi stock solution was aspirated and added to MRS medium for enrichment culture for 24 hours. The plate streak method was used to streak on an MRS medium plate containing bromocresol purple and cultured at 37°C for 24 hours. Single colonies that can cause bromocresol purple to turn yellow were picked for microscopic examination, and strains that were Gram-positive were selected and cultured at 37°C for 24 hours. The streaking inoculation was continued for separation and purification until a purified product with a single colony morphology was obtained. It was further inoculated in aesculin screening medium (used to isolate strains that produce β-glucosidase, 1 g / L of aesculin and 0.5 g / L of ferric citrate were added to the MRS agar solid medium and sterilized at high pressure at 121°C for 20 minutes). The strains were surrounded by brown-red to dark brown circles, which were positive strains (such as Figure 1 The pure cultured strain was inoculated into liquid MRS medium for further culture, 0.8 ml of bacterial solution was added to 0.2 ml of glycerol and mixed, and stored in a -80°C refrigerator. One of the lactic acid bacteria was named QM70-3.

[0024] 2. Identification of strains

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

[0026] Gram staining is positive. Among the 11 carbohydrate patterns, esculin, cellobiose, salicin and 1% sodium hippurate are positive. However, maltose, sucrose, glycosides, sorbitol, raffinose, inulin and lactose are negative. The optimal growth temperature is 37℃; the suitable pH is 6.0-7.0; it tolerates 6.5% NaCl; it grows uniformly turbid in liquid MRS medium, and the bacteria will show white precipitation after long-term storage.

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

[0028] The target strain was inoculated in fresh MRS liquid medium and cultured for 24 hours. The bacterial DNA was extracted using the kit of Tiangen Biochemical Technology Co., Ltd., and the 16S rDNA sequence was amplified. The primers used were species-universal primers composed of 1492R and 27F. The electrophoresis results of the 16S rDNA PCR product of strain QM70-3 showed that a band with good specificity was obtained at a molecular weight of about 1500 bp, which was consistent with the expected results. The sequence was sequenced, and the sequence was shown in SEQ ID NO.1 in the sequence table. The sequencing sequence was compared with the 16S rDNA gene sequences of some strains registered on the website http: / / www.ncbi.nlm.nih.gov. The results 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] Wherein, the primer pair sequence information is as follows:

[0030] 27sF: 5′-agagttgatcctggctcag-3′; 1492R: 5′-ggttaccttgttacgactt-3′; wherein, the PCR amplification conditions adopted are as follows: preliminary denaturation: 94°C 2 min; denaturation: 94°C 30 s, annealing: 55°C 30 s, extension: 72°C 30 s, for a total of 30 cycles; final extension: 72°C 2 min, and storage at 4°C.

[0031] 3. Preservation of strains

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

[0033] 4. Preparation of Pediococcus acidilactici QM70-3 bacterial suspension

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

[0035] 5. Preparation of rare ginsenosides by fermentation of ginsenoside Re by Pediococcus acidilactici QM70-3

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

[0037] 2. Fermentation transformation: Dissolve ginsenoside Re in 2 mL of dimethyl sulfoxide, add it to the liquid fermentation medium after 10 min in a 50°C water bath. The amount of ginsenoside Re added is 0.5 g / L. Filter and sterilize with a 0.22 μM microporous filter membrane. 7 CFU / mL inoculation amount inoculated with Pediococcus acidilactici QM70-3 bacterial suspension, anaerobic fermentation at 37 ° C for 14 days. After the fermentation, the fermentation product was extracted three times with water-saturated n-butanol, then concentrated under reduced pressure at 50 ° C and freeze-dried at -80 ° C, 5 Pa. The product was extracted with methanol, centrifuged at 10000 rpm, 15 min, 4 ° C, and the supernatant was vacuum-freeze-dried at -80 ° C, 5 Pa to obtain a dry product, which contained rare ginsenosides. The dry product contains rare ginsenosides Rg2, Rg6, F4 and Rh1.

[0038] 6. Identification of rare ginsenosides by high performance liquid chromatography (HPLC)

[0039] Detection of rare ginsenosides Rg2, Rg6, F4 and Rh1 by high performance liquid chromatography (HPLC): Ginsenoside Re and the dried product obtained above were dissolved in chromatographic methanol, filtered through a 0.22 μm microporous filter membrane and used for HPLC chromatographic analysis. The HPLC chromatographic analysis method was: chromatographic column Agilent pursuit5 SB C18 column, injection volume 20 μL, elution rate 1.0 mL / min, column temperature 30°C, detection wavelength 203 nm. The mobile phase was water (A) and acetonitrile (C) with gradient elution 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.

[0040] HPLC chromatographic identification results are as follows Figure 2 As shown in the figure, through the qualitative and quantitative analysis of ginsenoside components in the products before and after fermentation, it can be seen that the retention time of the conversion product of ginsenoside Re is consistent with that of the rare ginsenosides Rg2, Rg6, F4 and Rh1 standards. This proves that ginsenoside Re can be converted into rare ginsenosides Rg2, Rg6, F4 and Rh1 through fermentation by Pediococcus acidilactici QM70-3 producing β-glucosidase.

[0041] In the process of preparing rare ginsenosides Rg2, Rg6, F4 and Rh1 by fermentation transformation of Pediococcus acidilactici QM70-3 bacterial suspension, the biotransformation synthesis route is as follows: Figure 3 As shown: Ginsenoside Re hydrolyzes glucose at the C-20 position under the action of Pediococcus acidilactici QM70-3 to produce rare ginsenoside Rg2, which is dehydrated to produce rare ginsenosides F4 and Rg6. At the same time, rare ginsenoside Rg2 removes a rhamnose at the C-6 position to produce rare ginsenoside Rh1.

[0042] The present invention discloses a strain of Pediococcus acidilactici QM70-3 and its application in the fermentation and conversion preparation of rare ginsenosides. Those skilled in the art can refer to 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 considered to be included in the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products described herein without departing from the content, spirit and scope of the present invention to realize 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 in the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025298.

2. Use of the strain Pediococcus acidilactici QM70-3 as claimed in claim 1 in the fermentation and conversion of ginsenoside Re to prepare rare ginsenosides.

3. The use according to claim 2, characterized in that: The rare ginsenosides include Rg2, Rg6, F4 and Rh1.

4. The use according to claim 3, characterized in that: The biotransformation synthesis route of the rare ginsenosides Rg2, Rg6, F4 and Rh1 is as follows: ginsenoside Re is hydrolyzed with glucose at the C-20 position under the action of Pediococcus acidilactici QM70-3 to generate rare ginsenoside Rg2, which is then dehydrated to generate rare ginsenosides F4 and Rg6. At the same time, rare ginsenoside Rg2 removes a rhamnose at the C-6 position to generate rare ginsenoside Rh1.

5. The use according to claim 2, characterized in that: The following steps are involved: Ginsenoside Re was dissolved in 2 mL of dimethyl sulfoxide and added to the liquid fermentation medium after a 50°C water bath for 10 min. The amount of ginsenoside Re added was 0.5 g / L. The mixture was sterilized by filtration with a 0.22 μM microporous filter membrane. Pediococcus acidilactici QM70-3 was prepared into a bacterial suspension and the bacterial suspension was heated to 2 × 10 7 The inoculum amount of CFU / ml is inoculated into a liquid fermentation medium added with ginsenoside Re, and anaerobically fermented at 37±0.5°C for 7 to 14 days. After the fermentation, the fermentation product is extracted with water-saturated n-butanol, then concentrated under reduced pressure at 50°C and freeze-dried at -80°C, 5PA, the residue is dissolved in methanol and centrifuged at 10000rpm, 15min, 4°C, the supernatant is decompressed and the solvent is recovered, and then vacuum freeze-dried at -80°C, 5PA to obtain a dry product, which contains rare ginsenosides Rg2, Rg6, F4 and Rh1.

6. The use according to claim 5, characterized in that: The specific process of preparing Pediococcus acidilactici QM70-3 into bacterial suspension is as follows: Pediococcus acidilactici QM70-3 is inoculated into liquid MRS medium, cultured anaerobically at 37°C for 24-48 hours, centrifuged at 4°C and 8000-12000 rpm for 5-10 minutes, and then the bacterial precipitate is collected and suspended with sterile PBS to adjust the viable bacterial count to 1.0×10 7 ~1.0×10 8 CFU / ml, and obtain Pediococcus acidilactici QM70-3 bacterial suspension.

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