Pediococcus acidilactici N6-3 and its application in fermentation and conversion of ginsenoside Rb1 to prepare rare ginsenosides

Abstract

The invention relates to the field of probiotic fermentation of Pediococcus acidilactici N6‑3 and its application in fermenting and transforming ginsenoside Rb1 to prepare rare ginsenosides. Pediococcus acidilactici N6‑3 was deposited in the China Center for Type Culture Collection on September 18, 2024, with a deposit number of CCTCC NO:M 20241999. The self-isolated and identified high-yielding β-glucosidase Pediococcus acidilactici N6‑3 of the present invention can be used for fermenting and transforming ginsenoside monomer Rb1. In the process of preparing rare ginsenosides, the present invention does not need to be combined with alkaline hydrolysis, high temperature and high pressure, steam treatment and other treatment methods, and has the advantages of mild reaction, green environmental protection, low cost and short fermentation time. After fermentation, ginsenoside monomer Rb1 can be completely converted into rare ginsenosides 20(R)‑Rg3, 20(S)‑Rg3, Rk1 and Rg5, providing a new way for Pediococcus acidilactici to ferment and transform ginsenoside Rb1 to prepare rare ginsenosides.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic fermentation, and specifically to a strain of Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 and its application in the fermentation and conversion of ginsenoside Rb1 to prepare rare ginsenosides. Background Art

[0002] Ginseng( Panax ginseng C am ey.) is a traditional Chinese herbal medicine with unique characteristics in my country. Pharmacological studies have shown that ginseng has anti-fatigue, anti-tumor, hypoglycemic and anti-inflammatory properties. Research reports have found that rare ginsenosides have more obvious biological activity than primary ginsenosides, but the content of rare saponins naturally produced in ginseng is extremely low, which is difficult to meet the needs of industrial production and market products. At present, the preparation methods of rare ginsenosides mainly include: acid hydrolysis, alkaline hydrolysis, heating hydrolysis and enzymatic hydrolysis. The reaction conditions for preparation by chemical methods are severe, and the hydrolysis products are complex and diverse and not easy to control. Although the enzymatic method has certain advantages, a large amount of crude enzymes need to be operated and purified during the enzyme reaction process, and the biological enzymes are very easy to inactivate, which greatly increases the industrial cost. Ginsenosides are a class of heat-sensitive substances, and high-temperature and high-pressure steam treatment is very likely to cause structural changes or degradation, thereby affecting its biological activity and pharmacological effects. In recent years, the use of probiotic fermentation to transform primary saponin components in ginseng has not only strong specificity, high yield, but also high safety, and has become one of the important ways to improve the biological activity of ginseng products.

[0003] As a probiotic, Pediococcus acidilactici has multiple functional properties, including promoting intestinal health, preventing diseases, and improving food quality. It is a recommended strain in the "List of Bacteria that Can Be Used in Food". At present, there are few reports on the biotransformation of ginsenosides by Pediococcus acidilactici. Only one patent, "Pediococcus acidilactici BT425 and its application in the preparation of rare ginsenosides by fermentation transformation combined with high temperature and high pressure steam treatment", was found. The difference between the present invention and the above patents is that 1) the fermentation substrate is different: the above patent uses total ginsenosides as the fermentation substrate. The total ginsenosides have complex components, and primary ginsenosides can also be converted into rare ginsenosides after high-temperature and high-pressure steam treatment, which makes it impossible to clearly define the transformation pathway of ginsenosides, which is not conducive to industrial production. The present invention uses ginsenoside monomer Rb1 as the fermentation substrate, has a clear transformation pathway, and can completely transform ginsenoside monomer Rb1 to prepare rare ginsenosides, which is more conducive to later industrial purification; 2) Different fermentation methods: In the comparative patent, the acid production performance of lactic acid bacteria is combined with high-temperature and high-pressure steam treatment to prepare ginseng rare saponins. Due to the differences between strains, the present invention uses high-yield β-glucosidase lactic acid Pediococcus N6-3, which can completely transform ginsenoside monomer Rb1 without combining with high-temperature and high-pressure steam treatment, and has the advantages of green environmental protection, low cost, and low energy consumption. In summary, the present invention is different from the existing reported patents. For the first time, the high-yield β-glucosidase-producing Pediococcus lactis N6-3 was used to ferment and transform the primary ginsenoside Rb1 monomer to prepare rare ginsenosides. This process is suitable for large-scale industrial production, and the prepared rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 have wide applications and broad application prospects. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a strain of β-glucosidase-producing Pediococcus acidilactici N6-3 and its application in fermenting and converting ginsenoside Rb1 to prepare rare ginsenosides. The present invention uses ginsenoside Rb1 as a substrate and utilizes the screened Pediococcus acidilactici ( Pediococcus acidilactici )N6-3 was fermented and converted to prepare rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The Pediococcus acidilactici N6-3 provided by the present invention has been deposited in the China Center for Type Culture Collection on September 18, 2024, with the deposit number: CCTCC NO: M 20241999.

[0007] The β-glucosidase-producing Pediococcus lactis provided by the present invention ( Pediococcus acidilactici)Application of N6-3 in the fermentation and conversion of ginsenoside Rb1 to prepare rare ginsenosides.

[0008] As a preferred embodiment, the rare ginsenosides include 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5.

[0009] As a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides is: Pediococcus acidilactici ( Pediococcus acidilactici )N6-3 hydrolyzes the glucose at the C-20 position of ginsenoside Rb1 to generate ginsenoside Rd, and then removes one glucose to generate rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, which are then dehydrated to generate rare ginsenosides Rk1 and Rg5.

[0010] As a preferred embodiment, the present invention provides Pediococcus acidilactici ( Pediococcus acidilactici )The application of N6-3 in fermentation and conversion of ginsenoside Rb1 to prepare rare ginsenosides mainly includes the following steps:

[0011] Step 1: Preparation of Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 bacterial suspension;

[0012] Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 was inoculated into liquid MRS medium and cultured anaerobically at 37-39°C for 18-24 h. The culture was centrifuged at 8000-12000 rpm for 5-10 min at 4-10°C. The bacterial precipitate was collected and suspended in sterile 0.85% saline solution to adjust the viable count to 1.0×10 8 ~1.0×10 9 CFU / ml, obtained Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 bacterial suspension;

[0013] Step 2: Fermentation conversion;

[0014] Prepare liquid fermentation medium, add ginsenoside Rb1 (0.5 mg / mL) and add 2×10 7 CFU / mL inoculation volume inoculated with Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 bacterial suspension, anaerobically fermented at 37°C for 7 to 14 days; after the fermentation, the fermentation product was extracted with water-saturated n-butanol, then concentrated under reduced pressure and freeze-dried at low temperature, the residue was dissolved in methanol and centrifuged (10000rpm, 15min, 4°C), the supernatant was decompressed and the solvent was recovered, then vacuum freeze-dried to obtain a dry product, which contained rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5.

[0015] 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.

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

[0017] In order to achieve biotransformation to prepare rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5, the present invention first screened a strain N6-3 from a sample of traditional fermented spicy cabbage made by farmers in Yanji, and identified it as Pediococcus acidilactici. It was deposited in the China Center for Type Culture Collection on September 18, 2024, with the preservation number: CCTCC NO:M 20241999. Taking ginsenoside Rb1 as raw material and utilizing the β-glucosidase-producing property of Pediococcus acidilactici N6-3, the ginsenoside monomer Rb1 was transformed by biofermentation and rare ginsenosides 20(R)-Rg3, 20(S)-Rg3 and Rg5 were obtained. The transformed rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 were identified by HPLC chromatography. The results showed that the retention time of the conversion product of ginsenoside Rb1 was consistent with that of the rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 standards. Therefore, the fermentation and conversion of ginsenoside Rb1 by the β-glucosidase-producing Pediococcus acidilactici N6-3 of the present invention can obtain high-content rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5. This method has the advantages of mild reaction, low cost and safe process, and the prepared rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 are widely used and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the color development diagram of Pediococcus acidilactici N6-3 on β-glucosidase screening medium.

[0019] Figure 2 HPLC chromatograms of ginsenoside Rb1 on days 0, 7, and 14 after fermentation by Pediococcus acidilactici N6-3.

[0020] Figure 3 It is a biotransformation synthesis route for converting Rb1 into Rd and then into rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5. DETAILED DESCRIPTION

[0021] The Pediococcus acidilactici N6-3 provided by the present invention has been deposited in the China Center for Type Culture Collection on September 18, 2024, with the deposit number: CCTCC NO: M 20241999.

[0022] The present invention provides an application of β-glucosidase-producing Pediococcus acidilactici N6-3 in fermenting and transforming ginsenoside Rb1 to prepare rare ginsenosides, which mainly comprises the following steps:

[0023] Step 1: Preparation of Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 bacterial suspension;

[0024] Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24 h. The bacterial precipitate was collected and suspended in sterile 0.85% saline solution to adjust the viable bacterial count to 1.0 × 10 8 ~1.0×10 9 CFU / ml, obtained Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 bacterial suspension;

[0025] Step 2: Fermentation conversion;

[0026] Liquid fermentation medium was prepared, which included 1 g / L glucose, 1 g / L tryptone, 0.5 g / L yeast extract and 1 g / L sodium chloride; pH 6.0-7.0; ginsenoside Rb1 (0.5 mg / mL) was added to the liquid fermentation medium, and 2×10 7 CFU / mL inoculation volume inoculated with Pediococcus acidilactici ( Pediococcus acidilactici ) N6-3 bacterial suspension, anaerobically fermented at 37°C for 7 to 14 days; after the fermentation, the fermentation product was extracted with water-saturated n-butanol, then concentrated under reduced pressure and freeze-dried at low temperature, the residue was dissolved in methanol and centrifuged (10000rpm, 15min, 4°C), the supernatant was decompressed and the solvent was recovered, then vacuum freeze-dried to obtain a dry product, which contained rare ginsenosides.

[0027] Among them, rare ginsenosides mainly include 20(R)-Rg3, 20(S)-Rg3, Rg5 and a small amount of Rk1.

[0028] The biotransformation synthesis route of rare ginsenosides is as follows: the ginsenoside Rb1 is synthesized in Pediococcus acidilactici ( Pediococcus acidilactici)N6-3 hydrolyzes the glucose at the C-20 position of ginsenoside Rb1 to generate primary ginsenoside Rd, and then removes one glucose to generate rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, which are then dehydrated to generate rare ginsenosides, Rk1 and Rg5.

[0029] 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.

[0030] Example 1 Isolation, identification and preservation of strains

[0031] 1. Isolation of strains

[0032] The sample used for isolating the strain was a sample of traditional fermented kimchi collected from a farmer's home in Yanji City, Jilin Province in April 2023. The collected traditional fermented kimchi samples were ground, and then diluted step by step with sterile saline in multiples of 10 to obtain dilutions; each level of dilution was spread on an MRS medium plate supplemented with bromocresol purple, and cultured at 37°C for 48 hours. According to the morphology of single colonies, colonies that could turn bromocresol purple yellow were picked for microscopic examination, and strains with positive Gram staining were selected. After repeated streaking and purification, the strain was further inoculated into aesculin screening medium (used to isolate strains producing β-glucosidase, 1 g / L aesculin and 0.5 g / L ferric citrate were added to MRS agar and sterilized under high pressure at 121°C for 20 minutes). The strains were surrounded by reddish brown to dark brown circles, which were positive strains (such as Figure 1 The pure cultured bacteria were inoculated into liquid MRS medium for further culture, and after adding 80% glycerol, they were stored in a -80°C refrigerator. One of the lactic acid bacteria was named N6-3.

[0033] 2. Identification of strains

[0034] (1) Physiological and biochemical identification results of strain N6-3: Gram staining is positive, and it can utilize esculin, cellobiose, salicin, sucrose and 1% sodium hippurate, and it is negative in maltose, mannitol, sorbitol, raffinose, inulin and lactose. 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.

[0035] (2) Molecular biological identification: The target strain was inoculated in fresh MRS liquid medium and cultured for 24 h. 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 the species-universal primers composed of 1492R and 27F (1492r: 5′-ggttaccttgttacgactt-3′; 27f: 5′-agagttgatcctggctcag-3′) for PCR amplification. The PCR amplification conditions were: pre-denaturation: 94℃ 2min; denaturation: 94℃ 30s, annealing: 55℃ 30s, extension: 72℃ 30s, 30 cycles in total; final extension: 72℃ 2min, stored at 4℃. The electrophoresis results of the 16S rDNA PCR product of strain N6-3 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 is 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 strain N6-3 was similar to Pediococcus acidilactici. Pediococcus acidilactici The homology of NGRI 0510Q (NR 041640.1) reached 100%. Based on the above results, the strain N6-3 was identified as belonging to Pediococcus acidilactici ( Pediococcus acidilactici ).

[0036] 3. Preservation of strains

[0037] The Pediococcus acidilactici N6-3 of the present invention has been deposited in the China Center for Type Culture Collection on September 18, 2024, abbreviated as CCTCC NO: M 20241999, and 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 20241999.

[0038] Example 2 Using Pediococcus acidilactici ( Pediococcus acidilactici )N6-3 fermentation conversion of ginsenoside Rb1 to prepare rare ginsenosides

[0039] 1. Pediococcus acidilactici ( Pediococcus acidilactici ) Preparation of N6-3 bacterial suspension

[0040] Pediococcus acidilactici ( Pediococcus acidilactici) N6-3 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24 h. The culture was centrifuged at 4°C and 12,000 rpm for 10 min, and the bacterial precipitate was collected. The culture was suspended in sterile 0.85% saline solution and the viable count was adjusted to 1.0 × 10 9 CFU / mL, obtained Pediococcus acidilactici ( Pediococcus acidilactici )N6-3 bacterial suspension.

[0041] 2. Prepare ginsenoside Rb1

[0042] Commercially available ginsenoside Rb1 was purchased from Chengdu Aifa Biotechnology Co., Ltd., and its HPLC purity was greater than 98%.

[0043] 3. Fermentation conversion

[0044] Liquid fermentation medium was prepared, including 1 g / L glucose, 1 g / L tryptone, 0.5 g / L yeast extract powder and 1 g / L sodium chloride, pH 6.6, and sterilized at 121°C for 20 min. In the liquid fermentation medium, ginsenoside Rb1 was added in an amount of 0.5 mg / mL, filtered and sterilized with a 0.22 μM microporous filter membrane, and then 2 × 10 7 CFU / mL inoculation volume inoculated with Pediococcus acidilactici ( Pediococcus acidilactici )N6-3 bacterial suspension, anaerobic fermentation at 37℃ for 14 days. After the fermentation, the fermentation product was extracted three times with water-saturated n-butanol, and the fermentation product was concentrated under reduced pressure at low temperature (50℃) and vacuum freeze-dried (-80℃, 5pa). The product was extracted with methanol, centrifuged (10000rpm, 15min, 4℃), and the supernatant was vacuum-freezed and recovered by solvent after vacuum freeze-drying (-80℃, 5pa) to obtain a dry product containing rare ginsenosides. The dry product contains rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rg5 and a small amount of Rk1.

[0045] Identification of 3 rare ginsenosides

[0046] Detection of rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 by high performance liquid chromatography (HPLC): Ginsenoside Rb1 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.

[0047] The HPLC chromatographic analysis method was as follows: 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.

[0048] 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 ginsenoside Rb1 conversion product is consistent with that of rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 standards. This proves that ginsenoside Rb1 can be transformed into β-glucosidase-producing Pediococcus lactis ( Pediococcus acidilactici )N6-3 is fermented and converted into rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rg5 and a small amount of Rk1. And, Figure 3 As shown, in the use of Pediococcus acidilactici ( Pediococcus acidilactici )The biotransformation synthesis route for preparing rare ginsenosides by fermentation transformation of N6-3 is as follows: Ginsenoside Rb1 is hydrolyzed by Pediococcus acidilactici N6-3 at the C-20 position of glucose to generate primary ginsenoside Rd, and then one glucose is removed to generate rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, which are then dehydrated to generate rare ginsenosides Rk1 and Rg5.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Pediococcus acidilactici N6-3, characterized in that It was deposited in the China Center for Type Culture Collection on September 18, 2024, with the deposit number: CCTCC NO:M 20241999.

2. The use of Pediococcus acidilactici N6-3 as claimed in claim 1 in the fermentation and conversion of ginsenoside Rb1 to prepare rare ginsenosides, characterized in that: The Pediococcus acidilactici has the characteristic of producing beta-glucosidase, and the rare ginsenosides are 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5.

3. The use according to claim 2, characterized in that: The biotransformation synthesis route of the rare ginsenosides is as follows: the ginsenoside Rb1 is hydrolyzed by Pediococcus acidilactici N6-3 to form the primary ginsenoside Rd, and then one glucose is removed to form the rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, which are then dehydrated to form the rare ginsenosides Rk1 and Rg5.

4. The use according to claim 2, characterized in that: The following steps are involved: Step 1: preparing a Pediococcus acidilactici N6-3 bacterial suspension; Pediococcus acidilactici N6-3 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24 h. The bacterial precipitate was collected and suspended in sterile 0.85% saline solution to adjust the viable bacterial count to 1.0×10 9 CFU / mL, obtain the Pediococcus acidilactici N6-3 bacterial suspension; Step 2: Fermentation conversion; Prepare liquid fermentation medium, add 0.5 mg / mL ginsenoside Rb1, and 7 The method comprises the following steps: inoculating a suspension of Pediococcus acidilactici N6-3 at an inoculum amount of 1.4477 CFU / mL and anaerobically fermenting the suspension at 37°C for 7 to 14 days; extracting the obtained fermentation product with water-saturated n-butanol, then concentrating under reduced pressure at low temperature and freeze-drying the product; dissolving the residue in methanol and centrifuging the product at 10,000 rpm, 15 min, and 4°C; recovering the solvent from the supernatant under reduced pressure and performing vacuum freeze-drying to obtain a dry product, which contains rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5.

Images