Lactobacillus clavuliformis CY-06 and application thereof in preparing rare ginsenoside by fermenting and converting ginsenoside Re

Abstract

The invention relates to a strain of Lactobacillus corynosus CY-06 and application thereof in preparation of rare ginsenoside by fermentation and conversion of ginsenoside Re, and belongs to the field of microbial fermentation, the Lactobacillus corynosus CY-06 is preserved in China Center for Type Culture Collection on February 25, 2025, and the preservation number is CCTCC NO: M2025299. The Lactobacillus corynosus CY-06 is preserved in China Center for Type Culture Collection on February 25, 2025. The lactobacillus coryniformis CY-06 can be used for fermenting and converting ginsenoside Re, so that the rare ginsenoside can be prepared, and the lactobacillus coryniformis CY-06 can be used for preparing the rare ginsenoside. According to the invention, ginsenoside Re is used as a raw material, and the rare ginsenosides Rg2, Rh1, Rg6 and F4 are prepared by fermentation and conversion of Lactobacillus corynosus CY-06. The invention further provides a preparation method of the rare ginsenosides Rg2, Rh1, Rg6 and F4. After fermentation, ginsenoside Re can be completely converted into Rg2, Rh1, Rg6 and F4, methods such as alkaline hydrolysis, high temperature and high pressure, steam treatment and the like are not needed, and the method has the advantages of being mild in reaction, environmentally friendly, low in cost and high in conversion product content. The invention provides a new way for the directional synthesis path of rare ginsenosides Rg2, Rh1, Rg6 and F4, and the medicinal research and development and industrialization conversion values of the rare ginsenosides are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to the use of a strain of Loigolactobacillus coryniformis CY-06 in the fermentation conversion of ginsenoside Re to prepare rare ginsenosides. Background Art

[0002] Ginseng (Panax ginseng C.A.Mey.), as a medicinal plant of the Araliaceae family, its key pharmacodynamic ingredient, ginsenoside, has biological activities such as relieving physical fatigue, regulating glucose and lipid metabolism, and immune function. Compared with primary ginsenosides, rare ginsenosides are more likely to penetrate biological membranes due to their lipophilic characteristics to exert anti-tumor, immunomodulatory and other effects, but it is difficult to obtain them on a large scale due to their extremely low natural content. Currently, industrial preparation mainly relies on acid-base hydrolysis and thermal cracking technologies, but the high-temperature and high-pressure environment is prone to cause the cleavage of the saponin skeleton and the inactivation of functional groups. Although the traditional enzymatic method can cleave sugar chains directionally, it faces application bottlenecks such as the difficulty in purifying enzyme preparations and high operating costs. Although the intestinal flora can catalyze the conversion of ginsenosides, the heterogeneity of the individual microbiome leads to insufficient stability of the biotransformation pathway. Based on the microbial in vitro fermentation technology, by simulating the intestinal metabolism mechanism, precise regulation of glycosyl hydrolysis can be achieved at normal temperature and pressure. Among them, the probiotic fermentation technology shows its unique advantages: its substrate-specific recognition ability can selectively retain the active nucleus of ginsenoside, avoiding the generation of common by-products in chemical methods; the environmentally friendly process does not require the addition of toxic reagents, significantly reducing the pressure of "three wastes" treatment. Compared with the traditional enzymatic hydrolysis process, this technology through the synergistic action of intracellular enzyme systems of microorganisms not only maintains the structural integrity of saponins but also improves the bioavailability of fermentation products. This economically feasible biocatalytic mode provides a new path for the development of high-purity rare saponin preparation.

[0003] Loigolactobacillus coryniformis is a microorganism with extremely strong ecological adaptability. It is widely distributed and can be isolated from fermented foods, animal intestines, and natural environments. A large number of literature have confirmed that Loigolactobacillus coryniformis exhibits potential probiotic functions in many aspects such as acid resistance, bile salt resistance, adhesiveness, antioxidant activity, antibacterial activity, anti-inflammatory activity, and lipid-lowering effect, and is expected to be widely used in the fields of food, medicine, etc. However, it is worth noting that although Loigolactobacillus coryniformis shows probiotic characteristics in many aspects, there is no report on its application in the biotransformation of ginsenosides. Therefore, in this invention, Loigolactobacillus coryniformis strain CY-06 was first used to carry out directional fermentation with ginsenoside Re as the substrate. After 14 days of biotransformation, the biotransformation to rare ginsenosides of the Rg2, Rg6, F4, and Rh1 series was successfully achieved. This 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 have broad market prospects in the fields of treating cardiovascular diseases, developing functional foods, etc. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of Loigolactobacillus coryniformis CY-06 in the fermentation and transformation of ginsenoside Re to prepare 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] Loigolactobacillus coryniformis CY-06 provided by the present invention was deposited at the China Center for Type Culture Collection on February 25, 2025, and the deposit number is: CCTCC NO: M 2025299, and the deposit address is: Wuhan, China.

[0007] The use of a β-glucosidase-producing Loigolactobacillus coryniformis CY-06 provided by the present invention in the fermentation and transformation of ginsenoside Re to prepare rare ginsenosides.

[0008] As a preferred embodiment, the rare ginsenosides include Rg2, Rg6, F4, and Rh1.

[0009] As a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides Rg2, Rg6, F4, and Rh1 is as follows: Ginsenoside Re is hydrolyzed at the C-20 position of glucose under the action of Loigolactobacillus coryniformis CY-06 to generate the rare ginsenoside Rg2, which is further dehydrated to generate the rare ginsenoside F4 and Rg6. At the same time, the rare ginsenoside Rg2 can remove a rhamnose at the C-6 position to generate the rare ginsenoside Rh1.

[0010] As a preferred embodiment, the present invention provides the use of a strain of Loigolactobacillus coryniformis CY-06 in the fermentation conversion of ginsenoside Re to prepare rare ginsenosides, which specifically includes the following steps:

[0011] Dissolve ginsenoside Re in 2 mL of dimethyl sulfoxide, heat it in a water bath at 50 °C for 10 min, add it to the sterilized liquid fermentation medium according to the addition amount of 0.5 g / L, and then sterilize it through a microporous filter membrane. Prepare a bacterial suspension of Loigolactobacillus coryniformis CY-06, and inoculate the bacterial suspension into the liquid fermentation medium added with ginsenoside Re according to the inoculation amount of 2×10 7 CFU / ml, and perform anaerobic fermentation at 37 ± 0.5 °C for 7 to 14 days; after the fermentation is completed, the obtained fermentation product is extracted with water-saturated n-butanol, followed by low-temperature (50 °C) vacuum concentration and freeze-drying (-80 °C, 5 Pa). The residue is dissolved in methanol and centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatant is vacuum-concentrated to recover the solvent and then vacuum freeze-dried (-80 °C, 5 Pa) to obtain a dry product, which contains dry products of rare ginsenosides Rg2, Rh1, Rg6, and F4.

[0012] As a preferred embodiment, the liquid fermentation medium includes: 1 g / L of glucose, 1 g / L of tryptone, 0.5 g / L of yeast extract powder, and 1 g / L of sodium chloride; pH 6.0 - 7.0.

[0013] As a preferred embodiment, the specific process of preparing a bacterial suspension of Loigolactobacillus coryniformis CY-06 is as follows:

[0014] Inoculate Loigolactobacillus coryniformis CY-06 into liquid MRS medium, anaerobically culture at 37 °C for 24 - 48 h, centrifuge at 4 °C and 8000 - 12000 rpm for 5 - 10 min, then collect the cell precipitate, suspend it with sterile PBS, and adjust the viable cell count to 1.0×10 7 ~1.0×10 8 CFU / ml to obtain a Loigolactobacillus coryniformis CY-06 bacterial suspension.

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

[0016] One strain of Loigolactobacillus coryniformis CY-06 that produces β-glucosidase and can biotransform ginsenoside Re into rare ginsenosides Rg2, Rh1, Rg6, and F4 was screened from a sample of traditional fermented pickled Chinese cabbage made by farmers in Jilin City, Jilin Province. At the same time, this strain CY-06 was identified and the result was Loigolactobacillus coryniformis. It was deposited in the China Center for Type Culture Collection on February 25, 2025, and the deposit number is: CCTCC NO: M 2025299. The present invention determined a new process for fermenting and transforming ginsenoside Re with Loigolactobacillus coryniformis CY-06 to prepare rare ginsenosides Rg2, Rh1, Rg6, and F4. At the same time, the transformed rare ginsenosides Rg2, Rh1, Rg6, and F4 were identified by HPLC chromatographic analysis. This process utilizes the property of Loigolactobacillus coryniformis CY-06 to produce β-glucosidase, uses ginsenoside Re as the raw material, and ferments and transforms with Loigolactobacillus coryniformis CY-06 to prepare rare ginsenosides Rg2, Rh1, Rg6, and F4. At the same time, the transformed rare ginsenosides Rg2, Rh1, Rg6, and F4 were identified by HPLC chromatographic analysis. The results showed that the retention times of the transformation products of ginsenoside Re were consistent with those of the standard products of rare ginsenosides Rg2, Rh1, Rg6, and F4. This process has the advantages of mild reaction conditions, environmental friendliness, high conversion efficiency, etc., and has significant differences compared with the existing biological transformation process routes. This process is suitable for large-scale industrial production, and the prepared rare ginsenosides Rg2, Rh1, Rg6, and F4 have a wide range of uses and broad application prospects. Brief Description of the Drawings

[0017] Figure 1 Chromogenic map of Loigolactobacillus coryniformis CY-06 with high β-glucosidase production on the screening medium.

[0018] Figure 2 HPLC chromatograms of ginsenoside Re on the 0th and 14th days of fermentation; among them, a is the HPLC chromatogram of ginsenoside Re on the 0th day of fermentation, and b is the HPLC chromatogram of ginsenoside Re on the 14th day of fermentation.

[0019] Figure 3 Biotransformation synthesis route for Loigolactobacillus coryniformis CY-06 to ferment and transform ginsenoside Re to produce rare ginsenosides Rg2, Rh1, Rg6, and F4. Detailed Embodiments

[0020] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] I. Isolation of Strains

[0022] A sauerkraut sample made by farmers in Jilin City, Jilin Province, and self-made through traditional fermentation in November 2024 was selected. The original sauerkraut liquid was dipped and inoculated on an MRS medium plate containing bromocresol purple by the streak plate method, and cultured at 37°C for 24 h. Colonies that could turn bromocresol purple yellow were selected according to the single colony morphology for microscopic examination, and strains with positive Gram staining were selected and cultured at 37°C for 24 h, and then continuously streaked and inoculated until a pure isolate was obtained. Further inoculated on a esculin screening medium (used to isolate strains producing β-glucosidase, adding 1 g / L esculin and 0.5 g / L ferric citrate to MRS agar, autoclaved at 121°C for 20 min), strains with a red-brown to dark brown circle around them were positive strains (as Figure 1 shown), indicating that the strain has the ability to produce β-glucosidase. The obtained pure cultured strains were inoculated into a liquid MRS medium for further culture, and after adding 80% glycerol, they were stored in a -80°C refrigerator. One of the strains, Loigolactobacillus coryniformis, was named CY-06.

[0023] II. Identification of Strains

[0024] 1. The physiological and biochemical identification results of strain CY-06 are as follows:

[0025] Gram-positive. Among 11 carbohydrate patterns, esculin, maltose, mannitol, sorbitol, sucrose, raffinose, and inulin show positive results. While cellobiose, salicin, lactose, and 1% sodium hippurate show negative reactions. The optimal growth temperature is 37 °C; the suitable pH is 6.0 - 7.0; it can tolerate 6.5% NaCl; it grows uniformly turbid in liquid MRS medium, and the cells form white precipitates after standing for a long time.

[0026] 2. Molecular biology identification and results are as follows: The target strain was inoculated into fresh MRS liquid medium and cultured for 24 h. The genomic DNA of the cells was extracted using a kit from Tiangen Biochemical Technology Co., Ltd., and the 16S rDNA sequence was amplified. The primers used for amplification were the universal primers for species composed of 1492R and 27F. The electrophoresis result of the 16S rDNA PCR product of strain CY-06 showed a specific band with a molecular weight of about 1500 bp, which was consistent with the expected result. Sequencing was performed, and the sequence is shown as SEQ ID NO.1 in the sequence listing. The sequenced sequence was compared with the 16S rDNA gene sequences of some strains already registered on the website http: / / www.ncbi.nlm.nih.gov. The results showed that the homology between strain CY-06 and Loigolactobacillus coryniformis subsp. torquens (NR_029018.1) reached 99.59%. Based on the above results, strain CY-06 was identified as Loigolactobacillus coryniformis.

[0027] Among them, the specific sequence information of the primer pair is as follows: 27sF: 5′-agagttgatcctggctcag-3′; 1492R: 5′-ggttaccttgttacgactt-3′; Among them, the PCR amplification conditions used are 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, stored at 4 °C.

[0028] III. Preservation of Strains

[0029] The rod-shaped Loigolactobacillus coryniformis CY-06 of the present invention was deposited at the China Center for Type Culture Collection, abbreviated as CCTCC, on February 25, 2025. The address is: Inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Preservation Center), and the deposit number is: CCTCC NO: M 2025299.

[0030] IV. Preparation of Loigolactobacillus coryniformis CY-06 bacterial suspension

[0031] The obtained Loigolactobacillus coryniformis CY-06 was inoculated into liquid MRS medium and cultured statically at 37°C for 24 h, centrifuged at 4°C and 8000 rpm for 10 min, and the cell precipitate was collected; it was suspended with sterilized 0.85% saline solution, and the viable cell count was adjusted to 1.0×10 9 CFU / mL to obtain a Loigolactobacillus coryniformis CY-06 bacterial suspension.

[0032] V. Fermentation and conversion of Loigolactobacillus coryniformis CY-06 to prepare rare ginsenosides from ginsenoside Re

[0033] 1. Preparation of liquid fermentation medium: glucose 1 g / L, tryptone 1 g / L, yeast extract powder 0.5 g / L, and sodium chloride 1 g / L; pH 6.0 - 7.0, prepared by sterilization at 121°C for 15 min.

[0034] 2. Fermentation and conversion: Ginsenoside Re (purchased from Chengdu Efa Biotechnology Co., Ltd., HPLC≥98%) was added to the liquid fermentation medium and dissolved in 2 mL of dimethyl sulfoxide. The addition amount of ginsenoside Re was 0.5 g / L, and it was filtered and sterilized through a 0.22 μM microporous filter membrane, and then inoculated at 2×10 7Inoculate the suspension of Loigolactobacillus coryniformis CY-06 at an inoculum size of CFU / mL and carry out anaerobic fermentation at 37°C for 14 days. After the fermentation is completed, the obtained fermentation product is extracted three times with n-butanol saturated with water. After the obtained fermentation product is concentrated under reduced pressure at a low temperature (50°C) and vacuum freeze-dried (-80°C, 5 Pa), the product is extracted with methanol, centrifuged at 10,000 rpm for 15 min at 4°C. After the solvent in the supernatant is recovered under reduced pressure, it is vacuum freeze-dried (-80°C, 5 Pa) to obtain a dried product, and the dried product contains rare ginsenosides. The dried product contains rare ginsenosides Rg2, Rh1, Rg6 and F4.

[0035] VI. Identification of rare ginsenosides by high performance liquid chromatography (HPLC)

[0036] Detect rare ginsenosides Rg2, Rh1, Rg6 and F4 by high performance liquid chromatography (HPLC): Dissolve ginsenoside Re and the above-obtained dried product in chromatographic methanol respectively, and filter through a 0.22 μm microporous filter membrane for HPLC chromatographic analysis. The HPLC chromatographic analysis method is as follows: The chromatographic column is Agilent pursuit5 SB C18 chromatographic column, the injection volume is 20 μL, the elution rate is 1.0 mL / min, the column temperature is 30°C, and the detection wavelength is 203 nm. The mobile phase is water (A) and acetonitrile (C), and gradient elution is carried out 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.

[0037] In the process of fermenting and transforming ginsenoside Re with the suspension of Loigolactobacillus coryniformis CY-06 to prepare rare ginsenosides Rg2, Rh1, Rg6 and F4, its biotransformation synthesis route is as Figure 2As shown in the figure: Under the action of Loigolactobacillus coryniformis CY-06, ginsenoside Re hydrolyzes the glucose at the C-20 position to generate rare ginsenoside Rg2, which is further dehydrated to generate rare ginsenoside F4 and Rg6. At the same time, rare ginsenoside Rg2 can also remove a rhamnose at the C-6 position to generate rare ginsenoside Rh1.

[0038] The HPLC chromatographic identification results are as Figure 3 shown. Through the qualitative and quantitative analysis of ginsenoside components in the products before and after fermentation, it can be seen that the retention times of the conversion products of ginsenoside Re are consistent with those of the standard products of rare ginsenosides Rg2, Rg6, F4 and Rh1. This proves that ginsenoside Re can be fermented and converted into rare ginsenosides Rg2, Rg6, F4 and Rh1 by Loigolactobacillus coryniformis CY-06 producing β-glucosidase. This proves that ginsenoside Re can be fermented and converted into rare ginsenosides Rg2, Rg6, F4 and Rh1 by Loigolactobacillus coryniformis CY-06.

[0039] The present invention discloses the use of Loigolactobacillus coryniformis CY-06 in fermenting and converting ginsenoside Re to prepare rare ginsenosides. Those skilled in the art can draw on 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 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 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. Loigolactobacillus coryniformis CY-06, characterized in that: This strain was deposited in the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025299.

2. Use of the coryneform lactobacillus (Loigolactobacillus coryniformis) CY-06 as claimed in claim 1 in fermenting and converting 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 2, 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 Loigolactobacillus coryniformis CY-06 to generate rare ginsenoside Rg2, which is further dehydrated to generate rare ginsenosides F4 and Rg6. At the same time, rare ginsenoside Rg2 can remove 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 then added to the sterilized liquid fermentation medium at a rate of 0.5 g / L. After sterilization, the culture medium was filtered through a microporous membrane. Loigolactobacillus coryniformis CY-06 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, and then concentrated under reduced pressure at low temperature (50°C) and freeze-dried (-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 (-80°C, 5Pa) is obtained to obtain a dry product, which contains rare ginsenosides Rg2, Rh1, Rg6 and F4.

6. The use according to claim 5, characterized in that: The specific process of preparing the bacterial suspension of Loigolactobacillus coryniformis CY-06 is as follows: Loigolactobacillus coryniformis CY-06 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 4000-8000 rpm for 5-10 min and suspended in sterile PBS to adjust the viable bacterial count to 1.0 × 10 7 ~1.0×10 8 CFU / ml, and obtain the bacterial suspension of Loigolactobacillus coryniformis CY-06.

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