Lactobacillus plantarum and its application in the preparation of drugs for the prevention and / or treatment of vaginitis

Abstract

This invention relates to the field of microbiology, and discloses a strain of *Lactobacillus plantarum* and its application in the preparation of drugs for the prevention and / or treatment of vaginitis. The *Lactobacillus plantarum* strain has the accession number CGMCC No. 33152. The *Lactobacillus plantarum* strain provided by this invention has a strong antibacterial effect against common vaginal pathogens such as *Atopobacterium*, *Staphylococcus aureus*, *Candida albicans*, *Escherichia coli*, and *Gardnerella vaginalis*. It can also reduce the degree of infection by inhibiting the colonization and reproduction of pathogens, and decrease the levels of pro-inflammatory factors and increase the levels of anti-inflammatory factors in diseased vaginitis tissue.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically to a strain of Lactobacillus plantarum and its use in the preparation of drugs for the prevention and / or treatment of vaginitis. Background Technology

[0002] Bacterial vaginosis (BV) is one of the most common lower genital tract diseases in women, primarily caused by Gardnerella vaginalis (Gardnerella vaginalis). Gardnerella vaginalis ), vaginal atobacillus ( Atopobium minutum This is caused by a significant increase in the number of pathogenic bacteria such as Gardnerella vaginalis and Candida albicans. Besides BV, vulvovaginal candidiasis (VVC) is also one of the most common vaginal diseases in women. These vaginal diseases disrupt the vaginal microecological balance, causing significant harm to women's physical and mental health. Traditional clinical treatments mainly use antibiotics and estrogen to treat vaginal inflammation, but these methods often have adverse reactions and are prone to recurrence. Microorganisms are frequently used to treat or prevent vaginitis caused by pathogenic bacteria, but existing strains can only inhibit Gardnerella vaginalis and Candida albicans. Candida albicans There are no strains that specifically inhibit Atobococcus.

[0003] Therefore, there is an urgent need to develop strains that can simultaneously inhibit Atobococcus and other pathogenic bacteria, and that can be used to prepare drugs for the prevention and / or treatment of vaginitis. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and to provide a strain of Lactobacillus plantarum and its use in the preparation of drugs for the prevention and / or treatment of vaginitis.

[0005] To achieve the above objectives, the first aspect of the present invention provides a strain of *Lactobacillus plantarum* (… Lactobacillus plantarum The preservation number of the *Lactobacillus plantarum* is CGMCC No. 33152.

[0006] A second aspect of the present invention provides a microbial agent containing *Lactobacillus plantarum* as described above.

[0007] A third aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of pharmaceutical plants for the prevention and / or treatment of pathogenic bacterial colonization.

[0008] The fourth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for the prevention and / or treatment of the proliferation of pathogenic bacteria.

[0009] The fifth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for the prevention and / or treatment of vaginitis.

[0010] The sixth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for reducing the level of pro-inflammatory factors.

[0011] The seventh aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for increasing the level of anti-inflammatory factors.

[0012] The beneficial effects obtained by the present invention through the above technical solution include at least the following:

[0013] (1) The strain provided by the present invention has a strong ability to produce acid and H2O2, and has a strong antibacterial effect on common vaginal pathogens such as Atobococcus, Staphylococcus aureus, Candida albicans, Escherichia coli and Gardnerella vaginalis. It can also reduce the degree of infection of pathogens by inhibiting the colonization and reproduction of pathogens.

[0014] (2) The *Lactobacillus plantarum* provided by this invention can reduce the level of pro-inflammatory factors and increase the level of anti-inflammatory factors in tissues with vaginitis, reduce the degree of tissue disease, and does not cause cell hemolysis. It is also sensitive to common antibiotics, so it can be used to prepare microbial preparations and has the potential to prepare drugs for the prevention and / or treatment of vaginal diseases.

[0015] Biological Preservation

[0016] The strain provided by this invention is classified and named *Lactobacillus plantarum*. Lactobacillus plantarum It was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33152 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum CCNH263 provided by the present invention;

[0018] Figure 2 This is a Gram micrograph of Lactobacillus plantarum CCNH263 provided by the present invention;

[0019] Figure 3 These are the cell adhesion microscopy results from the negative control group in Example 9;

[0020] Figure 4 These are the cell adhesion microscopy results of Lactobacillus plantarum CCNH263 in Example 9;

[0021] Figure 5These are the cell adhesion microscopy results of UREX-1 in Example 9;

[0022] Figure 6 These are the cell adhesion microscopy results of the negative control group in Example 10 that inhibited Candida albicans;

[0023] Figure 7 This is the microscopic result of cell adhesion inhibition of Candida albicans by Lactobacillus plantarum CCNH263 in Example 10;

[0024] Figure 8 These are the microscopic results of UREX-1 inhibiting Candida albicans cell adhesion in Example 10;

[0025] Figure 9 This refers to the vaginal redness and swelling score results in mice in Example 12;

[0026] Figure 10 These are pathological sections of vaginal tissue from the model group, control group, and UREX-1 group in Example 12;

[0027] Figure 11 These are pathological sections of vaginal tissue from groups CCNH263L, CCNH263M, and CCNH263H in Example 12. Detailed Implementation

[0028] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] The first aspect of this invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarum The preservation number of the *Lactobacillus plantarum* is CGMCC No. 33152.

[0030] The *Lactobacillus plantarum* strain with accession number CGMCC No. 33152 provided by this invention was isolated from fermented mare's milk in Xinjiang. After cultivation, milky-white, smooth, round or oval single colonies were obtained. The colony morphology and Gram microscopic images on the culture medium plates are shown below. Figure 1 and Figure 2 As shown. Further experiments have demonstrated that the *Lactobacillus plantarum* strain with accession number CGMCC No. 33152 provided by this invention possesses excellent growth performance, resistance to gastric acid, resistance to bile salts, and acid production performance. It can inhibit the colonization of pathogenic bacteria in the gastrointestinal tract through its high self-aggregation and hydrophobic properties. Furthermore, this strain does not cause hemolysis.

[0031] "CCNH263" is the strain number assigned by the inventors during the strain screening process, and "CGMCC No.33152" is the preservation number of the strain. Both represent the same strain, and their numbers can be used interchangeably in the following text.

[0032] A second aspect of the present invention provides a microbial agent containing *Lactobacillus plantarum* as described above.

[0033] The present invention does not impose any particular limitation on the type of microbial agent, and it can be any type of microbial agent existing in the art. According to a preferred embodiment of the present invention, the microbial agent is selected from at least one of liquid microbial agents, concentrated microbial agents, and solid microbial agents.

[0034] In this invention, the bacterial agent may also contain excipients, preferably selected from protectants (e.g., freeze-drying protectants, such as skim milk powder, maltodextrin, trehalose, dextran and glycerol, etc.) and / or buffers (e.g., buffer solutions remaining from the preparation of solid bacterial agents).

[0035] According to the present invention, the excipients may be selected from glycerin and / or corn oil.

[0036] Preferably, the viable count of the *Lactobacillus plantarum* is 10 relative to 1 mL of excipient. 4 -10 12 CFU.

[0037] The *Lactobacillus plantarum* provided by this invention can produce a large number of live *Lactobacillus plantarum* cells through liquid culture. The culture method is not particularly demanding, as long as it enables the *Lactobacillus plantarum* to proliferate. For example, live *Lactobacillus plantarum* cells can be inoculated into a culture medium at an inoculum volume of 1-5 vol%, and cultured at 30-40°C for 9-50 hours to obtain a culture solution. The culture medium can be any culture medium suitable for *Lactobacillus plantarum* culture known in the art, such as MRS medium.

[0038] This invention can further isolate live cells of Lactobacillus plantarum from the above-mentioned culture medium. The method of separation is not particularly limited, as long as it can enrich the cells from the culture medium. For example, it can be achieved by centrifugation and / or filtration. The conditions for centrifugation and filtration can be known conditions, which will not be elaborated here.

[0039] A third aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting the colonization of pathogenic bacteria.

[0040] The fourth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting the growth of pathogenic bacteria.

[0041] In this invention, the pathogenic bacteria may include Escherichia coli (Escherichia coli) Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella Candida albicans ( Candida albicans Gardnerella vaginalis Gardnerella vaginalis ) and Atobococcus ( Atopobium minutum At least one of the following, preferably Atobococcus.

[0042] According to some embodiments of the present invention, the Salmonella preferably includes Salmonella Typhimurium (Salmonella Typhimurium). Salmonella typhimurium ) and Salmonella enteritidis ( Salmonella enteritidis ).

[0043] The fermentation supernatant prepared from *Lactobacillus plantarum* provided by the present invention can inhibit the reproduction of *Atopobacterium*. According to a preferred embodiment of the present invention, the supernatant of *Lactobacillus plantarum* provided by the present invention can achieve an inhibition rate of 99% against *Atopobacterium*. Therefore, *Lactobacillus plantarum* provided by the present invention is particularly suitable for inhibiting *Atopobacterium*.

[0044] The fifth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for the prevention and / or treatment of vaginitis.

[0045] The sixth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for reducing the level of pro-inflammatory factors.

[0046] According to the present invention, the *Lactobacillus plantarum* provided by the present invention can inhibit the increase in the levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β and IL-8) caused by *Candida albicans* infection and pro-inflammatory factors (TNF-α and IL-1β) caused by *Gardnerella vaginalis* infection.

[0047] The seventh aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for increasing the level of anti-inflammatory factors.

[0048] According to the present invention, the *Lactobacillus plantarum* provided by the present invention can inhibit the decrease in the levels of anti-inflammatory factors IL-4 and IL-10 caused by *Candida albicans* infection.

[0049] According to the present invention, the drug may include pharmaceutically acceptable excipients.

[0050] According to the present invention, the drug preferably includes the fermentation supernatant of Lactobacillus plantarum as described above and / or a bacterial solution containing live bacteria.

[0051] The present invention will be described in detail below through embodiments.

[0052] Unless otherwise specified, all chemical / biological reagents used in the following examples were purchased from legitimate chemical / biological reagent suppliers.

[0053] Escherichia coli ( Escherichia coli (1) ATCC 25922, purchased from the American Center for Type Culture Collection; (2) CICC 10421, purchased from the China Industrial Microbial Culture Collection Center.

[0054] Staphylococcus aureus ( Staphylococcus aureus The items, numbered CMCC(B)26001 and CMCC(B)26003, were purchased from the China Medical Bacteriological Culture Collection Center.

[0055] Salmonella, (1) Salmonella typhimurium ( Salmonella typhimurium (2) Salmonella enteritidis (ATCC 14028), purchased from the American Center for Type Culture Collection; Salmonella enteritidis (Item number CVCC 3378), purchased from the National Veterinary Microbial Culture Collection Center;

[0056] Candida albicans ( Candida albicans (1) CICC 1965, purchased from China Industrial Microbial Culture Collection Center; (2) ATCC 10231, purchased from the American Type Culture Collection Center.

[0057] Gardnerella vaginalis ( Gardnerella vaginalis (ATCC 14018), purchased from the American Center for Type Culture Collection;

[0058] Atobococcus ( Atopobium minutum (ATCC 33267), purchased from the American Center for Type Culture Collection;

[0059] MRS liquid medium: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 20g glucose, 1mL Tween-80, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1L deionized water, pH 6.5, autoclaved; add 18g agar before final volume adjustment to obtain MRS solid medium.

[0060] Human vaginal epithelial cells (VK2 / E6E7) were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number BFN60808401;

[0061] Double-antibiotic cell culture medium: VK2 / E6E7 cell-specific culture medium, purchased from Shanghai Jinyuan Biotechnology Co., Ltd., product number JY-Y1196;

[0062] Penicillin-Streptomycin: Purchased from Thermo Fisher Scientific, catalog number 15140-122;

[0063] K-SFM complete culture medium: purchased from Guangzhou Kelujie Biotechnology Co., Ltd., product number M1017;

[0064] PBS buffer was purchased from Solarbio, product number P1003;

[0065] Reference strain: (1) Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), with the serial number ATCC 53103, purchased from the American Center for Type Culture Collection, hereinafter referred to as LGG; (2) Lactobacillus reuteri ( Lactobacillus reuteri The compound bacterial agent is a combination of Lactobacillus reuteri and Lactobacillus rhamnosus, wherein the ratio of viable bacteria of Lactobacillus reuteri and Lactobacillus rhamnosus is 1:1. Both strains were isolated from UREX® bacterial powder produced by Chr. Hansen in Denmark (purchased from Shaanxi Mufan Biotechnology Co., Ltd., catalog number MF-00395). The compound bacterial agent will be referred to as UREX-1 below.

[0066] Example 1

[0067] Frozen glycerol tubes of strain CCNH263 and LGG were inoculated at 2% in fresh MRS liquid medium and cultured overnight at 37°C for 18 h. OD 600 Normalization was performed to obtain activated bacterial suspensions, which were then inoculated into fresh MRS liquid medium at a 2% inoculum and cultured at 37°C and 200 rpm for 18 h to obtain expanded bacterial suspensions of CCNH263 and LGG. The OD values ​​of the CCNH263 and LGG expanded bacterial suspensions were measured. 600 The values ​​were 8.27±0.05 and 8.13±0.02, respectively, indicating that CCNH263 had better growth performance than LGG.

[0068] Example 2

[0069] The amplified cultures of Lactobacillus plantarum CCNH263 and LGG were used to evaluate the gastric acid resistance and bile salt resistance of the strains according to T / CNHFA435-2024 "Test Method for Gastric Juice Tolerance of Probiotics".

[0070] In the gastric acid tolerance test, the pH was 3, and the incubation period was 2 hours; in the bile salt tolerance test, the bile salt content was 0.2 wt%, and the incubation period was 4 hours. OD 600 Measure the absorbance of the bacterial solution before and after the experiment.

[0071] Survival rate: (OD before experiment) 600 Value - OD after experiment 600 Value) / OD before experiment 600 Value × 100%.

[0072] The gastric acid resistance survival rates of CCNH263 and LGG were 94.51% and 93.33%, respectively, and the bile salt resistance survival rates were 62.4% and 53.13%, respectively. It can be seen that CCNH263 is superior to LGG in both gastric acid resistance and bile salt resistance.

[0073] Example 3

[0074] The activated bacterial solutions CCNH263 and LGG were inoculated into 30 mL of fresh MRS liquid medium at a 2% inoculum. After incubation at 37°C for 18 h, the supernatant was collected by centrifugation, filtered through a membrane (membrane pore size of 0.22 μm), and the lactic acid yield was detected by high performance liquid chromatography (HPLC).

[0075] The lactic acid production of strain CCNH263 was 16.52 g / L, while that of LGG was 15.1 g / L. This indicates that strain CCNH263, when activated under the same conditions, has higher lactic acid production performance, suggesting that strain CCNH263 can metabolize more sugars during cultivation and reduce the sugar content in the environment.

[0076] Example 4

[0077] Take the expanded bacterial culture, wash twice with physiological saline, and then resuspend in 0.1M PBS buffer to adjust the OD of the bacterial suspension. 600 =1, aspirate 100 μL of bacterial suspension into the ELISA plate and measure the initial OD. 600 The value is recorded as A0; after the bacterial suspension has stood for 4 hours, 100 μL of the upper bacterial solution is transferred to an ELISA plate, and its OD value after standing is measured. 600 The value is denoted as A1, and the self-aggregation rate of the strain is calculated. 900 μL of the above bacterial suspension is taken, 180 μL of dodecane is added, and the mixture is vortexed until homogeneous. After standing for 20 min to separate into layers, the lower aqueous phase is taken and its OD value is measured. 600 The value is denoted as A2, and the hydrophobicity of the strain is calculated.

[0078] Strains’ self-aggregation rate or hydrophobicity = (A0 - A1 or A2) / A0 × 100%.

[0079] The self-aggregation rates of strains CCNH263 and LGG were 53.21% and 54.55%, respectively, and their hydrophobicities were 46.74% and 43.85%, respectively. These two data combined indicate that CCNH263 has excellent adhesion properties, can bind to intestinal epithelial cells and colonize the intestine, thereby inhibiting the colonization of pathogens in the gastrointestinal tract.

[0080] Example 5

[0081] Weigh 44g of Columbia blood agar medium powder (purchased from Qingdao Haibo Biotechnology, catalog number HB9295), add 1000mL of water, sterilize at 121°C for 15min, then add 10vol% sterile defibrinated sheep blood to obtain Columbia blood agar plates.

[0082] 10 μL of the expanded bacterial culture was inoculated onto the Columbia blood agar plate mentioned above. In addition, Escherichia coli ATCC25922 and Staphylococcus aureus CMCC(B)26001 with the same number of viable bacteria were used as control strains.

[0083] The results showed that CCNH263 did not exhibit hemolysis, and Escherichia coli did not show hemolysis, while Staphylococcus aureus had a β-hemolytic zone around its white colonies. This indicates that the strain CCNH263 provided by this invention will not cause hemolysis when applied to organisms, thus making it possible for in vivo application.

[0084] Example 6

[0085] H₂O₂ solutions with concentrations of 0, 0.5, 1, 1.5, 2, 5, 8, 10, 25, and 50 μg / mL were prepared, and the OD values ​​at different concentrations were measured. 505 The absorbance was used to establish a standard curve of absorbance-concentration.

[0086] Take 30 mL of cultured bacteria, centrifuge to obtain the supernatant, measure its absorbance, and calculate the H2O2 yield based on the absorbance-concentration standard curve.

[0087] The H2O2 production of Lactobacillus plantarum CCNH263 and LGG was 4.38 μg / mL and 4.33 μg / mL, respectively, indicating that both strains could non-specifically inhibit the growth of pathogenic bacteria by generating H2O2.

[0088] Example 7

[0089] *Escherichia coli* (CICC10421 and ATCC25922), *Staphylococcus aureus* (CMCC(B)26001 and CMCC(B)26003), *Salmonella* (ATCC14028), and *CVCC3378* were inoculated into LB liquid medium, respectively. *Candida albicans* (CICC1965 and ATCC10231) were inoculated into YPD solid medium. *Gardnerella vaginalis* (ATCC14018) and *Atopobia ulmoides* (ATCC33267) were inoculated into blood agar plates. Except for *Gardnerella vaginalis*, which was cultured anaerobically, all other indicator strains were cultured aerobically. After incubation at 37°C for 18 h to activate the culture, the cultures were transferred to their respective fresh media and cultured until a viable count of 10⁻⁶ was achieved. 5 -10 7 CFU / mL, diluted to 10 with the appropriate culture medium. 5CFU / mL was used as the indicator bacterial solution.

[0090] The activated bacterial cultures of Lactobacillus plantarum CCNH263 and LGG were inoculated into 30 mL of fresh MRS liquid medium at a 2% inoculum. After incubation at 37°C for 18 h, the supernatant was collected by centrifugation and then filtered through a membrane (membrane pore size of 0.22 μm) to obtain the fermentation supernatant.

[0091] Three experimental groups were set up: S1: 100 μL fermentation supernatant, 100 μL indicator culture; S2: 50 μL fermentation supernatant, 150 μL indicator culture; S3: 25 μL fermentation supernatant, 175 μL indicator culture. The fermentation supernatant was replaced with an equal volume of sterile MRS liquid culture medium as a negative control group, and the fermentation supernatant was replaced with 10 mL of kanamycin (100 μg / mL) as a positive control group. The OD values ​​of the experimental groups and the negative control group were measured. 600 The values ​​are A and A respectively. 0， The inhibition rate was calculated according to the following formula, as shown in Table 1, where the unit of inhibition rate is %.

[0092] Inhibition rate (%) = (A0-A) / A0×100%.

[0093] Table 1

[0094]

[0095] As shown in Table 1 above, strain CCNH263 exhibits a certain inhibitory rate against Escherichia coli, Staphylococcus aureus, Salmonella, and common vaginal pathogens such as Candida albicans, Gardnerella vaginalis, and Atobococcus aureus, especially against Gardnerella vaginalis and Candida albicans. This result indicates that strain CCNH263 has the potential to prevent or alleviate BV and VVC diseases caused by Candida albicans, Gardnerella vaginalis, and Atobococcus aureus.

[0096] Example 8

[0097] Take the expanded bacterial culture and dilute it with MRS medium to a viable count of 1.5 × 10⁻⁶. 5 CFU / mL.

[0098] Antibiotic samples with different initial concentrations (INC) were prepared according to Table 2. The antibiotics were diluted using the two-fold dilution method, and the growth of bacterial strains was measured until growth was observed. This concentration is the minimum inhibitory concentration (MIC). The MIC was compared with the breakpoint value in the "General Rules for Probiotics for Food Use" (T / CIFST 009-2022) to determine antibiotic sensitivity. A MIC lower than the cutoff concentration indicates strong sensitivity to the antibiotic, meaning low concentrations are sufficient to inhibit growth. A MIC higher than the breakpoint value indicates strong resistance to the antibiotic, requiring higher concentrations to inhibit growth. "nr" indicates no resistance. The test results and standard values ​​are shown in Table 2. MRS liquid culture medium was used as a blank control.

[0099] Table 2

[0100]

[0101] Previous studies have shown that vancomycin and streptomycin have poor inhibitory effects on *Lactobacillus plantarum*, with MICs typically greater than 64. However, as shown in Table 2, compared to other conventional *Lactobacillus plantarum* strains, the *Lactobacillus plantarum* strain provided by this invention exhibits significantly improved sensitivity to streptomycin. Lower concentrations of vancomycin and streptomycin can also inhibit the growth of *Lactobacillus plantarum* CCNH263. Furthermore, *Lactobacillus plantarum* CCNH263 shows higher or no lower sensitivity to antibiotics other than erythromycin than the standard. These results indicate that strain CCNH263 is safe and has the potential to be used in the development of probiotic additives or as a food fermentation strain.

[0102] Example 9

[0103] VK2 / E6E7 cells were activated using a cell culture medium containing penicillin and antibiotics. The activation method involved rapidly thawing frozen VK2 / E6E7 cells at 37°C, centrifuging at 1000 rpm for 5 min, discarding the supernatant, resuspending the cells in VK2 / E6E7 culture medium containing 10 vol% fetal bovine serum and 1 vol% penicillin and antibiotics, adjusting the cell density, seeding them into 6 cm culture dishes, and culturing at 37°C with 5 vol% CO2. The medium was changed every 36 hours. When the cells reached 90% surface area in the cell culture dish, they were passaged (every 3 days).

[0104] Human vaginal epithelial cells were divided into groups of 10. 6 Cells / mL were seeded into six-well plates with sterile coverslips. After 24 hours of cell adhesion and growth, the cell culture medium containing antibiotics was removed, and the bacterial suspension was diluted with K-SFM complete medium. 2 mL of 10% K-SFM solution was added to each well. 8CFU / mL of expanded bacterial culture was used, with UREX-1 as a positive control and physiological saline as a negative control. The culture was co-cultured with cells at 5 vol% CO2 and 37°C for 2 h. After co-culture, unattached strains were washed with PBS buffer and fixed with methanol at room temperature for 20 min. The fixative was washed twice with deionized water and dried. Gram staining was performed, and the coverslips were dried and placed on a glass slide, which was then fixed with neutral resin. The adhesion of different groups of strains to cells was observed under a microscope (e.g., ...). Figure 3 , Figure 4 and Figure 5 (as shown in the figure), and randomly select 20 cells to count the number of strains adhering to each cell.

[0105] The cell adhesion count of Lactobacillus plantarum CCNH263 was 13.2±3.8 CFU / cell, while the cell adhesion count of the positive control group was 9±2.7 CFU / cell. This result indicates that strain Lactobacillus plantarum CCNH263 can colonize better in vivo, thereby exerting a probiotic effect.

[0106] Example 10

[0107] Candida albicans suspension was prepared according to the methods in Examples 1 and 4, and cell adhesion assay was performed according to the method in Example 9. Specifically, when adding the expanded bacterial culture to the six-well plate, an additional 2 mL of a 10% concentration was added to each well. 7 CFU / mL Candida albicans suspension. Observe the adhesion of Candida albicans to cells under a microscope (e.g., Figure 6 , Figure 7 and Figure 8 (As shown). Count the number of Candida albicans adhering to each cell.

[0108] The number of Candida albicans cells adhering to the Lactobacillus plantarum CCNH263 group was 14.4±3.9 CFU / cell, while the number of cells adhering to the negative control group and the positive control group were 41.7±13.1 CFU / cell and 23.1±5.9 CFU / cell, respectively. Lactobacillus plantarum CCNH263 can significantly reduce the probability of Candida albicans colonization in tissues, thereby treating or alleviating vaginitis.

[0109] Example 11

[0110] VK2 / E6E7 cells were cultured in a double-antibiotic medium at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 12-well plates. After 24 hours of cell culture and adherence, the antibiotic-free medium was replaced with antibiotic-free medium. A final concentration of 1×10⁻⁶ cells was added to the antibiotic-free medium containing VK2 / E6E7 cells. 5 CFU / mL Candida albicans, 1×10 6Gardnerella vaginalis CFU / mL and 1×10 6 Atobacillus acetiferus CFU / mL was cultured at 37°C for 2 h at 5 vol% CO2.

[0111] Dilute the bacterial culture to 1×10⁻⁶ with KSFM complete medium. 9 CFU / mL, add bacterial culture to each well to achieve a final concentration of 10. 7 CFU / mL. A similar model group (no bacterial strain), a negative control group (no pathogenic bacteria or bacterial strain), and a positive control group (with pathogenic bacteria, and strain CCNH263 replaced with UREX-1) were set up. After co-incubation at 5% CO2 and 37℃ for 6 h, the cell culture medium was removed, and the cells were washed three times with PBS buffer. Cell RNA samples were extracted from each group using a cell RNA extraction kit. The expression levels of TNF-α, IL-6, and IL-8 in the *Candida albicans* infection group, TNF-α and IL-1β in the *Gardnerella vaginalis* infection group, and TNF-α, IL-1β, IL-6, and IL-8 in the *Atopobacterium* infection group relative to the control group (control group = 1) were detected using an RNA detection kit. The results are shown in Table 3.

[0112] Table 3

[0113]

[0114] As shown in Table 3, both *Lactobacillus plantarum* CCNH263 and UREX-1 inhibited the expression of inflammatory factors induced by *Candida albicans*, *Gardnerella vaginalis*, and *Atopobacter* infections. This indicates that *Lactobacillus plantarum* CCNH263 provided by this invention can significantly reduce inflammatory factors following *Candida albicans*, *Gardnerella vaginalis*, and *Atopobacter* infections, thereby reducing cellular inflammation and demonstrating the potential to improve vaginal inflammation caused by these bacteria.

[0115] Example 12

[0116] Seven-week-old female BALB / c mice (purchased from Vital Rivers) were selected and acclimatized for four days. They were then housed in a constant temperature (22℃) and constant humidity (50% relative humidity) environment with a 12-hour light-dark cycle, and provided with free access to standard feed and sterile water. Ninety mice were randomly divided into six groups: a control group, a model group, and four experimental groups.

[0117] Except for the control group, mice in the model group and experimental group were subcutaneously injected with 100 μL of estradiol valerate (5 mg / mL). Then, the mice were vaginally inoculated with live bacteria of 10... 9 20 μL of CFU / mL Candida albicans suspension was inoculated for 5 consecutive days. An equal volume of PBS buffer was inoculated into the vagina of control mice.

[0118] The experimental group was divided into a low-dose CCNH263 group (CCNH263L, 1×10⁻⁶). 8 CFU / mL), medium-dose CCNH263 group (CCNH263M, 1×10 9 CFU / mL), high-dose CCNH263 group (CCNH263H, 1×10) 10 CFU / mL) and URES-1 group (1×10) 9 Mice in each group (CFU / mL) were administered 200 μL of bacterial suspension via gavage daily; the control and model groups received an equal volume of PBS via gavage. Gavage was continued for 12 consecutive days, during which the vaginal redness and swelling of the six groups of mice were scored daily (maximum score 10 points, higher scores indicating more severe redness and swelling). Specific scoring details are as follows: Figure 9 As shown.

[0119] The day before the strain intervention, except for the control group, the vaginal redness and swelling scores of mice in all groups were relatively high. The vaginal score of the model group mice that did not receive strain intervention remained at a high level. After treatment with strains CCNH263 and UREX-1, the vaginal redness and swelling scores showed a decreasing trend. This result indicates that strain CCNH263 can effectively alleviate vaginal redness and swelling caused by Candida albicans infection.

[0120] Vaginal tissue was collected from mice and stained using the Diff-Quik staining kit. The stained tissue slides were then observed under a 400x optical microscope. Five microscopic fields were randomly selected, and the number of detached epithelial cells in each field was counted. The number of detached epithelial cells in the control group, model group, and UREX-1 group were 10.3±1.2, 66.0±6.5, 39.3±4.0, 23.0±1.6, 12.3±2.4, and 15.0±1.7, respectively. The model group mice showed severe vaginal epithelial cell detachment, which significantly decreased after CCNH263 intervention. p <0.0001), among which there was no significant difference in the number of cells shed between the medium-dose and high-dose CCNH263 groups and the control group ( p >0.05). This result indicates that Lactobacillus plantarum CCNH263 has a significant therapeutic effect on vaginal epithelial cell damage caused by Candida albicans infection, and can reduce vaginal epithelial cell damage caused by pathogen infection.

[0121] Mouse vaginal tissue was taken, sectioned, and stained with hematoxylin and eosin (HE) to observe the epithelial morphology. Figure 10 and Figure 11As shown in the figure, the vaginal mucosal epithelium in the control group was smooth and continuous, with intact tissue structure and no obvious inflammatory cell infiltration. In the model group, the vaginal mucosal epithelium showed poor continuity, superficial erosion, pore formation, hyperemia of the submucosal stroma, and extensive infiltration of inflammatory cells into the epithelial layer and stroma. After bacterial intervention, the inflammatory cell infiltration in the vaginal tissue of mice in the CCNH263H and UREX-1 groups decreased to varying degrees, and mucosal shedding was improved. These results indicate that an appropriate dose of strain CCNH263 can effectively promote the recovery of the vaginal mucosa in mice, alleviate inflammatory symptoms in the vaginal tissue, and improve the damaged vaginal structure.

[0122] Mouse vaginal tissue samples were placed in 200 μL of radioimmunoprecipitation lysis buffer (RIPA lysis buffer) containing 2 vol% of a protease inhibitor mixture and 2 vol% of a phosphatase inhibitor mixture, and homogenized using a high-throughput tissue homogenizer to obtain a homogenate. The homogenate was obtained by centrifugation at 12000×g for 15 min at 4°C. The concentrations of pro-inflammatory factors (TNF-α, IL-6, and IL-1β) and anti-inflammatory factors (IL-4 and IL-10) in the supernatant were determined using a kit according to the instructions (in ng / g supernatant), as shown in Table 4.

[0123] Table 4

[0124]

[0125] The levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the vaginal tissue of mice infected with Candida albicans were significantly higher than those in the control group. p <0.05), while the content of pro-inflammatory factors in the vaginal tissue of mice treated with strain CCNH263 was significantly lower than that in the model group ( p <0.05). The levels of anti-inflammatory factors IL-4 and IL-10 in the vaginal tissue of the model group mice were significantly lower than those in the control group mice. p <0.05), while its content increased significantly after intervention with strain CCNH263 compared to the model group ( p <0.05). The results of inflammatory factor detection in vaginal tissue showed that, except for the low-dose group of strain CCNH263, the anti-inflammatory and pro-inflammatory factors in mice treated with strain CCNH263 were significantly improved, indicating a strong potential to improve vaginal inflammation caused by Candida albicans.

[0126] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum ), characterized in that, The preservation number of the Lactobacillus plantarum is CGMCC No. 33152.

2. A microbial agent, characterized in that, The microbial agent contains *Lactobacillus plantarum* as described in claim 1.

3. The microbial agent according to claim 2, wherein, The microbial agent contains excipients selected from glycerin and / or corn oil.

4. The use of the *Lactobacillus plantarum* according to claim 1 in the preparation of a medicament for inhibiting the colonization of *Candida albicans*.

5. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria, wherein, The pathogenic bacteria are Escherichia coli, Staphylococcus aureus, Salmonella, Candida albicans, Gardnerella vaginalis, or Atoborobacter.

6. The use of the *Lactobacillus plantarum* according to claim 1 in the preparation of a medicament for the prevention and / or treatment of vaginitis.

7. The application according to claim 6, wherein, The plant lactobacillus is used to reduce the level of pro-inflammatory factors.

8. The application according to claim 6, wherein, The plant lactobacillus is used to increase the level of anti-inflammatory factors.

9. The application according to claim 7 or 8, wherein, The pro-inflammatory factor is at least one of TNF-α, IL-6, IL-1β and IL-8; Alternatively, the anti-inflammatory factor is IL-4 and / or IL-10.

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