Phytobacterium plantarum RET215 and application thereof in preparing rare ginsenoside by fermenting and converting ginsenoside Re

Abstract

The invention discloses a plant lactobacillus RET215 and application thereof in preparing rare ginsenoside by fermenting and converting ginsenoside Re, and belongs to the field of microbial fermentation, the plant lactobacillus RET215 is preserved in China Center for Type Culture Collection on May 21, 2024, and the preservation number is CCTCC NO: M20241024. The plant lactobacillus RET215 can be used for preparing rare ginsenosides 20 (R)-Rg2 and 20 (S)-Rg2 through fermentation and conversion by taking ginsenosides Re as a substrate. The method has the advantages of simple process, high conversion efficiency, high content of the obtained conversion product and the like, can ensure that the fermentation product takes 20 (R)-Rg2 and 20 (S)-Rg2 as main components, has higher specificity, and can be applied to ginseng deep processing products with unique requirements on ginsenosides 20 (R)-Rg2 and 20 (S)-Rg2.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Lactobacillus plantarum RET215 and application thereof in fermenting and transforming ginsenoside Re to prepare rare ginsenosides. Background Art

[0002] Ginsenosides are the main active ingredients in ginseng and have a variety of pharmacological effects, such as anti-inflammatory, antioxidant, and anti-tumor. Among them, rare ginsenosides have gradually become a hot topic in contemporary ginseng research due to their low natural content and strong pharmacological activity. Ginsenoside Re is a ginsenoside with a high natural reserve in ginseng and is often used as a substrate for the preparation of other rare ginsenosides.

[0003] Microbial fermentation conversion is an emerging biosynthesis technology in recent years. At present, many microorganisms have been confirmed to have the ability to change the structure of compounds through fermentation, hydrolysis of glycoside chains, induction of compound cyclization, etc. Among them, lactic acid bacteria, as a type of beneficial microorganism, have been proven by existing studies to be able to ferment and convert ginsenosides into rare ginsenosides using total ginsenosides as substrates. However, due to the specificity of the fermentation process of different strains and the differences in the hydrolysis position of the glycosidic bond of saponins, there are not many reports that can specifically convert ginsenoside Re into rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus plantarum RET215 and its use in fermenting and transforming ginsenoside Re to prepare rare ginsenosides. The present invention develops a new method for preparing rare ginsenosides by lactic acid bacteria fermentation and transformation, providing a new way for the preparation and acquisition of rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2.

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

[0006] The plant lactobacillus (Lactiplantibacillus plantarum) RET215 provided by the present invention has been deposited in the China Center for Type Culture Collection on May 21, 2024, and the deposit number is: CCTCC NO: M20241024.

[0007] The invention provides a use of a Lactobacillus plantarum RET215 in fermenting and transforming ginsenoside Re to prepare rare ginsenosides.

[0008] As a preferred embodiment, the rare ginsenosides are 20(R)-Rg2 and 20(S)-Rg2.

[0009] As a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 is as follows: the ginsenoside Re is hydrolyzed with glucose at the C-20 position under the action of Lactiplantibacillus plantarum RET215 to generate rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2.

[0010] The present invention provides a method for preparing rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 by using the strain Lactiplantibacillus plantarum RET215, which comprises the following steps:

[0011] Rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 were prepared by fermentation and transformation of ginsenoside Re by Lactobacillus plantarum RET215.

[0012] The beneficial effects of the present invention are:

[0013] The present invention screened and obtained a new lactobacillus strain RET215 that can bioconvert ginsenoside Re into rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2. The strain RET215 was identified as Lactobacillus plantarum.

[0014] The obtained Lactobacillus plantarum RET215 is preserved in the China Center for Type Culture Collection, abbreviated as CCTCC, with an address of: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center), with a preservation number of: CCTCC NO: M20241024, and a preservation date of: May 21, 2024.

[0015] The present invention utilizes a screened strain of Lactiplantibacillus plantarum RET215 to ferment and transform ginsenoside Re to prepare rare ginsenosides, wherein the obtained rare ginsenosides are 20(R)-Rg2 and 20(S)-Rg2. The method of utilizing Lactiplantibacillus plantarum RET215 to ferment and transform ginsenoside Re to prepare rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 can ensure that the fermentation product is mainly composed of rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2, has high specificity, and can be applied to ginseng deep-processing products that have unique requirements for rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2.

[0016] In addition, the preparation of rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 by fermenting and converting ginsenoside Re with Lactiplantibacillus plantarum RET215 has the characteristics of simple process. The preparation of rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 can be completed directly through a simple fermentation process, laying the foundation for the large-scale production of rare ginsenosides.

[0017] At the same time, the preparation of rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 by fermenting and transforming ginsenoside Re with Lactobacillus plantarum RET215 has the advantages of high conversion efficiency and high content of the obtained conversion products. Rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 have a wide range of uses and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 (A) is the HPLC chromatogram of ginsenoside Re before fermentation, (B) is the HPLC chromatogram of ginsenoside 20(S)-Rg2 standard, (C) is the HPLC chromatogram of ginsenoside 20(R)-Rg2 standard, and (D) is the HPLC chromatogram of 20(R)-Rg2 and 20(S)-Rg2 generated after fermentation.

[0019] Figure 2 The biotransformation roadmap of rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2. DETAILED DESCRIPTION

[0020] 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 making creative work are within the scope of protection of the present invention.

[0021] Example 1 Isolation, identification and preservation of strains

[0022] 1. Isolation of strains

[0023] In November 2021, sauerkraut samples were collected from the vegetable market in Tonghua City, Jilin Province. 0.1g of sauerkraut 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 morphology and appearance of the MRS solid culture medium plate were 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 isolates were inoculated into liquid MRS culture medium for culture, and after adding 60% glycerol, they were stored in a -80°C refrigerator. At the same time, one of the strains was named RET215.

[0024] 2. Identification of strains

[0025] The physiological and biochemical identification results of strain RET215 are as follows: Gram staining positive, catalase test negative, benzidine test negative, indole test negative, and acetylmethyl carbinol test positive; it does not hydrolyze starch, liquefy gelatin, or produce hydrogen sulfide, and ferments glucose to produce acid but not gas; it is a non-motile bacillus; it can grow at 15°C and 45°C, with the optimum growth temperature being 37-42°C; its suitable pH is 5.0-7.0; it tolerates 6.5% NaCl; it grows evenly turbidly in liquid MRS culture medium, and the bacteria present a white precipitate after long-standing storage.

[0026] The 16S rDNA sequence homology analysis process and results are as follows: extract the genomic DNA of strain RET215, use the primer pair consisting of 16sF and 16sR to perform PCR amplification, and then sequence the obtained amplification product, wherein the 16s rDNA sequence is shown in SEQ ID NO:1. The 16s rDNA sequence is subjected to homology comparison analysis in the GenBank library by the BLAST program, and the homology with Lactiplantibacillus plantarum reaches 100%. Therefore, according to the above identification results, the extracted strain RET215 is identified as Lactiplantibacillus plantarum.

[0027] Wherein, the primer pair sequence information is as follows:

[0028] 16sF: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0029] 16sR: 5′-AGAAAGGAGGTGATCCAGC-3′.

[0030] The PCR amplification conditions used are as follows:

[0031] Pre-denaturation: 95℃5min;

[0032] Cycle: denaturation at 1.95°C for 15 s;

[0033] Anneal at 2.55°C for 15 seconds;

[0034] 3.72℃ extension for 90s;

[0035] Steps 1-3 were repeated for 35 cycles;

[0036] Last extension: 72°C for 7 min.

[0037] 3. Preservation of strains

[0038] The plant lactobacillus (Lactiplantibacillus plantarum) RET215 of the present invention has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on May 21, 2024, with an address of: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center), and the preservation number is: CCTCC NO: M20241024.

[0039] Example 2: Fermentation of Ginsenoside Re by Lactobacillus plantarum RET215

[0040] 1. Preparation of Lactobacillus plantarum RET215 bacterial suspension

[0041] The obtained Lactobacillus plantarum RET215 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 and suspended with sterile distilled water to adjust the viable bacterial count to 1.0×10 9 CFU / mL, and obtain the bacterial suspension of Lactobacillus plantarum RET215.

[0042] 2. Fermentation conversion

[0043] The liquid fermentation medium contains 3g / L of glucose and 1g / L of yeast extract powder, and the pH is adjusted to 6.6 by hydrochloric acid, and sterilized at 121°C for 15min to obtain the product. Ginsenoside Re (purchased from Chengdu Aifa Biotechnology Co., Ltd., HPLC≥95%) is added to the liquid fermentation medium, wherein the amount of ginsenoside Re added is 1g / L, and a Lactobacillus plantarum RET215 bacterial suspension is inoculated at an inoculum volume of 10ml / L, and the product is fermented at 37°C for 15d. After the fermentation is completed, the fermentation product is concentrated under reduced pressure, and vacuum freeze-dried (-80°C, 5pa) to obtain the ginsenoside Re fermentation product.

[0044] Example 3 Identification of Ginsenoside Re Fermentation Products

[0045] 1. High performance liquid chromatography (HPLC) analysis

[0046] The ginsenoside Re fermentation product was dissolved in methanol and filtered through a 0.22 μm microporous filter membrane for HPLC chromatography analysis.

[0047] The specific operation process of the high performance liquid chromatography (HPLC) analysis method is as follows: the chromatographic column is Agilent pursuit5SB-C18 chromatographic column, the injection volume is 20 μL, the flow rate is 1 mL / min, the column temperature is 30°C, and the detection wavelength is 203 nm. The mobile phase is A: water, B: acetonitrile, 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).

[0048] 2. Identification results

[0049] The results of high performance liquid chromatography (HPLC) analysis were as follows Figure 1As shown, the retention time under high performance liquid chromatography (HPLC) conditions is consistent with that of the reference standards of ginsenoside 20(R)-Rg2 and 20(S)-Rg2 (purchased from Shanghai Yuanye Biotechnology Co., Ltd., HPLC ≥ 98%). This proves that ginsenoside Re can be fermented and transformed into rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 by Lactiplantibacillus plantarum RET215.

[0050] The biotransformation synthetic route for the fermentation of ginsenoside Re by the cell suspension of Lactiplantibacillus plantarum RET215 to prepare rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 is as follows Figure 2 shown: Ginsenoside Re is hydrolyzed at the C-20 glucose position under the action of Lactiplantibacillus plantarum RET215 to generate rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2.

[0051] The present invention discloses a strain of Lactiplantibacillus plantarum RET215 and its use in the fermentation and transformation of ginsenoside Re to prepare rare ginsenosides. Those skilled in the art can draw on the content of this article and appropriately modify 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 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 Lactobacillus plantarum RET215, characterized in that: It was deposited in the China Center for Type Culture Collection on May 21, 2024, with the deposit number: CCTCC NO: M20241024.

2. Use of the Lactobacillus plantarum RET215 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 are 20(R)-Rg2 and 20(S)-Rg2.

4. The use according to claim 3, characterized in that The biotransformation synthesis route of the rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 is as follows: the ginsenoside Re is hydrolyzed with glucose at the C-20 position under the action of Lactiplantibacillus plantarum RET215 to generate rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2.

5. A method for preparing rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 using the strain Lactiplantibacillus plantarum RET215 of claim 1, characterized in that: The following steps are involved: Rare ginsenosides 20(R)-Rg2 and 20(S)-Rg2 were prepared by fermentation and transformation of ginsenoside Re by Lactobacillus plantarum RET215.

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