Fermented lactobacillus mucus NJ-10 and application thereof in preparation of rare ginsenoside-rich metagen through fermentation conversion

Abstract

The invention discloses fermented lactobacillus mucus NJ-10 and application thereof in preparation of rare ginsenoside-rich metagen through fermentation conversion, and relates to the field of probiotic fermentation. The lactobacillus fermentum NJ-10 is preserved in the China Center for Type Culture Collection on February 25, 2025, and the preservation number of the lactobacillus fermentum NJ-10 is CCTCC (China Center for Type Culture Collection) NO: M 2025300. According to the present invention, the fermentation lactobacillus mucus NJ-10 capable of producing beta-glucosidase, which is subjected to autonomous separation and identification, can be used for fermentation and conversion of ginseng stem and leaf saponin, and then is subjected to post-generation preparation in a high-temperature and high-pressure steam manner, such that the beta-glucosidase producing beta-glucosidase can be produced, and the beta-glucosidase producing beta-glucosidase producing beta-glucosidase producing beta-glucosidase producing beta-glucosidase producing beta-glucosidase can be produced. According to the method, ginseng stem and leaf saponin is converted through two-step fermentation to prepare the postbiotic fermentation product rich in rare ginsenoside, resource utilization of non-medicinal parts of ginseng is expanded, and the postbiotic fermentation product has wide application prospects in the fields of food industry, pharmaceutical industry, cosmetics, animal breeding and the like.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic fermentation, and in particular to a strain of Limosilactobacillus fermentum NJ-10 and application thereof in preparing postbiotics rich in rare ginsenosides through fermentation and conversion. Background Art

[0002] Ginseng (Panax ginseng CAMey.) is a traditional Chinese herbal medicine with unique characteristics in my country. Pharmacological studies have confirmed that ginseng (Panax ginseng) has multi-dimensional biological activities. In addition, the structural differences of ginsenosides significantly affect their bioavailability. Modern studies have found that rare ginsenosides (such as Rg3, Rh2, CK, etc.) show stronger pharmacological activity than primary saponins (such as Rb1, Re, Rg1, etc.). However, the content of rare saponins in natural ginseng is less than 0.01%, and the traditional extraction process has a technical bottleneck of low conversion rate. At present, through biotechnology means such as microbial transformation, enzyme catalysis and biomimetic synthesis, the directional conversion rate of rare saponins 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 are mainly concentrated on its roots. Ginseng plants have strict requirements on the growth environment and grow slowly. It usually takes 5-10 years to obtain ginseng roots. In addition, ginseng has the problem of continuous cropping and is very sensitive to soil. A large number of studies have shown that the chemical composition of non-medicinal parts of ginseng is similar to that of traditional medicinal parts and has strong biological functions. Therefore, non-medicinal parts of ginseng (such as stems, leaves, flowers, and fruits) are ideal resources for preparing rare ginsenosides.

[0003] At present, the preparation methods of rare ginsenosides mainly include heating, acid hydrolysis, alkaline hydrolysis, and microbial and enzyme transformation. Compared with heat treatment, acid hydrolysis and alkaline hydrolysis, microbial and enzyme biotransformation is more environmentally friendly and efficient. In recent years, probiotic fermentation of ginseng has become an important way to enhance its biological activity. Fermentation can convert primary saponins in ginseng into rare ginsenosides, such as Rg3, Rh2, etc. These ingredients have higher biological activity and are easier to absorb. In addition, the fermentation process is mild, highly safe, and can improve the taste of ginseng, which has attracted widespread attention.

[0004] As a probiotic, fermented mucus lactobacillus has multiple functional properties, including promoting intestinal health, preventing diseases, improving feed utilization and improving food quality. It is a recommended strain in the "List of Bacteria that Can Be Used in Food". Postbiotics is a general term for probiotic metabolites after specific processing of probiotics, mainly including probiotic bodies and metabolites. The processing methods mainly include: heat treatment, physical treatment, high hydrostatic pressure treatment, ultrasonic vibration, etc. A large number of studies have shown that postbiotic ingredients still retain high physiological activity even after high temperature or gastrointestinal digestive fluid treatment, and have significant benefits to host health. Therefore, screening a high-yield β-glucosidase lactic acid bacteria strain that can realize the fermentation transformation of ginseng stem and leaf saponins and the preparation of postbiotics is of great significance for the preparation of postbiotics rich in high activity and high content of rare ginsenosides. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a strain of fermented Lactobacillus fermentum NJ-10 that produces β-glucosidase and its application in fermentation and conversion to prepare postbiotics rich in rare ginsenosides. The present invention uses total saponins from ginseng stems and leaves as substrates, and uses the screened fermented Lactobacillus fermentum NJ-10 to ferment and convert ginseng stem and leaf saponins to prepare postbiotic fermentation products 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-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The fermented mucus lactobacillus (Limosilactobacillus fermentum) NJ-10 provided by the present invention has been deposited in the China Center for Type Culture Collection on February 25, 2025, with the deposit number: CCTCC NO: M 2025300.

[0008] The invention provides an application of β-glucosidase-producing fermented Lactobacillus fermentum NJ-10 in fermentation and conversion to prepare 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 postbiotics were prepared by inactivation 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 ginsenosides of ...

[0011] As a preferred embodiment, the use of fermented Lactobacillus fermentum NJ-10 provided by the present invention in the fermentation and conversion preparation of rare ginsenoside-rich postbiotics mainly comprises the following steps:

[0012] Step 1: preparing a suspension of Limosilactobacillus fermentum NJ-10;

[0013] Limosilactobacillus fermentum NJ-10 was inoculated into liquid MRS medium and cultured anaerobically at 37-39°C for 18-24 h. The culture was centrifuged at 4-10°C and 8000-12000 rpm for 5-10 min. 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 Limosilactobacillus fermentum NJ-10;

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

[0015] Prepare liquid fermentation medium, add 0.5 mg / mL total saponins from ginseng stems and leaves, filter and sterilize with a microporous filter membrane with a pore size of 0.22 μm, and 7The inoculum amount of CFU / mL was inoculated with a suspension of Limosilactobacillusfermentum NJ-10 and anaerobically fermented at 37°C for 14 days. After the fermentation, the obtained fermentation product was inactivated 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 glucose, 1 g / L tryptone, 0.5 g / L yeast extract powder and 1 g / L 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 spicy cabbage made by farmers in Yanji, and identified it. The identification result was Limosilactobacillus fermentum, and it was preserved in the China Center for Type Culture Collection on February 25, 2025, with a preservation number of CCTCC NO: M 2025300. Then, using ginseng stem and leaf total saponins as raw materials, the fermented mucus lactobacillus (Limosilactobacillus fermentum) NJ-10 has the characteristic of producing β-glucosidase, and the ginseng stem and leaf saponins are biofermented and transformed; further, the fermentation product is inactivated at 105°C for 20 minutes to obtain a postbiotic fermentation product rich in rare ginsenosides. At the same time, HPLC chromatography was used to identify the rare ginsenosides in the fermentation products before and after fermentation and postbiotic fermentation. The results showed that the postbiotic fermentation products rich in rare ginsenosides prepared by fermentation of Limosilactobacillus fermentum NJ-10 were consistent with the retention time of the rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 standards. Therefore, the fermentation of ginseng stem and leaf saponins by β-glucosidase-producing Limosilactobacillus fermentum NJ-10 combined with postbiotic treatment can obtain high content 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, fermentation products can also be used as postbiotic products in the food industry, pharmaceutical industry, cosmetics field, animal husbandry and other fields with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a color development picture on the screening culture medium for the fermentation of Lactobacillus mucilaginosus NJ-10 producing β-glucosidase.

[0020] Figure 2 HPLC chromatograms of ginseng stem and leaf saponin fermentation products on days 0 and 14 and postbiotic fermentation products.

[0021] Figure 3 The invention discloses a biotransformation synthesis route for the fermentation of total saponins of ginseng stems and leaves by fermentation of Lactobacillus mucilaginosus NJ-10 to generate rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5. The invention also ... DETAILED DESCRIPTION

[0022] The present invention provides a strain of Limosilactobacillus fermentum NJ-10, which has been deposited in the China Center for Type Culture Collection on February 25, 2025, with a deposit number of CCTCC NO: M 2025300.

[0023] The present invention provides an application of fermented Lactobacillus fermentum NJ-10 producing β-glucosidase in fermentation and conversion to prepare postbiotics rich in rare ginsenosides, which mainly comprises the following steps:

[0024] Step 1: preparing a suspension of Limosilactobacillus fermentum NJ-10;

[0025] Limosilactobacillus fermentum NJ-10 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, to obtain the bacterial suspension of Limosilactobacillus fermentum NJ-10;

[0026] Step 2: Fermentation conversion and postbiotic preparation;

[0027] Liquid fermentation medium was prepared, which included 1g / L glucose, 1g / L tryptone, 0.5g / L yeast extract and 1g / L sodium chloride, with a pH of 6.0-7.0; 0.5 mg / mL total saponins from ginseng stems and leaves were added thereto, sterilized by filtration with a microporous filter membrane with a pore size of 0.22μM, and inoculated with a suspension of Limosilactobacillus fermentum NJ-10 at an inoculum of 2×107CFU / mL, and anaerobically fermented at 37℃ for 14 days. After the fermentation, the obtained fermentation product was inactivated at 105℃ for 20min to obtain a postbiotic fermentation product rich in rare ginsenosides.

[0028] Among them, 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: 1) the primary saponins Rb1, Rc, and Rb2 in the ginsenosides of ginsenosides of ginsenosides Rb1, Rc, and Rb2 are hydrolyzed by fermentation of Lactobacillus fermentum NJ-10 at the C-20 position 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; 2) the primary ginsenoside Re in the ginsenosides of ginsenosides of ginsenosides of ginsenosides Re are hydrolyzed by fermentation of Lactobacillus fermentum NJ-10 at the C-20 position to generate primary ginsenoside Rg2, which is then dehydrated to generate rare ginsenosides Rg6 and F4.

[0030] 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.

[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 traditional fermented kimchi collected from a farmer's home in Yanji City, Jilin Province in October 2024. The collected traditional fermented kimchi sample was 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 24 hours. According to the morphology of single colonies, colonies that can make bromocresol purple turn 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 of aesculin and 0.5 g / L of ferric citrate were added to MRS agar and sterilized at 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 NJ-10.

[0034] 2. Identification of strains

[0035] (1) Physiological and biochemical identification results of strain NJ-10: Gram staining is positive, and it can utilize glucose, maltose, lactose, sucrose, sucrose, fructose, galactose, cellobiose, trehalose, xylose, arabinose, salicin and esculin, and is negative in rhamnose, mannitol, sorbitol, amygdalin and melezitose. 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.

[0036] (2) Molecular biological identification: The target strain was inoculated into 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, a total of 30 cycles; final extension: 72℃ 2min, stored at 4℃. The electrophoresis results of the 16S rDNA PCR product of strain NJ-10 showed that a band with good specificity was obtained at a molecular weight of about 1500 bp, which was consistent with the expected results, and the sequence was sequenced, and the sequence was 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, and the results 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 strains

[0038] The fermented mucus lactobacillus (Limosilactobacillus fermentum) NJ-10 of the present invention has been deposited in the China Center for Type Culture Collection on February 25, 2025, abbreviated as CCTCC N5O: M 2025300, 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: M2025300.

[0039] Example 2 Preparation of postbiotics rich in rare ginsenosides by fermenting and converting ginseng stem and leaf saponins using Limosilactobacillus fermentum NJ-10

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

[0041] Limosilactobacillus fermentum NJ-10 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24 h. The bacterial precipitate was collected by centrifugation at 4°C and 8000 rpm for 10 min. The bacterial precipitate was suspended in sterile 0.85% saline solution and the viable bacterial count was adjusted to 1.0×10 9 CFU / mL, and obtain the Limosilactobacillusfermentum NJ-10 bacterial suspension.

[0042] 2. Fermentation conversion and postbiotic preparation

[0043] Preparation of ginseng stem and leaf saponins: Ginseng stem and leaf total saponins (HPLC purity ≥ 80%) were purchased from Chengdu Aifa Biotechnology Co., Ltd. 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 6.0-7.0, and sterilized at 121°C for 20 min. In the liquid fermentation medium, the amount of ginseng stem and leaf total saponins added was 0.5 mg / mL, filtered and sterilized with a 0.22 μM microporous filter membrane, and 2 × 10 7 CFU / mL inoculation amount of Limosilactobacillus fermentum NJ-10 bacterial suspension, anaerobic fermentation at 37℃ for 14 days; after the fermentation, the obtained fermentation product was inactivated at 105℃ for 20min to obtain a postbiotic fermentation product rich in rare ginsenosides. After inactivation, the plate was spread to ensure that the bacteria were completely inactivated. The prepared postbiotic fermentation product was packaged and stored at -80℃ for later use.

[0044] Example 3 Identification of rare ginsenosides

[0045] The postbiotic fermentation product was filtered through a microporous membrane with a pore size of 0.22 μm, extracted with water-saturated n-butanol, and then concentrated under reduced pressure and vacuum freeze-dried (-80 ° C, 5 pa). The residue was dissolved in methanol and centrifuged at 4 ° C at 10000 rpm for 15 min. The supernatant was vacuum-freezed and the solvent was recovered to obtain a dry product. High-performance liquid chromatography (HPLC) was used to detect the rare ginsenosides Rg6, F4, 20 (R) -Rg3, 20 (S) -Rg3, Rk1 and Rg5: Ginseng stem and leaf saponins and the above-obtained dry products were dissolved in chromatographic methanol, filtered through a 0.22 μm microporous membrane, and used for HPLC chromatographic analysis.

[0046] HPLC chromatographic analysis method: chromatographic column Agilent pursuit5 SB C18 column, injection volume 20μL, elution rate 1.0 mL / min, column temperature 30℃, detection wavelength 203nm. The mobile phase is water (A) and acetonitrile (C), and gradient elution is performed as follows: 0min, 81.50%A, 18.50%C; 20min, 79.50%A, 20.50%C; 30min, 70%A, 30%C; 45min, 65%A, 35%C; 60min, 55%A, 45%C; 70min, 40%A, 60%C; 80min, 30%A, 70%C; 90min, 20%A, 80%C; 91min, 81.50%A, 18.50%C; 95min, 81.50%A, 18.50%C.

[0047] HPLC chromatographic identification results are as follows Figure 2 As shown in the figure, through the qualitative and quantitative analysis of ginsenoside components before, after and in postbiotic fermentation products, it can be seen that the retention time of ginsenoside fermentation conversion products and postbiotic fermentation products of ginseng stem and leaf saponins is consistent with that of rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5 standards. This proves that ginsenosides of ginseng stem and leaf can be fermented and transformed by β-glucosidase-producing Lactobacillus fermentum NJ-10, and the postbiotic fermentation products prepared by it are rich in rare ginsenosides Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rg5 and Rk1. In addition, as Figure 3 As shown in the figure, the biotransformation synthesis route for preparing rare ginsenosides by fermentation transformation using Limosilactobacillus fermentum NJ-10 is as follows: the primary saponins Rb1, Rc, and Rb2 in ginsenosides of ...

[0048] 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. Limosilactobacillus fermentum NJ-10, characterized in that: It was deposited in the China Center for Type Culture Collection on January 25, 2025, with the deposit number: CCTCC NO: M 2025300.

2. Use of the fermented mucus lactobacillus (Limosilactobacillus fermentum) NJ-10 as claimed in claim 1 in the fermentation and conversion preparation of postbiotics rich in rare ginsenosides.

3. The use according to claim 2, characterized in that: The fermented mucus lactobacillus (Limosilactobacillus fermentum) NJ-10 has the characteristic of producing β-glucosidase, and the rare ginsenosides include Rg6, F4, 20(R)-Rg3, 20(S)-Rg3, Rk1 and Rg5.

4. The use according to claim 2, characterized in that: The biotransformation synthesis route of the rare ginsenosides is as follows: 1) the glucose at the C-20 position of the primary ginsenosides ginsenosides Rb1, Rc and Rb2 in the ginsenosides of ginsenosides of ginsenosides Rb1, Rc and Rb2 is hydrolyzed by fermentation of Lactobacillus fermentum NJ-10 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; 2) the glucose at the C-20 position of the primary ginsenoside Re in the ginsenosides of ginsenosides of ginsenosides Re is hydrolyzed by fermentation of Lactobacillus fermentum NJ-10 to generate primary ginsenoside Rg2, which is then dehydrated to generate rare ginsenosides Rg6 and F4.

5. The use according to claim 2, characterized in that: The following steps are involved: Step 1: preparing a suspension of Limosilactobacillus fermentum NJ-10; Limosilactobacillus fermentum NJ-10 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 24 h. The culture was centrifuged at 4°C and 8000 rpm for 10 min. 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, obtained Limosilactobacillusfermentum NJ-10 bacterial suspension; Step 2: Fermentation conversion and postbiotic preparation; Liquid fermentation medium was prepared, with 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; 0.5 mg / mL total saponins from ginseng stems and leaves were added, and the resulting solution was sterilized by filtering with a microporous membrane with a pore size of 0.22 μm and then 2 × 10 7 The fermented Lactobacillus fermentum NJ-10 bacterial suspension was inoculated with an inoculum size of 500 CFU / mL and anaerobically fermented at 37°C for 14 days. After the fermentation, the obtained fermentation product was inactivated at 105°C for 20 min to obtain a postbiotic fermentation product rich in rare ginsenosides.

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