A strain of Pediococcus acidilactici N67-2 and its use in fermentation and conversion of ginsenoside Rg1 to prepare rare ginsenosides

Abstract

A strain of Pediococcus acidilactici N67‑2 and its use in fermenting and transforming ginsenoside Rg1 to prepare rare ginsenosides, belonging to the field of probiotic fermentation. The Pediococcus acidilactici N67‑2 of the present invention has been deposited in the China Center for Type Culture Collection on September 18, 2024, with a deposit number of CCTCC NO: M 20241998. The present invention uses ginsenoside Rg1 as a raw material and utilizes Pediococcus acidilactici N67‑2 to ferment and transform to prepare rare ginsenosides 20(R)‑Rh1, 20(S)‑Rh1, Rh4 and Rk3. After fermentation, the ginsenoside monomer Rg1 can be completely transformed, and the method has the advantages of mild reaction, green environmental protection, low cost and high content of transformation products. The present invention provides a new method for preparing rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic fermentation, and particularly relates to a strain of Pediococcus acidilactici N67-2 and use thereof in fermenting and transforming ginsenoside Rg1 to prepare rare ginsenosides. Background Art

[0002] Ginseng (Panax ginseng C.A.Mey.) is a treasure among traditional Chinese herbal medicines. Its efficacy covers anti-fatigue, anti-tumor, hypoglycemic and anti-inflammatory effects. In-depth studies have found that rare ginsenosides show more significant biological activity than primary ginsenosides. However, the content of rare saponins in natural ginseng is very small, which is difficult to meet the growing industrial production and market demand. At present, the preparation technology of rare ginsenosides mainly includes acid hydrolysis, alkaline hydrolysis, heating hydrolysis and enzymatic hydrolysis. Although these methods have their own characteristics, the chemical preparation method is often accompanied by drastic reaction conditions, resulting in complex and changeable hydrolysis products that are difficult to accurately control. Although the enzymatic method has advantages in some aspects, it faces many challenges in the implementation process, such as the need for cumbersome purification of a large amount of crude enzymes, and the biological enzymes are very easy to inactivate, which greatly increases the cost of industrial production. In recent years, the technology of preparing rare ginsenosides by probiotic fermentation transformation has become one of the important ways to improve the biological activity of ginseng products.

[0003] As a widely recognized probiotic, lactic acid bacteria not only play an important role in food fermentation, probiotic product development and pharmaceutical manufacturing. Studies have shown that some types of lactic acid bacteria, such as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus acidophilus, can use ginsenosides as substrates and convert them into a series of rare ginsenosides with rare natural content, such as Rg6, Rh2, F2 and CK. These conversion products have significant value in the field of medicine. However, there are few reports on the biotransformation of ginsenosides by Pediococcus acidilactici. As a probiotic, Pediococcus acidilactici has multiple functional properties, including promoting intestinal health, preventing diseases, improving feed utilization and improving food quality. In addition, there are no reports on the preparation of rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 by fermenting ginsenoside Rg1 monomer by Pediococcus acidilactici. The present invention adopts high-yield β-glucosidase lactic acid Pediococcus N67-2, takes ginsenoside monomer Rg1 as substrate, and can completely transform ginsenoside monomer Rg1 after 14 days of fermentation to prepare rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3. The process is suitable for large-scale industrial production, and the prepared rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 have wide applications and broad application prospects. Summary of the invention

[0004] The present invention aims to provide a strain of Pediococcus acidilactici N67-2 and its use in fermenting and transforming ginsenoside Rg1 to prepare rare ginsenosides, and to provide a new method for preparing rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3.

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

[0006] The Pediococcus acidilactici N67-2 provided by the present invention has been deposited in the China Center for Type Culture Collection on September 18, 2024, with a deposit number of: CCTCC NO: M 20241998.

[0007] The present invention provides a use of a β-glucosidase-producing Pediococcus lactis N67-2 in fermenting and transforming ginsenoside Rg1 to prepare rare ginsenosides.

[0008] As a preferred embodiment, the rare ginsenosides include 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3.

[0009] As a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 is as follows: ginsenoside Rg1 is hydrolyzed with glucose at the C-20 position under the action of Pediococcus acidilactici N67-2 to generate rare ginsenosides 20(R)-Rh1 and 20(S)-Rh1, which are further dehydrated to generate rare ginsenosides Rh4 and RK3.

[0010] As a preferred embodiment, the use of a strain of Pediococcus acidilactici N67-2 provided by the present invention in fermenting and transforming ginsenoside Rg1 to prepare rare ginsenosides specifically comprises the following steps:

[0011] Pediococcus acidilactici N67-2 was prepared into a bacterial suspension and the bacterial suspension was diluted to 2×10 7The inoculum amount of CFU / ml is inoculated into the sterilized liquid fermentation medium, and ginsenoside Rg1 sterilized by microporous filter membrane is added at an addition amount of 0.5g / L, and anaerobically fermented at 37±0.5°C for 7-14 days. After the fermentation, the obtained fermentation product is extracted with water-saturated n-butanol, and then concentrated and freeze-dried under reduced pressure at low temperature, the residue is dissolved in methanol and centrifuged (10000rpm, 15min, 4°C), the supernatant is decompressed and the solvent is recovered, and then vacuum freeze-dried to obtain a dry product, which contains rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3.

[0012] 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.

[0013] As a preferred embodiment, the specific process of preparing Pediococcus acidilactici N67-2 into a bacterial suspension is as follows:

[0014] Pediococcus acidilactici N67-2 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 then suspended in sterile 0.85% saline solution to adjust the viable count to 1.0×10 7 ~1.0×10 8 CFU / ml, and obtain the Pediococcus acidilactici N67-2 bacterial suspension.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention screened out a lactic acid bacterium N67-2 that produces β-glucosidase and can bioconvert ginsenoside Rg1 into rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 from a sample of traditionally fermented cabbage made by farmers in Yanji City, Jilin Province. The strain N67-2 was identified as Pediococcus acidilactici and was deposited in the China Center for Type Culture Collection on September 18, 2024, with the deposit number: CCTCCNO: M 20241998. The invention determines a new process for preparing rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 by fermentation and transformation of Pediococcus acidilactici N67-2, and identifies the transformed rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 by HPLC chromatography analysis. The process utilizes the property of Pediococcus acidilactici N67-2 to produce β-glucosidase, takes ginsenoside Rg1 as raw material, and uses Pediococcus acidilactici N67-2 to ferment and transform to prepare rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3. At the same time, the transformed rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 are identified by HPLC chromatography. The results show that the retention time of the conversion product of ginsenoside Rg1 is consistent with that of the standards of rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3. The process has the advantages of mild reaction conditions, environmental friendliness, high conversion efficiency, etc., and has significant differences compared with the existing biotransformation process route. The process is suitable for large-scale industrial production, and the prepared rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 have a wide range of uses and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the color development diagram of β-glucosidase-producing Pediococcus lactis N67-2 on the screening culture medium.

[0018] Figure 2 HPLC chromatograms of ginsenoside Rg1 on days 0, 7, and 14 of fermentation.

[0019] Figure 3 The biotransformation synthesis route for the fermentation and transformation of Rg1 by Pediococcus acidilactici N67-2 to produce rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and RK3. 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 of strains

[0022] In April 2024, the cabbage samples were selected from the homemade traditional fermented cabbage samples from farmers in Yanji City, Jilin Province. Dip the kimchi stock solution and inoculate it on the MRS medium plate containing bromocresol purple by the plate streak method. Incubate it at 37°C for 48 hours, pick the colonies that can make bromocresol purple turn yellow according to the morphology of single colonies for microscopic examination, select the strains that are Gram-positive, incubate at 37°C for 24 hours, and continue streaking inoculation until the pure isolate is isolated and purified. Further inoculate on the esculin screening medium (used to isolate strains that produce β-glucosidase, add esculin 1g / L and ferric citrate 0.5g / L to MRS agar, and autoclave at 121°C for 20min), the strains are surrounded by reddish brown to dark brown circles, which are positive strains (such as Figure 1 The pure cultured strain was inoculated into liquid MRS medium for further culture, and after adding 80% glycerol, it was stored in a -80°C refrigerator. One of the lactic acid bacteria was named N67-2.

[0023] Example 2 Identification of strains

[0024] 1. The physiological and biochemical identification results of strain N67-2 are as follows:

[0025] Gram staining is positive. Among 11 carbohydrate patterns, esculin, cellobiose, salicin and 1% sodium hippurate are positive. However, maltose, sucrose, mannitol, 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.

[0026] 2. Molecular biological identification and results are as follows:

[0027] The target strain was inoculated in fresh MRS liquid culture medium and cultured for 24 hours. The kit of Tiangen Biochemical Technology Co., Ltd. was used to extract bacterial DNA and amplify the 16S rDNA sequence. The primers used were 1492R and 27F, which were species-universal primers. The electrophoresis results of the 16S rDNA PCR product of strain N67-2 showed that a band with good specificity was obtained at a molecular weight of about 1500bp, 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 sequence was compared with the 16S rDNA gene sequence of some strains that have been logged on the website http: / / www.ncbi.nlm.nih.gov. The results showed that the homology between strain N67-2 and Pediococcus acidilactici NGRI 0510Q (NR 041640.1) reached 100%. According to the above results, strain N67-2 was identified as Pediococcus acidilactici.

[0028] Wherein, the primer pair sequence information is as follows:

[0029] 27sF: 5′-agagttgatcctggctcag-3′;

[0030] 1492R: 5′-ggttaccttgttacgactt-3′.

[0031] 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 storage at 4°C.

[0032] Example 3 Preservation of strains

[0033] The Pediococcus acidilactici N67-2 of the present invention has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on September 18, 2024. 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 20241998.

[0034] Example 4 Preparation of Pediococcus acidilactici N67-2 bacterial suspension

[0035] The obtained Pediococcus acidilactici N67-2 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 sterilized 0.85% physiological saline solution and the viable cell count was adjusted to 1.0×10 9 CFU / mL, and obtain the Pediococcus acidilactici N67-2 bacterial suspension.

[0036] Example 5 Fermentation and transformation of ginsenoside Rg1 by Pediococcus acidilactici N67-2 to prepare rare ginsenosides

[0037] 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 15 min.

[0038] 2. Fermentation transformation: Ginsenoside Rg1 (purchased from Chengdu Aifa Biotechnology Co., Ltd., HPLC ≥ 98%) was added to the liquid fermentation medium, wherein the addition amount of ginsenoside Rg1 was 0.5 g / L, and the medium was sterilized by filtration with a 0.22 μM microporous filter membrane, and the mixture was heated to 2 × 10 7 CFU / mL inoculation amount inoculated lactic acid Pediococcus acidilactici N67-2 bacterial suspension, 37 ℃ anaerobically fermented for 14 days. After the fermentation, the fermentation product was extracted three times with water-saturated n-butanol, the fermentation product was concentrated under reduced pressure at low temperature (50 ℃) and vacuum freeze-dried (-80 ℃, 5 pa), and the product was extracted with methanol, centrifuged (10000 rpm, 15 min, 4 ℃), the supernatant was vacuum-freeze-dried (-80 ℃, 5 pa) after decompression recovery of solvent, and the dry product was obtained, which contained rare ginsenosides. The dry product contains the rare ginsenosides 20 (R) -Rh1, 20 (S) -Rh1, Rh4 and Rk3.

[0039] Example 6 Identification of rare ginsenosides by high performance liquid chromatography (HPLC)

[0040] Detection of rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 by high performance liquid chromatography (HPLC): Ginsenoside Rg1 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), and gradient elution was performed 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] In the process of preparing rare ginsenosides 20(R)-Rh1 and 20(S)-Rh1, Rk3 and Rh4 by fermentation transformation of Pediococcus acidilactici N67-2 bacterial suspension, the biotransformation synthesis route is as follows: Figure 2 As shown: Under the action of Pediococcus acidilactici N67-2, ginsenoside Rg1 hydrolyzes glucose at the C-20 position to produce rare ginsenosides 20(R)-Rh1 and 20(S)-Rh1, and further dehydrates to produce rare ginsenosides Rh4 and RK3.

[0042] HPLC chromatographic identification results are as follows Figure 3 As shown in the figure, through the qualitative and quantitative analysis of the ginsenoside components in the products before and after fermentation, it can be seen that the retention time of the conversion product of ginsenoside Rg1 is consistent with that of the rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 standards. This proves that ginsenoside Rg1 can be converted into rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 by fermentation with β-glucosidase-producing Pediococcus acidilactici N67-2. This proves that ginsenoside Rg1 can be converted into rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 by fermentation with Pediococcus acidilactici N67-2.

[0043] The present invention discloses a strain of Pediococcus acidilactici N67-2 and its use in fermenting and transforming ginsenoside Rg1 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 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. Pediococcus acidilactici N67-2, characterized in that This strain was deposited in the China Center for Type Culture Collection on September 18, 2024, with the deposit number: CCTCC NO: M 20241998.

2. The use of Pediococcus acidilactici N67-2 as claimed in claim 1 in the fermentation and conversion of ginsenoside Rg1 to prepare rare ginsenosides, characterized in that: The rare ginsenosides are 20(R)-Rh1, 20(S)Rh1, Rh4 and Rk3.

3. The use according to claim 2, characterized in that: The biotransformation synthesis route of the rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3 is as follows: ginsenoside Rg1 is hydrolyzed with glucose at the C-20 position under the action of Pediococcus acidilactici N67-2 to generate rare ginsenosides 20(R)-Rh1 and 20(S)-Rh1, which are further dehydrated to generate rare ginsenosides Rh4 and RK3.

4. The use according to claim 2, characterized in that: The following steps are involved: Pediococcus acidilactici N67-2 was prepared into a bacterial suspension and the bacterial suspension was diluted to 2×10 7 The inoculum amount of CFU / ml is inoculated into the sterilized liquid fermentation medium, and ginsenoside Rg1 sterilized by microporous filter membrane is added at an addition amount of 0.5g / L, and anaerobically fermented at 37±0.5°C for 7-14 days. After the fermentation, the obtained fermentation product is extracted with water-saturated n-butanol, and then concentrated and freeze-dried under reduced pressure at low temperature, and the residue is dissolved in methanol and centrifuged at 10000rpm, 15min, 4°C, and the supernatant is decompressed and the solvent is recovered and then vacuum freeze-dried to obtain a dry product, which contains rare ginsenosides 20(R)-Rh1, 20(S)-Rh1, Rh4 and Rk3.

5. The use according to claim 4, characterized in that The specific process of preparing Pediococcus acidilactici N67-2 into a bacterial suspension is as follows: Pediococcus acidilactici N67-2 is inoculated into a liquid MRS medium, anaerobically cultured at 37°C for 24-48 hours, centrifuged at 4°C and 4000-8000 rpm for 5-10 minutes, and then the bacterial precipitate is collected and suspended in a sterilized 0.85% saline solution to adjust the viable bacterial count to 1.0×10 7 ~1.0×10 8 CFU / ml, and obtain the bacterial suspension of Pediococcus acidilactici N67-2.

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