A strain of Pediococcus pentosaceus TRE15 and its application in fermentation and conversion of ginsenoside Re to prepare rare ginsenosides

Abstract

A strain of Pediococcus pentosaceus TRE15 and its application in fermenting and transforming ginsenoside Re to prepare rare ginsenosides belong to the field of microbial fermentation. The Pediococcus pentosaceus TRE15 has been deposited in the China Center for Type Culture Collection on April 24, 2024, with a deposit number of CCTCC NO: M2024753. The Pediococcus pentosaceus TRE15 can be used in fermenting and transforming ginsenoside Re to prepare rare ginsenosides. Its biotransformation synthesis route is: ginsenoside Re hydrolyzes glucose at the C-20 position under the action of Pediococcus pentosaceus TRE15 to generate rare ginsenoside Rg2, and further dehydrates to generate rare ginsenosides Rg6 and F4. The present invention utilizes Pediococcus pentosaceus TRE15 to ferment and transform ginsenoside Re to prepare rare ginsenosides Rg2, Rg6 and F4, which can provide a new way for the preparation of rare ginsenosides and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Pediococcus pentosaceus TRE15 and application thereof in fermenting and transforming ginsenoside Re to prepare rare ginsenosides. Background Art

[0002] Saponins are representative bioactive components of ginseng. Saponins that exist naturally and in high content in ginseng are called primary saponins. Primary saponins need to be degraded by gastric acid or converted into secondary saponins or aglycones by intestinal flora in order to play a role. These secondary saponins are low in content or non-existent in ginseng and are also called rare saponins. Common rare ginsenosides include Rg3, CK, Rg5, Rh2, Rh4 and Rk3. Rare saponins have reduced water solubility and increased fat solubility, which can pass through the cell membrane more easily and play a stronger biological role. Primary saponins mainly play a role in vivo through the biotransformation of intestinal microorganisms to generate rare saponins, but due to the diversity of intestinal flora, there are also obvious individual differences in their transformation to rare saponins, which affects the bioavailability of ginsenosides. Therefore, deglycosylation of primary ginsenosides in vitro to generate easily absorbed rare saponins is an effective way to improve the utilization of ginsenosides.

[0003] At present, the transformation of rare saponins can be achieved through a variety of methods, including physical, chemical and biological methods. Among them, the biotransformation method based on microorganisms and enzymatic reactions has attracted much attention due to its mild reaction conditions, high selectivity, low carbon and environmental protection.

[0004] Lactic acid bacteria are a type of widely distributed microorganisms that can be isolated from animal intestines, fermented foods, and natural environments. Due to the characteristics of high safety, fast growth rate, clear metabolic mechanism, and few by-products, lactic acid bacteria are widely used in food, medicine, breeding, and environmental protection. Some lactic acid bacteria can produce β-glucosidase, which can be used for the bioconversion of primary ginsenosides into rare saponins. Existing studies have shown that the use of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus brevis, and Bifidobacterium can convert primary ginsenosides into rare ginsenosides, but there has been no report on the use of Pediococcus pentosaceus to convert ginsenosides into rare ginsenosides. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Pediococcus pentosaceus TRE15 and its application in fermenting and transforming ginsenoside Re to prepare rare ginsenosides. The present invention obtains a strain of Pediococcus pentosaceus TRE15, and utilizes the strain to ferment and transform ginsenoside Re to prepare rare ginsenosides Rg2, Rg6 and F4, which can provide a new way for the preparation of rare ginsenosides Rg2, Rg6 and F4, and has good application prospects.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] The strain of Pediococcus pentosaceus TRE15 provided by the present invention has been deposited in the China Center for Type Culture Collection on April 24, 2024, with the deposit number: CCTCC NO: M2024753.

[0008] The present invention provides an application of a Pediococcus pentosaceus TRE15 in fermenting and transforming ginsenoside Re to prepare rare ginsenosides.

[0009] Among them, as a preferred embodiment, the rare ginsenosides are Rg2, Rg6 and F4.

[0010] Among them, as a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides Rg2, Rg6 and F4 is: the ginsenoside Re is hydrolyzed with glucose at the C-20 position under the action of Pediococcus pentosaceus TRE15 to generate the rare ginsenoside Rg2, and further dehydrated to generate the rare ginsenosides Rg6 and F4.

[0011] The beneficial effects of the present invention are:

[0012] The present invention screened and obtained a beneficial strain TRE15 that can bioconvert ginsenoside Re into rare ginsenosides Rg2, Rg6 and F4, and simultaneously identified the strain TRE15, which turned out to be Pediococcus pentosaceus.

[0013] The present invention determines through experiments that a new process for preparing rare ginsenosides Rg2, Rg6 and F4 by fermenting and transforming ginsenoside Re by Pediococcus pentosaceus TRE15 is used, and HPLC chromatographic analysis is used to identify the transformed rare ginsenosides Rg2, Rg6 and F4. The new process uses ginsenoside Re as a raw material and utilizes the Pediococcus pentosaceus TRE15 of the present invention to ferment and transform and prepare rare ginsenosides Rg2, Rg6 and F4. Compared with the existing biological transformation process route, the new process has the advantages of strong specificity, mild reaction conditions, environmental friendliness, high transformation efficiency, etc., is suitable for large-scale preparation of mixed total rare saponins containing rare ginsenosides Rg2, Rg6 and F4, and the transformation products are all rare ginsenosides with better biological activity, which has a wider application prospect.

[0014] The present invention utilizes the screened Pediococcus pentosaceus TRE15 to ferment and transform ginsenoside Re to prepare rare ginsenosides Rg2, Rg6 and F4, which can provide a new way for the preparation of rare ginsenosides Rg2, Rg6 and F4 and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Biotransformation roadmap for rare ginsenosides Rg2, Rg6 and F4.

[0016] Figure 2 HPLC chromatograms of ginsenoside Re before and after fermentation by Pediococcus pentosaceus TRE15. In the figure, A is the HPLC chromatogram of ginsenoside Re before fermentation by Pediococcus pentosaceus TRE15, and B is the HPLC chromatogram of ginsenoside Re after fermentation by Pediococcus pentosaceus TRE15. DETAILED DESCRIPTION

[0017] 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.

[0018] 1. Isolation of strains

[0019] In September 2022, naturally fermented pickle samples sold at the West Market in Yanji City, Jilin Province were collected and quickly placed in an ice box and sent to the laboratory for strain isolation. 0.1g of pickle sample was accurately weighed and diluted with 99.9mL of sterile distilled water to obtain a suspension. 0.1mL of the suspension was inoculated into an MRS solid culture medium plate and incubated at 37°C for 48h. The colony morphology of the MRS solid culture medium plate was observed, and a single suspected colony with good growth was picked and continued to be streaked and inoculated into an agar culture plate. Different colonies were passaged until pure isolates were isolated and purified; the purified pure isolate was inoculated into liquid MRS culture medium for culture, and after adding 60% glycerol, it was stored in a -80°C refrigerator. At the same time, one of the strains was named TRE15.

[0020] 2. Identification of strains

[0021] 1. The physiological and biochemical identification results of strain TRE15 are as follows:

[0022] Positive for Gram staining, negative for catalase test, negative for benzidine test, negative for indole test, positive for acetylmethyl carbinol test; does not hydrolyze starch, does not liquefy gelatin, does not produce hydrogen sulfide, and ferments glucose to produce acid but not gas; non-motile cocci; can grow at 15°C and 45°C, with the optimum growth temperature being 37-42°C; suitable pH is 5.0-7.0; tolerates 6.5% NaCl; grows uniformly turbid in liquid MRS medium, and the bacteria show white precipitate after long storage.

[0023] 2. The process and results of 16S rDNA sequence homology analysis are as follows:

[0024] The genomic DNA of strain TRE15 was extracted, and a primer pair consisting of 16sF and 16sR was used for PCR amplification, and then the obtained amplified product was sequenced, wherein the 16s rDNA sequence is shown in SEQ ID NO: 1. The 16s rDNA sequence was subjected to homology comparison analysis in the GenBank library using the BLAST program, and it was found that the homology between strain TRE15 and Pediococcus pentosaceus strain 8368 (GenBank: MT538853.1) reached 100%. Therefore, according to the above identification results, the extracted strain TRE15 was identified as Pediococcus pentosaceus.

[0025] Wherein, the primer pair sequence information is as follows:

[0026] 16sF: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0027] 16sR: 5′-AGAAAGGAGGTGATCCAGC-3′.

[0028] The PCR amplification conditions used were as follows: pre-deformation: 95°C for 5 min; denaturation: 95°C for 15 s, annealing: 55°C for 15 s, extension: 72°C for 1.5 min, for a total of 35 cycles; final extension: 72°C for 7 min, and storage at 4°C.

[0029] 3. Preservation of strains

[0030] A strain of Pediococcus pentosaceus TRE15 of the present invention has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on April 24, 2024, with an address of: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center), and the collection number is: CCTCC NO: M2024753.

[0031] 4. Preparation of Pediococcus pentosaceus TRE15 bacterial suspension

[0032] The obtained Pediococcus pentosaceus TRE15 was inoculated into liquid MRS medium, cultured at 37°C for 16 h, centrifuged at 4°C and 6000 rpm for 8 min, and the bacterial precipitate was collected; after being suspended with fermentation sterilized medium aqueous solution, the viable bacterial count was adjusted to 1.0×10 10 CFU / mL, and obtain the bacterial suspension of Pediococcus pentosaceus TRE15.

[0033] 5. Fermentation and transformation of ginsenoside Re by Pediococcus pentosaceus TRE15 to prepare rare ginsenosides

[0034] 1. Preparation of liquid fermentation medium: The liquid fermentation medium contains 3 g / L glucose and 1 g / L soybean oligopeptide, has a pH of 6.6, and is prepared by sterilization at 121° C. for 15 min.

[0035] 2. Fermentation culture: Ginsenoside Re (purchased from Shanghai Yuanye Biotechnology Co., Ltd., HPLC ≥ 98%) was dissolved with a small amount of dimethyl sulfoxide, and water was added to prepare a 1 mg / mL stock solution. After filtering through a 0.22um microporous filter membrane, it was added to a sterilized liquid fermentation medium at an addition amount of 1g / L, and the bacterial suspension of Pediococcus pentosaceus TRE15 was inoculated into the liquid fermentation medium at an inoculum amount of 10ml / L, and co-fermented and cultured at 37°C for 7 days. After the fermentation was completed, the obtained fermentation product was concentrated under reduced pressure, vacuum freeze-dried (-80°C, 5pa), and the obtained product was extracted with methanol, centrifuged at 4°C, 10000rpm for 15min, and the supernatant was vacuum-freezed and the solvent was recovered and then vacuum freeze-dried (-80°C, 5pa) to obtain a dry product containing rare ginsenosides, which was a mixture of rare ginsenosides.

[0036] 6. Identification of rare ginsenosides by high performance liquid chromatography (HPLC)

[0037] The dried product containing rare ginsenosides obtained above was dissolved in methanol, filtered through a 0.22 μm microporous filter membrane and then used for HPLC chromatography analysis.

[0038] The specific HPLC chromatography analysis method is as follows:

[0039] The chromatographic column was an Agilentpursuit5SB-C18 column, the injection volume was 20 μL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 203 nm;

[0040] The mobile phase was A: water;

[0041] B: acetonitrile;

[0042] 0~40min 18~21%(B); 40~42min 21~26%(B); 42~46min 26~32%(B); 46~66min 32~34%(B); 66~71min 34~38%(B); 71~77.70min 38.0~49.1%(B); 77.70~82min 49.1%(B); 82~83min 49.1~50.6%(B); 83~88min 50.6~59.6%(B); 88~89.80min 59.6~65.0%(B); 89.80~97min 65%(B);97~102min 65~75%(B);102~110min 75~85%(B); 110~115min 85%(B); 115~125min 85~18%(B); 125~130min18.0%(B).

[0043] In the process of preparing rare ginsenosides Rg2, Rg6 and F4 by fermenting ginsenoside Re with Pediococcus pentosaceus TRE15 suspension, the biotransformation synthesis route is as follows: Figure 1 As shown, ginsenoside Re was hydrolyzed by Pediococcus pentosaceus TRE15 to produce rare ginsenoside Rg2 after glucose at C-20 position, and further dehydrated to produce rare ginsenosides Rg6 and F4.

[0044] HPLC chromatographic analysis results are as follows Figure 2As shown in the figure, by analyzing the changes in the content of ginsenoside Re in the products before and after fermentation, it can be seen that the retention time of rare ginsenosides Rg2, Rg6 and F4 under HPLC chromatographic conditions is consistent with the retention time of ginsenoside standard (purchased from Shanghai Yuanye Biotechnology Co., Ltd., HPLC ≥ 98%). This proves that ginsenoside Re can be converted into rare ginsenosides Rg2, Rg6 and F4 through fermentation by Pediococcus pentosaceus TRE15.

[0045] The present invention discloses a strain of Pediococcus pentosaceus TRE15 and its application in fermenting and converting ginsenoside Re to prepare 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 is particularly important to point out 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 pentosaceus ( Pediococcus pentosaceus ) TRE15, characterized in that It was deposited in the China Center for Type Culture Collection on April 24, 2024, with the deposit number: CCTCC NO: M2024753.

2. The Pediococcus pentosaceus according to claim 1 ( Pediococcus pentosaceus ) Application of TRE15 in fermentation and conversion of ginsenoside Re to prepare rare ginsenosides, characterized in that: The rare ginsenosides are Rg2, Rg6 and F4; the biotransformation synthesis route of the rare ginsenosides Rg2, Rg6 and F4 is: the ginsenoside Re is hydrolyzed with glucose at the C-20 position under the action of Pediococcus pentosaceus TRE15 to generate the rare ginsenoside Rg2, which is further dehydrated to generate the rare ginsenosides Rg6 and F4.

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