Lactobacillus mucilaginosus and application thereof in ginseng fermentation liquor

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses lactobacillus mucilaginosus and application thereof in ginseng fermentation liquor. The lactobacillus mucus LFG-228 is preserved in the Guangdong Province Microbial Culture Collection Center, the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 66778, and the preservation date is July 30, 2025. The lactobacillus mucus LFG-228 has the advantages that the lactobacillus mucus LFG-228 can be used for preparing the lactobacillus mucus LFG-228; according to the invention, the unique metabolic characteristics of the lactobacillus mucus LFG-228 are found and utilized for the first time. The strain can efficiently and directionally convert main saponins in ginseng into rare ginsenosides Rk1 and Rg5. Experimental data show that after the strain is fermented, the content of Rk1 and Rg5 in fermentation liquor is greatly increased, and the blank of targeted production of the two high-activity saponins in the prior art is filled.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of fermenting *Lactobacillus mucilaginosus* and its application in ginseng fermentation broth. Background Technology

[0002] Ginseng, a traditional and precious Chinese medicinal herb, is rich in various bioactive components, among which ginsenosides are its most important pharmacological basis. However, the major naturally occurring ginsenosides in ginseng (such as Rb1, Rc, and Rd) have low biological activity and poor absorption and utilization by the human body due to the high number of glycosyl groups in their molecular structure. Modern research shows that these major saponins need to undergo structural transformations such as deglycosylation to generate rarer ginsenosides (such as Rk1, Rg5, and Rg3) in order to be more effectively absorbed by the human body and exert stronger biological activities, such as antioxidant, antitumor, and anti-fatigue effects.

[0003] Microbial fermentation has become an important biotransformation technology for obtaining rare ginsenosides. Existing technologies include research on ginseng fermentation using lactic acid bacteria. However, current fermentation techniques still have certain limitations. First, the metabolic enzyme systems of different strains exhibit specificity, decisively influencing the transformation pathway and final products of ginsenosides. Current technologies largely focus on the transformation of saponins such as Rg3 and F2, while specific strains and related fermentation processes are lacking for the efficient and targeted transformation of equally active rare ginsenosides Rk1 and Rg5. Second, an excellent fermentation strain should not only possess high transformation capacity but also synergistically enhance the overall quality of the fermentation product, including the content of other active substances (such as total flavonoids and total polyphenols) and overall antioxidant activity. Current technologies still have room for improvement in this regard.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to solve at least one of the above technical problems, and provides a strain of fermenting *Lactobacillus mucilaginosus* and its application in ginseng fermentation broth.

[0006] To achieve the above objectives, the first technical solution adopted by the present invention is as follows: A fermenting lactobacillus ( Limosilactobacillus fermentum LFG-228 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66778 and deposit date of July 30, 2025.

[0007] The second technical solution adopted in this invention is: A microbial inoculant comprising at least one of the fermenting *Lactobacillus mucinus* LFG-228, its culture, or fermentation supernatant as described in the first technical solution.

[0008] The third technical solution adopted in this invention is: The application of the fermented Lactobacillus mucinus LFG-228 as described in the first technical solution or the microbial agent as described in the second technical solution in any of the following: Preparation of rare ginsenosides Rk1 and / or Rg5; Preparation of ginseng fermentation broth; Increase the content of rare ginsenosides Rk1 and / or Rg5 in ginseng fermentation liquid; Prepare drugs containing rare ginsenosides Rk1 and / or Rg5.

[0009] The fourth technical solution adopted in this invention is: A method for preparing ginseng fermentation liquid includes: fermenting treated ginseng raw materials with either the fermenting Lactobacillus mucilaginosus LFG-228 described in the first technical solution or the microbial agent described in the second technical solution.

[0010] Preferably, the inoculum size of the fermenting Lactobacillus mucinus LFG-228 is 1%-5%.

[0011] Preferably, the fermenting *Lactobacillus mucinus* LFG-228 is inoculated in the form of a seed culture, the concentration of which is 1×10⁻⁶. 8 CFU / mL and above.

[0012] Preferably, the fermentation temperature is 25℃-35℃ and the fermentation time is 1-5 days.

[0013] Preferably, the fermentation is carried out on a shaker at a speed of 150-300 rpm.

[0014] The present invention also provides a ginseng fermentation broth prepared using a fourth technical solution. The fermentation broth contains at least 0.44 mg / g of rare ginsenoside Rk1 and at least 0.52 mg / g of rare ginsenoside Rg5.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover and utilize the unique metabolic characteristics of *Lactobacillus fermentans* LFG-228. This strain can efficiently and directionally convert the main saponins in ginseng into rare ginsenosides Rk1 and Rg5. Experimental data show that after fermentation by this strain, the content of Rk1 and Rg5 in the fermentation broth is greatly increased, filling the gap in existing technologies for the targeted production of these two highly active saponins.

[0016] The ginseng fermentation broth prepared by this invention not only has an increased content of rare saponins, but also a comprehensive and significant improvement in the content of active substances such as total saponins, total flavonoids, and total polyphenols. This indicates that the LFG-228 strain has extensive biotransformation and dissolution-promoting capabilities during fermentation, endowing the fermentation broth with a richer material basis and higher nutritional and health benefits.

[0017] Thanks to the synergistic increase of antioxidant active ingredients such as Rk1, Rg5, flavonoids, and polyphenols, the ginseng fermentation broth prepared in this invention exhibits extremely strong free radical scavenging ability. In vitro antioxidant experiments confirmed that its DPPH free radical, hydroxyl free radical, and superoxide anion free radical scavenging rates were all much higher than those of the unfermented group and other fermentation groups of strains, indicating that this product has significant advantages in delaying aging and resisting oxidative stress. Attached Figure Description

[0018] Figure 1 This is a PCR amplification fragment map of the strain; Figure 2 Phylogenetic tree of Lactobacillus fermentans LFG-228; Figure 3 The colony morphology of Lactobacillus fermentans LFG-228; Figure 4 Microscopic morphology of Lactobacillus fermentans LFG-228; Figure 5 In the middle, (A) and (B) are SEM images of ginseng residue soaked in Comparative Example 1 at 1000×x and 5000×x magnification, respectively; (C) and (D) are SEM images of ginseng residue after fermentation in Example 2 at 1000×x and 5000×x magnification, respectively. Figure 6 (A) is an X-ray diffraction image of ginseng powder in Comparative Example 1, and (B) is an X-ray diffraction image of fermented ginseng residue in Example 2. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The first embodiment of the present invention provides a fermentation of *Lactobacillus mucilaginosus* (… Limosilactobacillus fermentum LFG-228 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66778 and deposit date of July 30, 2025.

[0021] It should be noted that fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum This is the current official name; its former name was Lactobacillus fermentum. Lactobacillus fermentum ).

[0022] The fermenting Lactobacillus mucinus LFG-228 provided in this embodiment can accurately identify and hydrolyze specific glycosidic bonds on the glycosyl structure of ordinary ginsenosides. Through a series of biotransformation reactions such as deglycosylation, dehydration and isomerization, it can convert ordinary ginsenosides into rare ginsenosides, achieving efficient and targeted conversion of ordinary ginsenosides into rare ginsenosides Rk1 and Rg5.

[0023] The second embodiment of the present invention provides a microbial inoculant comprising at least one of the fermenting Lactobacillus mucinus LFG-228, its culture, or fermentation supernatant as described in the first technical solution.

[0024] The third embodiment of the present invention provides the application of fermenting Lactobacillus mucinus LFG-228 or higher microbial inoculants in any of the following: Preparation of rare ginsenosides Rk1 and / or Rg5; Preparation of ginseng fermentation broth; Increase the content of rare ginsenosides Rk1 and / or Rg5 in ginseng fermentation liquid; Prepare drugs containing rare ginsenosides Rk1 and / or Rg5.

[0025] The third embodiment of the present invention provides a method for preparing ginseng fermentation liquid, comprising: fermenting treated ginseng raw materials with the fermenting Lactobacillus mucilaginosus LFG-228 described in the first technical solution or the microbial agent described in the second technical solution.

[0026] The specific fermentation method used is the microbial fermentation method commonly used in this field. Specific parameters can be selected by those skilled in the art based on the fermentation raw materials and the specific microorganisms' adaptability. For example, it may include pretreating ginseng raw materials, preparing the culture medium, activating the bacterial strain, inoculating the strain with seed liquid, and fermenting under suitable conditions.

[0027] As some preferred embodiments, the inoculum size of the fermenting Lactobacillus mucinus LFG-228 is 1%-5%.

[0028] In some preferred embodiments, the fermenting *Lactobacillus mucinus* LFG-228 is inoculated in the form of a seed culture with a concentration of 1 × 10⁻⁶. 8 CFU / mL and above.

[0029] As some preferred embodiments, the fermentation temperature is 25℃-35℃ and the fermentation time is 1-5 days.

[0030] As some preferred embodiments, the fermentation is carried out on a shaking table at a speed of 150-300 rpm.

[0031] The fermentation broth prepared according to the embodiments of the present invention contains a content of rare ginsenoside Rk1 of not less than 0.44 mg / g and a content of rare ginsenoside Rg5 of not less than 0.52 mg / g.

[0032] The following detailed descriptions of the screening, isolation, and identification of fermenting Lactobacillus mucinus LFG-228, as well as the preparation method and performance of ginseng fermentation broth, are provided through several specific examples.

[0033] Example 1: Screening, isolation and identification of strains Strain screening: Weigh 10g of cleaned ginseng tissue (purchased from Chengdu Jiankang Pharmaceutical Co., Ltd.), cut it into small pieces, place it in a sterile mortar, add 90mL of sterile physiological saline, and grind thoroughly into a homogenate to prepare a 10⁻¹ sample dilution. Using a sterile pipette, pipette 1mL of the 10⁻¹ dilution and add it to 9mL of sterile physiological saline, then serially dilute to 10⁻², 10⁻³, and 10⁻¹. 4 10⁻ 5 10⁻ 6 Prepare equal concentrations for later use. Take 0.1 mL of each dilution at different concentrations and spread it evenly on the surface of MRS solid medium (percentages can be replicated in triplicate). Invert the petri dishes and incubate them in a 37°C anaerobic incubator for 48-72 hours, until obvious colonies appear on the medium. Select colonies that are 1-3 mm in diameter, round, with neat edges, smooth surfaces, and a milky white or pale yellow color.

[0034] Strain purification: Pick the target colonies with a sterile inoculation loop and streak them on fresh MRS solid medium. Repeat the streak operation 2-3 times until a single colony with consistent morphology and purity is obtained.

[0035] Strain identification: DNA extraction and PCR amplification ( Figure 1 After sequencing (as shown in the PCR amplified fragment map), the 16S rRNA gene sequence of this bacterium is as shown in SEQ ID NO: 1. The assembled sequence file was compared with data in the NCBI S rRNA database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) using the NCBIL program, and it was identified as *Lactobacillus fermentatus*. Limosilactobacillus fermentum, Its phylogenetic tree is as follows Figure 2 As shown.

[0036] Strain Preservation: This strain was deposited on July 30, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; *Lactobacillus fermentatus* ( Limosilactobacillus fermentum The accession number for LFG-228 is GDMCC NO: 66778.

[0037] The colony morphology and microscopic morphology of *Lactobacillus fermentatus* LFG-228 are as follows: Figure 3 , 4 As shown.

[0038] Example 2: Preparation of Ginseng Fermentation Broth Ginseng is crushed to 60 mesh and sieved to obtain ginseng powder; Ginseng nutrient solution was prepared by adding 10 g of ginseng powder and 6.5 g of MRS culture medium dry powder to every 100 mL of distilled water. After sterilization, it was used as a fermentation medium and cooled to the inoculation temperature. Activated fermentation of Lactobacillus mucilage LFG-228 was prepared in MRS medium at a concentration of 1×10⁻⁶. 8 CFU / mL seed culture was inoculated into 3mL of ginseng nutrient solution and fermented in a shaker at 30℃ and 250rpm / min for 3 days. After fermentation, the fermentation broth was inactivated at 100℃, filtered to obtain the fermentation broth, centrifuged at 8000 rpm / min and the supernatant was collected to obtain the clear ginseng fermentation broth.

[0039] In this example, the SEM 1000× and SEM 5000× images of the ginseng residue obtained after fermentation are shown in the figures below. Figure 5 As shown in (C) and (D).

[0040] Example 3: Preparation of Ginseng Fermentation Broth The preparation method is the same as in Example 2, except that the ginseng powder is 40 mesh and the fermentation temperature is 25°C.

[0041] Example 4: Preparation of Ginseng Fermentation Broth The preparation method is the same as in Example 2, except that the ginseng powder is 80 mesh and the fermentation temperature is 35°C.

[0042] Example 5: Preparation of Ginseng Fermentation Broth The preparation method is the same as in Example 2, except that the ginseng powder is 40 mesh, the fermentation temperature is 35℃, and the fermentation time is 5 days.

[0043] Example 6 Preparation of Ginseng Fermentation Broth The preparation method is the same as in Example 2, except that the inoculation amount of the fermented Lactobacillus mucin LFG-228 seed liquid is 5 mL and the fermentation temperature is 35℃.

[0044] Comparative Example 1 Compared with Example 2, the only difference is that it was not inoculated with Lactobacillus fermentum LFG-228, but was directly soaked in water.

[0045] The SEM 1000× and SEM 5000× images of the ginseng residue obtained in this comparative example are shown below. Figure 5 As shown in (A) and (B).

[0046] Comparative Example 2 Compared with Example 2, the only difference is that an equal amount of Saccharomyces cerevisiae SCFF173 seed culture (deposited at the College of Food and Brewing Engineering, Sichuan University of Science and Engineering, published in Lujun Hu, Xiaodie Chen, Yulan Cao, Pei Gao, TengXu, Dake Xiong, Zhifeng Zhao. Lactiplantibacillus plantarum exerts strain-specific effects on malolactic fermentation, antioxidant activity, and aroma profile of apple cider[J]. Food Chemistry: X, 2024: 101575.) seed culture was used to replace the Lactobacillus mucilaginosus LFG-228 seed culture.

[0047] Performance testing of ginseng fermentation broth in experimental cases The active substance content, antioxidant activity, rare saponin composition and content of the samples prepared in Examples 2-6 and Comparative Examples 1-2 were detected.

[0048] Methods for detecting the content of active substances: The method for detecting total phenols shall be in accordance with GB / T 5009.11-2017. The method for detecting total flavonoids shall be in accordance with GB / T 5009.248-2016. The method for detecting total saponins shall be in accordance with DB22 / T 1668-2012.

[0049] Antioxidant activity detection methods: DPPH free radical scavenging rate detection, hydroxyl free radical scavenging rate detection, and superoxide anion free radical scavenging rate detection were performed according to CN105861381A.

[0050] Table 1 Content of active substances in each sample

[0051] Table 2 Antioxidant activity of each sample

[0052] Table 3. Composition and content of rare saponins in each sample .

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum LFG-228 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66778 and deposit date of July 30, 2025.

2. A microbial inoculant, characterized in that, at least one of the fermented Limosilactobacillus fermentum LFG-228, culture or fermentation supernatant thereof according to claim 1.

3. The fermented Limosilactobacillus fermentum LFG-228 according to claim 1 or the microbial inoculant according to claim 2 for use in any one of: preparing rare ginsenosides Rk1 and / or Rg5; preparing ginseng fermentation broth; increasing the content of rare ginsenosides Rk1 and / or Rg5 in ginseng fermentation broth; preparing a medicine containing rare ginsenosides Rk1 and / or Rg5.

4. A method for preparing ginseng fermentation liquid, characterized in that, comprising: fermenting the treated ginseng raw material with the fermented Limosilactobacillus fermentum LFG-228 according to claim 1 or the microbial inoculant according to claim 2.

5. The production method according to claim 4, wherein The inoculation amount of the Limosilactobacillus fermentum LFG-228 is 1%-5%.

6. The production method according to claim 4, wherein The fermenting L. m. LFG-228 is inoculated in the form of a seed broth, the concentration of which is 1 x 10 8 CFU / mL and above.

7. The production method according to claim 4, wherein The fermentation temperature is 25-35℃, and the fermentation time is 1-5d.

8. The production method according to claim 4, wherein The fermentation is carried out under shaking conditions at a rotation speed of 150-300 rpm.

9. A ginseng fermentation broth, characterized by, prepared by the preparation method according to claim 4 or 5.

10. The ginseng fermentation liquid as described in claim 9, characterized in that, The content of rare ginsenoside Rk1 in the fermentation broth is not less than 0.44 mg / g, and the content of rare ginsenoside Rg5 is not less than 0.52 mg / g.

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