Lactobacillus plantarum JSU32 and applications thereof

Abstract

The application discloses a lactobacillus plantarum JSU32, which has a preservation number of CGMCC No.39280 and a classification name of Lactiplantibacillus plantarum. As an endophytic fungus of ginseng, the strain is natural and safe, and is suitable for development and application in the fields of food and health food, and has a good industrialization prospect.

Description

Technical Field

[0001] This invention discloses a strain of Lactobacillus plantarum JSU32 and its applications, relating to the field of microbial fermentation technology. Background Technology

[0002] Ginsenoside Rb1, as the main active ingredient in ginsenosides, has anti-inflammatory and immunomodulatory effects. It can be transformed into different rare ginsenoside monomers. Among them, the enzymatic hydrolysis products of Rb1, ginsenosides CK and F2, play important roles in immunomodulation and oxidative stress.

[0003] Ginsenoside F2, as an antioxidant, can enhance the activity of antioxidant enzymes and reduce oxidative stress damage to hepatocytes. Anti-inflammatory activity is also an important biological activity of ginsenoside F2. Studies have shown that F2 can inhibit TPA-induced reactive oxygen species (ROS) generation and subsequently reduce IL-17A levels, thereby alleviating the inflammatory response.

[0004] Ginsenoside CK is one of the most promising anti-cancer ginsenosides, inducing apoptosis in cancer cells through both mitochondrial and death receptor pathways. It can activate Caspase-8, Caspase-9, and Caspase-3, leading to DNA fragmentation. It also enhances tissue sensitivity to insulin, inhibits gluconeogenesis, and promotes glycolysis, showing promising therapeutic effects on type 2 diabetes.

[0005] Studies have shown that macromolecular ginsenosides can be converted into smaller, rarer ginsenosides with higher bioactivity by hydrolyzing glucose residues through enzymes such as β-glucosidase secreted by lactic acid bacteria. Therefore, screening for and identifying new strains of lactic acid bacteria capable of producing ginsenosides will be of great value. Summary of the Invention

[0006] This application describes the isolation of a ginseng endophytic lactobacillus strain with deep transformation capabilities from ginseng, whose metabolic enzyme system may have a stronger adaptability to ginsenoside substrates.

[0007] The *Lactobacillus plantarum* JSU32 obtained by screening in this invention has the preservation number CGMCC No. 39280 and the classification name: *Lactiplantibacillus plantarum*. Date of deposit: June 5, 2026; Address of deposit: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing; Depository institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee.

[0008] The strain was identified using universal primers for 16S DNA: F5'-AGAGTTTGATCMTGGCTCAG-3' and R5'-GGTTACCTTGTTACGACTT-3' were used for identification. After PCR, the results were verified using 1% agarose gel.

[0009] Furthermore, the probiotics were compared with those in the NCBI database for homology. Lactobacillus plantarum With 100% homology, it belongs to the genus *Lactobacillus plantarum*, and is named *Lactobacillus plantarum* JSU32.

[0010] The beneficial effects achieved by this invention are as follows: *Lactobacillus plantarum* JSU32 can achieve a complete transformation chain of "Rb1→Rd→F2→CK", with the fermentation product containing 0.15 mg / g of F2 and 0.05 mg / g of CK, filling a technological gap in the field of deep transformation of this genus. *Lactobacillus plantarum* JSU32 is a food-grade lactobacillus that can be directly used in the development of functional foods, and its application threshold is lower compared to non-food-grade strains such as *Codonopsis pilosula* endophytic bacteria and *Aspergillus niger*. Attached Figure Description

[0011] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a photograph of Lactobacillus plantarum JSU32 plate; Figure 2 This is the microscopic morphology of Lactobacillus plantarum JSU32. Detailed Implementation

[0012] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0013] Example 1: Isolation, purification and identification of Lactobacillus fermentum (1) Rinse the ginseng sample with sterile water to remove surface dust, transfer the washed sample to 75% ethanol for 5 min for disinfection, and then place it in 5% sodium hypochlorite solution for another 5 min. After disinfection, rinse the sample with sterile water to thoroughly remove residual disinfectant, and place it on sterile gauze to drain surface moisture.

[0014] Place the ginseng sample in a clean bench, remove the ginseng skin and roots, cut the ginseng into small pieces of about 1 mm × 5 mm × 5 mm, embed them in MRS medium, and incubate at 30℃ for 2-7 days. Observe its growth, continuously pass single colonies, and observe the morphology of the strains under a microscope until the strain morphology is uniform.

[0015] The MRS culture medium formulation 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 80, with deionized water as the solvent, pH 6.2-6.8, and sterilized at 121℃ for 20 min.

[0016] (2) Identification of probiotics Single colonies were picked and added to sterile pure water. After repeated pipetting, they were identified using universal primers for 16S DNA: F5'-AGAGTTTGATCMTGGCTCAG-3', R5'-GGTTACCTTGTTACGACTT-3'. After PCR, the results were verified using a 1% agarose gel. The PCR products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequence is as follows: The sequencing results were compared with the NCBI database for homology, and the homology with the database was 100%, belonging to the genus *Lactobacillus plantarum*. It was named *Lactobacillus plantarum* JSU32.

[0017] Example 2

[0018] Lactobacillus plantarum JSU32 was inoculated into MRS medium and cultured statically at 37℃ for 24 h. The OD of the bacterial suspension was measured to be 0.6. After centrifugation at 6000 rpm for 8 min, the bacterial pellet was collected and resuspended in fresh liquid medium to obtain a bacterial suspension.

[0019] Ginseng powder was pulverized through a 1.5 mm sieve using a horizontal grinder for 5 minutes. The ginseng powder was then mixed with liquid MRS medium at a 1:10 ratio and sterilized. The activated probiotic seed culture was added at 10% to the ginseng mixture fermentation medium, and fermented at 37°C for 96 hours. After fermentation, the mixture was centrifuged at 8000 r / min for 5 minutes. The supernatant was collected and freeze-dried for 96 hours.

[0020] Comparative Example 1 The ginseng extract was prepared according to the decoction method specified in the Chinese Pharmacopoeia and served as a non-fermented control group. Powdered ginseng was placed in a decoction pot at a water-to-medicine ratio of 10:1, soaked for 1 hour, and then decocted for 40 minutes. The decoction was filtered, and 300 mL of water was added again to continue decocting the dregs for 45 minutes. The extracts were then filtered again, and both extracts were collected. The extracts were centrifuged at 8000 r / min for 5 minutes. The supernatant was collected and freeze-dried for 96 hours.

[0021] Comparative Example 2 To evaluate the technical advantages of the strain of the present invention, this comparative example was set with the exact same process conditions as Example 2, the only difference being that Lactobacillus paracasei was used instead of the strain of the present invention for fermentation.

[0022] Lactobacillus paracasei was inoculated into MRS medium and incubated at 37°C for 24 h. The OD of the bacterial suspension was measured to be 0.6. After centrifugation at 6000 rpm for 8 min, the bacterial pellet was collected and resuspended in fresh liquid medium to obtain a bacterial suspension.

[0023] The ginseng powder was pulverized through a 1.5 mm sieve using a horizontal grinder for 5 minutes. The ginseng powder was then mixed with liquid MRS medium at a 1:10 ratio and sterilized. The activated probiotic seed culture was added at 10% to the ginseng mixture fermentation medium, and the mixture was incubated at 37°C for 96 hours. After incubation, the mixture was centrifuged at 8000 r / min for 5 minutes. The supernatant was collected and freeze-dried for 96 hours.

[0024] The products obtained in Example 2 and Comparative Examples 1-2 were dissolved in methanol, and the contents of rare ginsenosides F2, CK, Rb1, Rd, and Re in the products were detected by HPLC and the yield was calculated.

[0025] The injection volume was 20 μL, the flow rate was 1 mL / min, the column temperature was 30°C, and the detection wavelength was 203 nm.

[0026] 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); 102–110 min 75~85% (B); 110~115 min 85% (B); 115~125 min 85~18% (B); 125~130 min 18.0% (B).

[0027] The results are shown in Table 1.

[0028] Rb1 is a key indicator of the degree of ginseng cell wall disruption and the efficiency of major saponin release. In Example 2, the Rb1 content reached 2.4 mg / g of the medicinal material, significantly higher than that of Comparative Example 1 (conventional water extraction method according to the Chinese Pharmacopoeia, 0.25 mg / g) and Comparative Example 2 (fermentation using Lactobacillus paracasei, 1.04 mg / g). Compared with Comparative Example 2, the Rb1 content in Example 2 increased by approximately 2.3 times, indicating that the strain of this invention has a significantly better effect than conventional Lactobacillus fermentation process in disrupting ginseng cell walls and releasing major saponins.

[0029] This invention's strain represents the first complete transformation chain in the *Lactobacillus* genus: "Rb1→Rd→F2→CK". In Example 2, this strain successfully converted Rb1 into intermediate F2 (0.15 mg / g) and the final product CK (0.05 mg / g), while neither F2 nor CK was detected in Comparative Example 2. The formation of F2, a highly active rare ginsenoside, signifies that this invention's strain possesses the unique ability to transform primary saponins into deep-seated rare saponins, filling a technological gap in the field of deep transformation within this genus.

[0030] Examples 2 all achieved superior saponin release and conversion effects compared to Comparative Examples 1 and 2, demonstrating good and stable fermentation performance. The strain of this invention is a food-grade *Lactobacillus plantarum*, exhibiting high safety and can be directly applied to the development of functional foods and health products, showing clear industrialization prospects.

[0031] In summary, the *Lactobacillus plantarum* JSU32 provided by this invention is significantly superior to conventional fermentation strains represented by *Lactobacillus paracasei* in terms of ginsenoside Rb1 release efficiency and deep conversion ability of rare ginsenosides F2 and CK, demonstrating outstanding technological advancement and practical application value.

[0032] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Lactiplantibacillus plantarum JSU32, with accession number CGMCC No.39280, is classified as Lactiplantibacillus plantarum. Date of deposit: June 5, 2026; Address of deposit: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing; Depository institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee.

2. The application of Lactobacillus plantarum JSU32 as described in claim 1 in the fermentation and conversion of ginsenosides.

3. The *Lactobacillus plantarum* JSU32 as described in claim 2, characterized in that, The ginsenosides mentioned are ginsenoside Rb1, ginsenoside F2, and ginsenoside CK.

4. The fermentation method of *Lactobacillus plantarum* JSU32 according to claim 1, characterized in that, Lactobacillus plantarum JSU32 was inoculated into MRS medium and cultured at 37°C for 24 h. The bacterial culture was then collected to obtain the activated bacterial culture solution. Ginseng powder was mixed with liquid MRS culture medium at a ratio of 1:10 and then sterilized. The activated bacterial culture was added to the ginseng mixture fermentation medium at a rate of 10%, and fermented at 37°C. After the culture was completed, the culture was centrifuged using a high-speed centrifuge. The supernatant was collected and freeze-dried.

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