Lactobacillus mucosae NJ-10 for fermentation and its application in fermentative conversion for preparing probiotics rich in rare ginsenosides

Abstract

Limosilactobacillus fermentum NJ-10 and its application in the fermentation conversion for preparing postbiotics rich in rare ginsenosides relate to the field of probiotic fermentation. Limosilactobacillus fermentum NJ-10 was deposited at the China Center for Type Culture Collection on February 25, 2025, with the deposit number of CCTCC NO: M 2025300. A strain of Limosilactobacillus fermentum NJ-10 producing β-glucosidase, which is isolated and identified independently in the present invention, can be used for the fermentation conversion of ginsenosides from ginseng stems and leaves, and then the postbiotics are prepared by means of high-temperature and high-pressure steam. The postbiotic fermented product rich in rare ginsenosides is prepared by the two-step fermentation conversion of ginsenosides from ginseng stems and leaves, which expands the utilization of non-medicinal parts of ginseng, and the postbiotic fermentation product has broad application prospects in many fields such as the food industry, the pharmaceutical industry, the cosmetics field and animal breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic fermentation, and particularly relates to a Limosilactobacillus fermentum NJ-10 and its application in fermentative conversion for preparing postbiotics rich in rare ginsenosides. Background Art

[0002] Ginseng (Panax ginseng C.A.Mey.) is a traditional Chinese herbal medicine with unique characteristics in China. Pharmacological studies have confirmed that Panax ginseng has multi-dimensional biological activities. In addition, the structural differences of ginsenosides significantly affect their bioavailability. Modern research has found that rare ginsenosides (such as Rg3, Rh2, CK, etc.) exhibit stronger pharmacological activities than primary ginsenosides (such as Rb1, Re, Rg1, etc.). However, the content of rare ginsenosides in natural ginseng is less than 0.01%, and the traditional extraction process has the technical bottleneck of low conversion rate. At present, through biotechnological means such as microbial transformation, enzymatic catalysis, and biomimetic synthesis, the directional conversion rate of rare ginsenosides has been increased to 15%-22%, but industrial production still faces key challenges such as strain safety, substrate specificity, and large-scale separation and purification. At present, the development and utilization of ginseng resources mainly focus on its roots. Ginseng plants have strict requirements for the growth environment and grow slowly. The acquisition of ginseng roots usually takes 5-10 years. In addition, ginseng has continuous cropping obstacles and extremely strong site specificity. A large number of studies have shown that the chemical components of non-medicinal parts of ginseng are similar to those of traditional medicinal parts and have strong biological functions. Therefore, non-medicinal parts of ginseng (such as stems and leaves, flowers, fruits) are ideal resources for preparing rare ginsenosides.

[0003] At present, the preparation methods of rare ginsenosides mainly include heating, acid hydrolysis, alkali hydrolysis, and microbial and enzymatic transformation. Compared with heat treatment, acid hydrolysis, and alkali hydrolysis, microbial and enzymatic biotransformation is more environmentally friendly and efficient. In recent years, fermenting ginseng with probiotics has become an important way to enhance its biological activity. Fermentation can convert primary ginsenosides in ginseng into rare ginsenosides, such as Rg3, Rh2, etc., which have higher biological activities and are more easily absorbed. In addition, the fermentation process is mild, has high safety, and can improve the taste of ginseng, which has attracted extensive attention.

[0004] As a probiotic, Limosilactobacillus fermentum has various functional characteristics, including promoting intestinal health, preventing diseases, improving feed utilization rate, and enhancing food quality. It is a recommended strain in the "List of Strains Applicable to Food". Postbiotics is a general term for the metabolite components of probiotics after specific processing, mainly including the cells and metabolites of probiotics. The processing methods mainly include: heat treatment, physical treatment, high hydrostatic pressure treatment, ultrasonic oscillation, etc. A large number of studies have shown that postbiotic components still retain high physiological activity even after being treated with high temperature or gastrointestinal digestive juices, and have significant benefits to the health of the host. Therefore, screening a high-yield β-glucosidase lactic acid bacteria strain that can combine the fermentation and transformation of ginsenosides from ginseng stems and leaves with the preparation of postbiotics is of great significance for the preparation of postbiotics rich in highly active and high-content rare ginsenosides. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a β-glucosidase-producing Limosilactobacillus fermentum NJ-10 and its application in the fermentation and transformation for the preparation of postbiotics rich in rare ginsenosides. The present invention uses the total ginsenosides from ginseng stems and leaves as a substrate, and utilizes the screened Limosilactobacillus fermentum NJ-10 to ferment and transform ginsenosides from ginseng stems and leaves to prepare a postbiotic fermentation product rich in rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5, providing a new way for the development and utilization of ginseng stem and leaf resources and the preparation of postbiotics rich in rare ginsenosides.

[0006] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The Limosilactobacillus fermentum NJ-10 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 2025300.

[0008] The application of the β-glucosidase-producing Limosilactobacillus fermentum NJ-10 provided by the present invention in the fermentation and transformation for the preparation of postbiotics rich in rare ginsenosides.

[0009] As a preferred embodiment, the rare ginsenosides include Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5. The preparation of postbiotics is inactivated at 105°C for 20 minutes.

[0010] As a preferred embodiment, the biotransformation synthesis route of the rare ginsenosides is as follows: 1) The primary ginsenosides Rb1, Rc, and Rb2 in the ginseng stem and leaf saponins are hydrolyzed by Limosilactobacillus fermentum NJ-10 at the C-20 glucose of ginsenosides Rb1, Rc, and Rb2 to generate the primary ginsenoside Rd, and then one glucose is removed to generate the rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, and then dehydrated to generate the rare ginsenosides Rk1 and Rg5; 2) The primary ginsenoside Re in the ginseng stem and leaf saponins is hydrolyzed by Limosilactobacillus fermentum NJ-10 at the C-20 glucose to generate the primary ginsenoside Rg2, and then dehydrated to generate the rare ginsenosides Rg6 and F4.

[0011] As a preferred embodiment, the application of Limosilactobacillus fermentum NJ-10 provided by the present invention in the fermentation conversion to prepare postbiotics rich in rare ginsenosides mainly includes the following steps:

[0012] Step 1: Prepare a Limosilactobacillus fermentum NJ-10 bacterial suspension;

[0013] Inoculate Limosilactobacillus fermentum NJ-10 into a liquid MRS medium, anaerobically culture at 37-39 °C for 18-24 h, centrifuge at 4-10 °C and 8000-12000 rpm for 5-10 min, collect the cell precipitate, suspend it with a sterilized 0.85% physiological saline solution, and adjust the viable cell count to 1.0×10 8 ~1.0×10 9 CFU / ml to obtain Limosilactobacillus fermentum NJ-10;

[0014] Step 2: Fermentation conversion and postbiotic preparation;

[0015] Prepare a liquid fermentation medium, add 0.5 mg / mL total ginsenosides from ginseng stems and leaves to it, filter and sterilize with a microporous filter membrane with a pore size of 0.22 μm, and use 2×10 7Inoculate the fermentation suspension of Limosilactobacillus fermentum NJ-10 at an inoculum size of CFU / mL and ferment anaerobically at 37°C for 14 days. After fermentation, inactivate the obtained fermentation product at 105°C for 20 min to obtain a postbiotic fermentation product rich in rare ginsenosides.

[0016] As a preferred embodiment, the liquid fermentation medium comprises: 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.

[0017] The beneficial effects of the present invention are as follows:

[0018] The present invention first screened a strain NJ-10 from a sample of traditional fermented kimchi homemade by a farmer in Yanji and identified it. The identification result was Limosilactobacillus fermentum, which was deposited at the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025300. Then, using total ginsenosides from ginseng stems and leaves as raw materials, and taking advantage of the property of Limosilactobacillus fermentum NJ-10 to produce β-glucosidase, the ginsenosides in ginseng stems and leaves were biochemically fermented and transformed. Further, the fermentation product was inactivated at 105°C for 20 min to obtain a postbiotic fermentation product rich in rare ginsenosides. At the same time, HPLC chromatographic analysis was used to identify the rare ginsenosides in the fermented product before and after fermentation and the postbiotic fermentation product. The results showed that the retention times of the postbiotic fermentation product rich in rare ginsenosides prepared by fermentation and transformation of Limosilactobacillus fermentum NJ-10 were consistent with those of the rare ginsenoside standards Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5. Therefore, the β-glucosidase-producing Limosilactobacillus fermentum NJ-10 of the present invention can ferment and transform ginsenosides from ginseng stems and leaves and combine with postbiotic treatment to obtain high contents of rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5, and the prepared rare ginsenosides have a wide range of uses. In addition, the fermentation product can also be used as a postbiotic product and has broad application prospects in multiple fields such as the food industry, pharmaceutical industry, cosmetics field, and animal breeding. Description of the Drawings

[0019] Figure 1 It is a color picture on the screening medium for β-glucosidase-producing Limosilactobacillus fermentum NJ-10.

[0020] Figure 2 HPLC chromatograms of ginsenosides from ginseng stem and leaf fermented for 0 and 14 days and the postbiotics fermentation products.

[0021] Figure 3 Bioconversion synthesis route for the fermentation of Limosilactobacillus fermentum NJ-10 to transform total ginsenosides from ginseng stem and leaf into rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5. It includes: the bioconversion synthesis route for the transformation of Rb1, Rc, and Rb2 to Rd and then to rare ginsenosides 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5; the bioconversion synthesis route for the transformation of Re to Rg2 and then to rare ginsenosides Rg6 and F4. Detailed implementation method

[0022] A strain of Limosilactobacillus fermentum NJ-10 provided by the present invention was deposited at the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025300.

[0023] The present invention provides the application of Limosilactobacillus fermentum NJ-10 producing β-glucosidase in the fermentation transformation for preparing postbiotics rich in rare ginsenosides, which mainly includes the following steps:

[0024] Step 1: Prepare a suspension of Limosilactobacillus fermentum NJ-10.

[0025] Inoculate Limosilactobacillus fermentum NJ-10 into liquid MRS medium, anaerobically culture at 37°C for 24 h, centrifuge at 4°C and 8000 rpm for 10 min, collect the cell precipitate, suspend it with sterilized 0.85% saline solution, and adjust the viable cell count to 1.0×10 8 ~1.0×10 9 CFU / ml to obtain a suspension of Limosilactobacillus fermentum NJ-10.

[0026] Step 2: Fermentation transformation and postbiotics preparation.

[0027] Prepare a liquid fermentation medium, which 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, with a pH of 6.0 - 7.0; add 0.5 mg / mL of total ginsenosides from ginseng stems and leaves, filter and sterilize it through a microporous membrane with a pore size of 0.22 μM, and inoculate a suspension of Limosilactobacillus fermentum NJ-10 at an inoculum size of 2×10⁷ CFU / mL, and ferment anaerobically at 37 °C for 14 days. After fermentation, inactivate the obtained fermentation product at 105 °C for 20 min to obtain a postbiotic fermentation product rich in rare ginsenosides.

[0028] Among them, the rare ginsenosides include Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rg5, and a small amount of Rk1.

[0029] Among them, the biotransformation synthesis route of rare ginsenosides is as follows: 1) The primary ginsenosides Rb1, Rc, and Rb2 in the ginsenosides from ginseng stems and leaves are hydrolyzed by Limosilactobacillus fermentum NJ-10 at the C-20 position of glucose in ginsenosides Rb1, Rc, and Rb2 to generate the primary ginsenoside Rd, and then one glucose molecule is removed to generate the rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, and then dehydrated to generate the rare ginsenosides, Rk1, and Rg5; 2) The primary ginsenoside Re in the ginsenosides from ginseng stems and leaves is hydrolyzed by Limosilactobacillus fermentum NJ-10 at the C-20 position of glucose to generate the primary ginsenoside Rg2, and then dehydrated to generate the rare ginsenosides Rg6 and F4.

[0030] 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 of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0031] Example 1 Isolation, Identification, and Preservation of Strains

[0032] 1. Isolation of Strains

[0033] The sample used for isolating the strain was a sample of homemade traditional fermented kimchi collected from Yanji City, Jilin Province in October 2024. The collected traditional fermented kimchi sample was ground, and then serially diluted by a factor of 10 with sterile physiological saline to obtain dilution solutions; each dilution solution was spread on an MRS medium plate supplemented with bromocresol purple 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. After repeated streak purification, the strain was further inoculated into an aesculin screening medium (used for isolating strains producing β-glucosidase, adding 1 g / L aesculin 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 the colonies were positive strains (as shown in Figure 1 ). The obtained pure culture of the strain was inoculated into a liquid MRS medium for continued culture. After adding 80% glycerol, it was stored in a -80 °C refrigerator. One of the lactic acid bacteria strains was named NJ-10.

[0034] 2. Identification of the strain

[0035] (1) Physiological and biochemical identification results of strain NJ-10: Gram-positive staining, capable of utilizing glucose, maltose, lactose, sucrose, fructose, galactose, cellobiose, trehalose, xylose, arabinose, salicin and aesculin, showing positive; negative in rhamnose, mannitol, sorbitol, amygdalin and melezitose. The optimum growth temperature is 37 °C; the suitable pH is 6.0 - 7.0; it can tolerate 6.5% NaCl; it grows uniformly turbid in the liquid MRS medium, and the cells form a white precipitate after standing for a long time.

[0036] (2) Molecular biological identification: The target strain was inoculated into fresh MRS liquid medium and cultured for 24 h. The genomic DNA of the strain was extracted using the kit from Tiangen Biochemical Technology Co., Ltd., and the 16S rDNA sequence was amplified. The universal primers for species identification composed of 1492R and 27F were used (1492r: 5′-ggttaccttgttacgactt-3′; 27f: 5′-agagttgatcctggctcag-3′) for PCR amplification. The PCR amplification conditions 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 stored at 4°C. The electrophoresis result of the 16S rDNA PCR product of strain NJ-10 showed a specific band at around 1500 bp in molecular weight, which was consistent with the expected result, and then it was sequenced. The sequence was shown as SEQ ID NO.1 in the sequence listing. The sequenced sequence was aligned with the 16S rDNA gene sequences of some strains registered on the website http: / / www.ncbi.nlm.nih.gov. The result showed that the homology between strain NJ-10 and Limosilactobacillus fermentum reached 100%. Based on the above results, strain NJ-10 was identified as Limosilactobacillus fermentum.

[0037] 3. Preservation of the strain

[0038] Limosilactobacillus fermentum NJ-10 of the present invention was deposited at the China Center for Type Culture Collection on February 25, 2025, abbreviated as CCTCC N5O: M 2025300, with the address being: Inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Preservation Center), and the deposit number being: CCTCC NO: M2025300.

[0039] Example 2 Preparation of postbiotics rich in rare ginsenosides by fermenting and transforming ginsenosides from ginseng stems and leaves with Limosilactobacillus fermentum NJ-10

[0040] 1. Preparation of Limosilactobacillus fermentum NJ-10 bacterial suspension

[0041] Inoculate Limosilactobacillus fermentum NJ-10 into liquid MRS medium, and culture it anaerobically at 37 °C for 24 h. Then centrifuge at 4 °C and 8000 rpm for 10 min to collect the cell precipitate. Suspend it with sterilized 0.85% saline solution and adjust the viable cell count to 1.0×10 9 CFU / mL to obtain a Limosilactobacillus fermentum NJ-10 bacterial suspension.

[0042] 2. Fermentation conversion and postbiotic preparation

[0043] Prepare ginsenosides from ginseng leaves and stems: Total ginsenosides from ginseng leaves and stems (HPLC purity ≥80%) were purchased from Chengdu Efa Biotechnology Co., Ltd. Prepare a liquid fermentation medium, which includes 1 g / L glucose, 1 g / L tryptone, 0.5 g / L yeast extract powder, and 1 g / L sodium chloride, with a pH of 6.0 - 7.0. Sterilize it at 121 °C for 20 min. In the liquid fermentation medium, the addition amount of total ginsenosides from ginseng leaves and stems is 0.5 mg / mL. Filter and sterilize it with a 0.22 μM microporous membrane, and inoculate the Limosilactobacillus fermentum NJ-10 bacterial suspension at an inoculation amount of 2×10 7 CFU / mL, and ferment anaerobically at 37 °C for 14 days. After the fermentation is completed, inactivate the obtained fermentation product at 105 °C for 20 min to obtain a postbiotic fermentation product rich in rare ginsenosides. After inactivation, perform plate coating to ensure that the bacteria have been completely inactivated. The prepared postbiotic fermentation product is aliquoted and stored at -80 °C for later use.

[0044] Example 3 Identification of rare ginsenosides

[0045] Filter the postbiotic fermentation product with a microporous membrane with a pore size of 0.22 μm, extract it with water-saturated n-butanol, then perform low-temperature reduced pressure concentration and vacuum freeze-drying (-80 °C, 5 Pa). Dissolve the residue in methanol and centrifuge at 4 °C with a centrifugal force of 10000 rpm for 15 min. After the supernatant is reduced in pressure to recover the solvent, perform vacuum freeze-drying to obtain a dry product. Use high-performance liquid chromatography (HPLC) to detect rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5: Dissolve ginsenosides from ginseng leaves and stems and the above-obtained dry product in chromatographic methanol respectively, and filter them through a 0.22 μm microporous membrane for HPLC chromatographic analysis.

[0046] The HPLC chromatographic analysis method is as follows: Agilent pursuit5 SB C18 chromatographic column, injection volume 20 μL, elution rate 1.0 mL / min, column temperature 30 °C, detection wavelength 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.

[0047] The HPLC chromatographic identification results are as Figure 2 shown. Through the qualitative and quantitative analysis of ginsenoside components in ginseng leaf and stem saponins before fermentation, after fermentation, and in postbiotic fermentation products, it can be seen that the retention times of the fermentation conversion products of ginseng leaf and stem saponins and postbiotic fermentation products are consistent with those of the rare ginsenoside standards Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1, and Rg5. This proves that ginseng leaf and stem saponins can be fermentatively converted by Limosilactobacillus fermentum NJ-10 producing β-glucosidase, and the postbiotic fermentation products prepared therefrom are rich in rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rg5, and Rk1. And, as Figure 3 shown, the biotransformation synthesis route for the preparation of rare ginsenosides by fermentative conversion using Limosilactobacillus fermentum NJ-10 is as follows: The primary saponins Rb1, Rc, and Rb2 in ginseng leaf and stem saponins are hydrolyzed by Limosilactobacillus fermentum NJ-10 to remove the glucose at the C-20 position of ginsenosides Rb1, Rc, and Rb2 to generate the primary ginsenoside Rd, and then one glucose is removed to generate rare ginsenosides 20(R)-Rg3 and 20(S)-Rg3, and then dehydrated to generate rare ginsenosides Rk1 and Rg5; The primary ginsenoside Re in ginseng leaf and stem saponins is hydrolyzed by Limosilactobacillus fermentum NJ-10 to remove the glucose at the C-20 position to generate the primary ginsenoside Rg2, and then dehydrated to generate rare ginsenosides Rg6 and F4.

[0048] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of rare ginsenoside postbiotics, characterized in that, Comprising the following steps: The rare ginsenoside postbiotics are selected from Rg6 and F4; Step 1: Prepare a suspension of Limosilactobacillus fermentum NJ-10; Limosilactobacillus fermentum NJ-10 was deposited at the China Center for Type Culture Collection on May 25, 2025, with the deposit number: CCTCC NO: M 2025300. NJ-10 was inoculated into liquid MRS medium and anaerobically cultured at 37 °C for 24 h, centrifuged at 8000 rpm for 10 min at 4 °C, the cell precipitate was collected, suspended in sterile 0.85% saline solution, and the viable cell count was adjusted to 1.0×10 9 CFU / mL to obtain a bacterial suspension of Limosilactobacillus fermentum NJ-10; Step 2: Fermentation conversion and preparation of postbiotics; Prepare a liquid fermentation medium containing 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; adjust the pH to 6.0 - 7.0; add 0.5 mg / mL of total ginsenosides from ginseng stems and leaves to it. Filter the resulting solution through a microporous membrane with a pore size of 0.22 μm to sterilize it, and inoculate it with a suspension of Limosilactobacillus fermentum NJ-10 at an inoculum size of 2×10 7 CFU / mL, and ferment anaerobically at 37°C for 14 days; after fermentation, inactivate the resulting fermentation product at 105°C for 20 min to obtain a postbiotic fermentation product rich in rare ginsenosides.

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