Lactobacillus mucilaginosus and application thereof in preparation of ginsenoside F4

Abstract

The invention relates to lactobacillus mucilaginosus and application thereof in preparation of ginsenoside F4, and belongs to the technical field of biotransformation of traditional Chinese medicinal materials. The technical problems that in the prior art, a biotransformation technology of rare ginsenoside F4 is insufficient in selectivity and environmental friendliness and high in cost are solved. The lactobacillus mucus disclosed by the invention is classified and named as LA729; the lactobacillus mucus is preserved in the China Center for Type Culture Collection (CCTCC) on August 4, 2025, and the preservation number of the lactobacillus mucus is CCTCC NO: M 20251765. The strain is safe in source and free of toxic and side effects, can secrete specific glycosidase in the fermentation process and selectively hydrolyze sugar residues on ginsenoside Re, so that structural reconstruction of the ginsenoside Re is achieved, target rare ginsenoside F4 is generated, selectivity is high, by-products are few, and the yield is stable.

Description

Technical Field

[0001] This invention belongs to the field of biotransformation technology of traditional Chinese medicine, specifically relating to a fermented Lactobacillus mucinus and its application in the preparation of ginsenoside F4. Background Technology

[0002] Biotransformation technology of Chinese medicinal materials is a green preparation method that uses microorganisms or their enzyme systems to selectively hydrolyze, desugar, redox, or rearrange natural medicinal components under mild conditions to obtain high-value-added active ingredients.

[0003] Rare ginsenosides have become a focus of biotransformation research due to their unique structure and significant biological activity. Among them, rare ginsenoside F4 has potential application value in cardiovascular protection, anti-inflammatory and immune regulation, anti-tumor and anti-aging.

[0004] However, existing chemical or physical transformation methods often require strong acids and bases, high temperature and high pressure, or complex separation and purification. They lack selectivity and environmental friendliness, and are also costly, thus limiting their large-scale application in the pharmaceutical and functional food fields. Summary of the Invention

[0005] In view of this, in order to solve the technical problems of insufficient selectivity and environmental friendliness and high cost of the biotransformation technology of rare ginsenoside F4 in the prior art, the present invention provides a fermentation of Lactobacillus myxoidis and its application in the preparation of ginsenoside F4. The strain is safe to obtain and has no toxic side effects. During the fermentation process, it can secrete specific glycosidases to selectively hydrolyze the sugar residues on ginsenoside Re, thereby realizing its structural reconstruction and generating the target rare ginsenoside F4.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a fermenting *Lactobacillus mucinus*, wherein the *Lactobacillus mucinus* is classified as... LA729; The fermenting Lactobacillus mucinus was deposited on August 4, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, accession number: CCTCC NO:M 20251765.

[0008] Secondly, the present invention also provides an application of fermented Lactobacillus mucinus, wherein the fermented Lactobacillus mucinus is capable of fermenting ginsenoside Re into ginsenoside F4.

[0009] Preferably, the fermentation of *Lactobacillus mucinus* is first prepared into a bacterial suspension, and then the bacterial suspension is inoculated into a liquid fermentation medium containing ginsenoside Re for co-fermentation. After fermentation, the fermentation product is dried to obtain ginsenoside F4.

[0010] More preferably, including:

[0011] Step 1: Preparation of bacterial suspension

[0012] First, *Lactobacillus fermentatus* was inoculated into liquid culture medium and incubated statically at 37–45°C for 16–25 h. Then, the bacterial pellet was collected by centrifugation and finally resuspended in liquid culture medium to achieve a viable count of 1.0 × 10⁻⁶ cells / mL. 9 ~1.0×10 10 CFU / mL, to obtain a bacterial suspension;

[0013] Step 2: Fermentation and Product Preparation

[0014] Inoculate 10-30 mL of the bacterial suspension from step one into 1 L of liquid fermentation medium containing ginsenoside Re, and let it ferment at 37-42℃ for 7-14 days. After fermentation, concentrate the obtained fermentation product under reduced pressure, freeze dry under vacuum, extract the dried product with an extractant, centrifuge, and freeze dry the supernatant under reduced pressure to obtain ginsenoside F4.

[0015] The liquid fermentation medium containing ginsenoside Re comprises glucose at a concentration of 2-5 g / L, yeast extract at a concentration of 1-3 g / L, and ginsenoside Re at a concentration of 5-10 g / L, using deionized water as a solvent, with a pH of 6.0-7.0.

[0016] The extractant is one or more of alcohols and ethyl acetate.

[0017] Preferably, in step one, the liquid culture medium is MRS liquid culture medium;

[0018] Most preferably, the formulation of the MRS liquid culture medium is as follows: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L ammonium citrate, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween 80L, using deionized water as solvent, with a pH of 6.2~6.8, and sterilized at 121℃ for 15~20 min.

[0019] Preferably, in step one, the centrifugation conditions are: centrifugation at 4000~8000 rpm for 5~10 min.

[0020] Preferably, in step two, the temperature of the vacuum concentration is 40~70℃;

[0021] Most preferably, the temperature for vacuum concentration is 50~60℃.

[0022] In particular, in step two, the vacuum freeze-drying process involves pre-freezing to -50°C to -30°C, with the vacuum freeze-drying time being 12 to 36 hours.

[0023] Preferably, in step two, the alcohol is one or more of methanol, ethanol, and n-butanol;

[0024] Most preferably, the alcohol is one or both of methanol and ethanol.

[0025] Preferably, in step two, the solid-liquid ratio of the extraction is 1g:5mL to 1g:20mL.

[0026] Preferably, in step two, the extraction temperature is 25~40℃, the extraction time is 30~120min, and the extraction is performed 1~3 times.

[0027] Particularly preferred is that, in step two, the centrifugation conditions are: temperature 4~10℃, speed 8000~12000rpm or 9000~12000×g, and time 10~20min;

[0028] The most preferred centrifugation conditions are: temperature 4°C, speed 10000 rpm, and time 15 min.

[0029] The principle of this invention is as follows:

[0030] The fermentation of *Lactobacillus mucilaginosus* of the present invention ( During fermentation, LA729 secretes specific glycosidases that selectively hydrolyze the sugar residues on ginsenoside Re. Therefore, ginsenoside Re is hydrolyzed by the glucosidase produced by fermenting *Lactobacillus myxobin* LA729 to generate rare ginsenoside F4, with the following structural formula: .

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] The fermentation of *Lactobacillus mucilaginosus* of the present invention ( LA729 belongs to the genus *Lactobacillus* and is included in the "List of Microbial Strains that Can Be Used in Food" (Announcement No. 4 of 2022) issued by the National Health Commission of China. This announcement lists *Lactobacillus fermentatus* under the genus *Lactobacillus*, clarifying that the *Lactobacillus fermentatus* used in this invention (…) The compliance status of LA729 in food production and processing provides a legal basis and safety guarantee for its application in the food or functional product fields.

[0033] The fermentation of *Lactobacillus mucilaginosus* of the present invention ( LA729 is a safe and non-toxic product. During fermentation, it secretes specific glycosidases that selectively hydrolyze sugar residues on ginsenoside Re, thereby reconstructing its structure and generating the target rare saponin. It can efficiently convert ginsenoside Re into saponin F4 through directional transformation. Based on being green, environmentally friendly, safe and controllable, it significantly improves the preparation efficiency of rare ginsenoside F4 and has good prospects for industrial application and market promotion value.

[0034] This invention achieves highly selective conversion of ginsenoside Re to rare ginsenoside F4 under mild conditions by optimizing parameters such as the composition of the liquid fermentation medium, pH, temperature, initial substrate concentration, and fermentation time. The result is low byproduct and stable yield. High-performance liquid chromatography (HPLC) is used to separate, purify, and confirm the structure of the reaction products, verifying the efficiency and controllability of the method. Attached Figure Description

[0035] Figure 1 This is the HPLC chromatogram of the ginsenoside Re standard used in Example 3 of the present invention;

[0036] Figure 2 This is the HPLC chromatogram of ginsenoside F4 standard in the prior art of this invention;

[0037] Figure 3 This is the HPLC chromatogram of ginsenoside F4 prepared in Example 3 of the present invention. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments.

[0039] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, and equipment used in the following embodiments are commercially available. The ginsenoside Re (HPLC ≥ 98%) of this invention was purchased from Chengdu EFA Biotechnology Co., Ltd.

[0040] Example 1: Isolation, Identification and Preservation of Strains

[0041] 1. Isolation of strains

[0042] Samples and source for strain isolation: Rice wine samples collected in Yanji City, Jilin Province in May 2025. The collected samples were ground, serially diluted, and spread onto MRS solid agar plates. After incubation at 37℃ for 48 hours, typical colonies (translucent milky white, round, with neat, raised edges, translucent, and smooth surface) were picked and streaked onto MRS agar plates to isolate pure colonies. The pure cultures were inoculated onto MRS liquid agar and cultured, then 60% glycerol was added, and the culture was stored at -80℃. One strain of *Lactobacillus fermentatus* was selected, hereinafter referred to as LA729.

[0043] 2. Identification of strains

[0044] Physiological and biochemical identification results of LA729: Gram staining positive, catalase test negative, benzidine test negative, indole test negative, acetylmethylmethanol test positive; does not hydrolyze starch, does not liquefy gelatin, does not produce hydrogen sulfide, ferments glucose to produce acid but not gas; a non-motile bacillus; can grow at 15℃ and 45℃, with an optimal growth temperature of 37–42℃; suitable pH of 5.0–7.0; tolerates 6.5% NaCl; LA729 grows uniformly turbidly in MRS liquid medium, and white precipitate forms after prolonged standing. The identification results are shown in Table 1.

[0045] Table 1. API 50CH (bioMérieux, France) Qualification Results of LA729

[0046]

[0047] In the table above, "+" indicates a positive reaction of the strain; "-" indicates a negative reaction of the strain.

[0048] 16S rDNA gene sequence amplification and alignment. Genomic DNA was extracted using the CTAB method. Based on conserved regions of the gene sequence, amplification primer sequences for 16S rDNA were designed (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (e.g., SEQ ID 1), 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (e.g., SEQ ID 2)) and sequencing primer sequences (V4-515F: GTGCCAGCAGCCGCGGTAA (e.g., SEQ ID 3), V4-806R: GGACTACCAGGGTATCTAA (e.g., SEQ ID 4)). After PCR amplification and product sequencing, the 16S rDNA sequence of the obtained strain was compared with the sequence information of known strains in the GenBank library using BLAST for homology analysis to identify the test strain. Genus and species classification was performed based on 16S rDNA sequence homology greater than 99%. The obtained 16S rDNA sequence is as follows:

[0049]

[0050] Homology analysis of the 16S rDNA sequence in the GenBank database using the BLAST program revealed that LA729 shared more than 99% homology with *Lactobacillus fermentatus*. Based on these results, LA729 was identified as belonging to the genus *Lactobacillus*, specifically *Lactobacillus fermentatus*.

[0051] 3. Preservation of bacterial strains

[0052] The fermentation of *Lactobacillus mucinus* of this invention is classified and named as follows: LA729 was deposited on August 4, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, accession number: CCTCC NO:M 20251765.

[0053] Example 2: Detection of LA729 glucosidase activity

[0054] First, LA729 was inoculated into MRS liquid medium and cultured statically at 37°C for 16 h. Then, it was centrifuged at 4°C and 6000 rpm for 8 min. The supernatant was taken as a liquid sample, which can be directly used for the determination of β-glucosidase (β-GC) activity.

[0055] Follow the instructions for the R41764 β-GC activity assay kit (visible spectrophotometry): Preheat the spectrophotometer for ≥30 min, set the wavelength to 400 nm, and zero the instrument with distilled water; dissolve R41764-A powder in distilled water immediately before use. Prepare one test tube and one control tube for each sample. Add 400 μL of R41764-A, 500 μL of R41764-B, and 100 μL of sample to the test tube; add 500 μL of R41764-B and 100 μL of sample (without R41764-A) to the control tube. Mix well and incubate at 37°C for 30 min, then at 95°C for 5 min to terminate the reaction and cool. Then, add 400 μL of R41764-A to the control tube. Centrifuge each tube at 4℃ and 8000g for 5 min, take 500 μL of the supernatant, mix with 1000 μL of R41764-C, let stand at room temperature for 2 min, and read the absorbance at 400 nm to obtain A determination and A control respectively; the standard is processed in the same way to obtain A standard and A blank.

[0056] For quantitative calculation, standard curves were established by preparing a series of 300 nmol / mL, 200 nmol / mL, 100 nmol / mL, 50 nmol / mL, 25 nmol / mL, 10 nmol / mL, and 0 nmol / mL using the 5 μmol / mL pNP standard solution provided in the kit. ΔA was calculated (ΔAdetermined = Adetermined − Acontrol; ΔAstandard = Astandard − Ablank) and the product concentration x (nmol / mL) was converted from the curves.

[0057] Table 2. LA729 glucosidase activity

[0058]

[0059] Example 3: Fermentation and preparation of ginsenoside F4

[0060] Step 1: Preparation of bacterial suspension

[0061] LA729 cells were first inoculated into liquid culture medium and incubated statically at 37°C for 16 hours. The cells were then collected by centrifugation (4°C, 6000 rpm, 8 min). Finally, the cell pellet was resuspended in liquid culture medium to achieve a viable count of 1.0 × 10⁻⁶ cells. 9 CFU / mL, to obtain a bacterial suspension;

[0062] The formulation of the MRS liquid culture medium is as follows: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L ammonium citrate, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween 80L, using deionized water as the solvent, with a pH of 6.2-6.8, and sterilized at 121°C for 15-20 min.

[0063] Step 2: Fermentation and Product Preparation

[0064] Inoculate 10 mL of the bacterial suspension from step one into 1 L of liquid fermentation medium containing ginsenoside Re, and ferment at 37 °C for 14 days. After fermentation, concentrate the fermentation product under reduced pressure at 70 °C, freeze-dry under vacuum, and extract the dried product with methanol (solid-liquid ratio 1 g: 10 mL, extraction temperature 30 °C, extraction time 60 min, extraction times 3 times). Centrifuge (10000 rpm, 15 min, 4 °C), and freeze-dry the supernatant under reduced pressure after solvent recovery to obtain ginsenoside F4.

[0065] The liquid fermentation medium containing ginsenoside Re comprises 3 g / L glucose, 1 g / L yeast extract, and 5 g / L ginsenoside Re, with deionized water as the solvent and pH = 6.0~7.0.

[0066] Example 4: Identification of Ginsenoside F4

[0067] The ginsenoside Re used in Example 3, the existing ginsenoside F4 standard, and the ginsenoside F4 prepared in Example 3 were dissolved in methanol, filtered through a 0.22 μm microporous membrane, and then used for chromatographic analysis.

[0068] HPLC chromatographic analysis method: The chromatographic column was an Agilent Pursuit 5 SB-C18 column, the injection volume was 20 μL, the flow rate was 1 mL / min, the column temperature was 30℃, and the detection wavelength was 203 nm. The mobile phase was A: water, B: acetonitrile. The concentrations were as follows: 0–40 min: 18–21% (B); 40–42 min: 21–26% (B); 42–46 min: 26–32% (B); 46–66 min: 32–34% (B); 66–71 min: 34–38% (B); 71–77.70 min: 38.0–49.1% (B); 77.70–82 min: 49.1% (B); 82–83 min: 49.1–50.6% (B); 83–88 min: 50.6–59.6% (B); 88–89.80 min: 59.6–65.0% (B); 89.80–97 min: 65% (B); 97–102 min… 65~75%(B); 102~110min 75~85%(B); 110~115min 85%(B); 115~125min 85~18%(B); 125~130min 18.0%(B).

[0069] The HPLC chromatograms of ginsenoside Re and ginsenoside F4 standards are as follows: Figure 1 and Figure 2 As shown, the HPLC chromatogram of ginsenoside F4 produced by fermentation of ginsenoside Re is as follows. Figure 3 As shown. From Figures 1-3 It can be seen that the retention time of ginsenoside F4 prepared in Example 3 under HPLC chromatographic conditions is consistent with that of ginsenoside F4 standard. The molecular ion peak of ginsenoside F4 prepared in Example 3 is 766.998, confirming that ginsenoside Re is converted into ginsenoside F4 by fermentation with Lactobacillus mucilaginosus LA729.

[0070] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the embodiments. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all embodiments here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Fermented Lactobacillus mucilaginosus, characterized in that, The fermenting *Lactobacillus mucinus* is classified as follows: LA729; The fermenting Lactobacillus mucinus was deposited on August 4, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, accession number: CCTCC NO:M 20251765.

2. The application of the fermented *Lactobacillus mucinus* according to claim 1, characterized in that, The fermented *Lactobacillus mucilaginosus* can ferment ginsenoside Re into ginsenoside F4.

3. The application of the fermented *Lactobacillus mucinus* according to claim 2, characterized in that, First, fermented Lactobacillus mucinus was prepared into a bacterial suspension. Then, the bacterial suspension was inoculated into a liquid fermentation medium containing ginsenoside Re and fermented together. After fermentation, the fermentation product was dried to obtain ginsenoside F4.

4. The application of the fermented *Lactobacillus mucinus* according to claim 3, characterized in that, include: Step 1: Preparation of bacterial suspension First, *Lactobacillus fermentatus* was inoculated into liquid culture medium and incubated statically at 37–45°C for 16–25 h. Then, the bacterial pellet was collected by centrifugation and finally resuspended in liquid culture medium to achieve a viable count of 1.0 × 10⁻⁶ cells / mL. 9 ~1.0×10 10 CFU / mL, to obtain a bacterial suspension; Step 2: Fermentation and Product Preparation Inoculate 10-30 mL of the bacterial suspension from step one into 1 L of liquid fermentation medium containing ginsenoside Re, and let it ferment at 37-42℃ for 7-14 days. After fermentation, concentrate the obtained fermentation product under reduced pressure, freeze dry under vacuum, extract the dried product with an extractant, centrifuge, and freeze dry the supernatant under reduced pressure to obtain ginsenoside F4. The liquid fermentation medium containing ginsenoside Re comprises glucose at a concentration of 2-5 g / L, yeast extract at a concentration of 1-3 g / L, and ginsenoside Re at a concentration of 5-10 g / L, using deionized water as a solvent, with a pH of 6.0-7.

0. The extractant is one or more of alcohols and ethyl acetate.

5. The application of the fermented *Lactobacillus mucinus* according to claim 4, characterized in that, In step one, all liquid culture media are MRS liquid culture media.

6. The application of the fermented *Lactobacillus mucinus* according to claim 5, characterized in that, The formulation of the MRS liquid culture medium is as follows: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L ammonium citrate, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween 80L, using deionized water as the solvent, with a pH of 6.2-6.8, and sterilized at 121°C for 15-20 min.

7. The application of the fermented *Lactobacillus mucinus* according to claim 4, characterized in that, In step one, the centrifugation conditions are: centrifugation at 4000~8000 rpm for 5~10 min.

8. The application of the fermented *Lactobacillus mucinus* according to claim 4, characterized in that, Step two has one or more of the following characteristics: The temperature for vacuum concentration is 40~70℃; Before vacuum freeze drying, the samples were pre-frozen to -50℃ to -30℃, and the vacuum freeze drying time was 12 to 36 hours. The alcohols are one or more of methanol, ethanol, and n-butanol; The solid-liquid ratio of the extraction is 1g:5mL to 1g:20mL; The extraction temperature is 25~40℃, the extraction time is 30~120min, and the extraction is performed 1~3 times. The centrifugation conditions are: temperature 4~10℃, speed 8000~12000rpm or 9000~12000×g, time 10~20min.

9. The application of the fermented *Lactobacillus mucinus* according to claim 8, characterized in that, The temperature for vacuum concentration is 50~60℃; The alcohols are one or both of methanol and ethanol; The centrifugation conditions were: temperature 4℃, speed 10000rpm, and time 15min.

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