Lactobacillus plantarum and application thereof in preparation of medicine for preventing and / or treating vaginitis

Abstract

The invention relates to the field of microorganisms, and discloses lactobacillus plantarum and application of the lactobacillus plantarum in preparation of a medicine for preventing and / or treating vaginitis, and the preservation number of the lactobacillus plantarum is CGMCC No.33152. The lactobacillus plantarum provided by the invention has a relatively strong bacteriostatic effect on common vaginal pathogenic bacteria such as Atortoria, staphylococcus aureus, Candida albicans, Escherichia coli and Gardneria, and also can reduce the infection degree of the pathogenic bacteria by inhibiting colonization and reproduction of the pathogenic bacteria; in addition, the proinflammatory factor level in sick vaginitis tissues is reduced, and the anti-inflammatory factor level is improved.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus plantarum and application thereof in preparing a medicine for preventing and / or treating vaginitis. Background Art

[0002] Bacterial vaginosis (BV) is the most common lower reproductive tract disease in women, mainly caused by Gardnerella vaginalis ( Gardnerella vaginalis ), Atopobium vaginalis ( Atopobium minutum ) and other pathogenic bacteria. In addition to BV, vulvovaginal candidiasis (Vulvovaginal Candidiasis, VVC) is also one of the common vaginal diseases in women. The occurrence of these vaginal diseases causes an imbalance in the female vaginal microecology, causing great harm to women's physical and mental health. Traditional clinical therapies mainly use antibiotics, estrogens and other methods to treat female vaginal inflammation, but there are many adverse reactions and easy recurrence. Microorganisms are often used to treat or prevent vaginitis caused by pathogenic bacteria, but the existing strains can only inhibit Gardnerella and Candida albicans ( Candida albicans ), but no specific inhibition of Atopobium strains was found.

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

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and to provide a strain of Lactobacillus plantarum and its use in preparing a medicine for preventing and / or treating vaginitis.

[0005] In order to achieve the above object, the present invention provides a plant lactobacillus ( Lactobacillus plantarum ), the preservation number of the plant lactobacillus is CGMCC No.33152.

[0006] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the aforementioned Lactobacillus plantarum.

[0007] The third aspect of the present invention provides the use of the aforementioned Lactobacillus plantarum in the preparation of a medicinal plant for preventing and / or treating colonization of pathogenic bacteria.

[0008] A fourth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for preventing and / or treating the proliferation of pathogenic bacteria.

[0009] A fifth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for preventing and / or treating vaginitis.

[0010] A sixth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for reducing the level of pro-inflammatory factors.

[0011] A seventh aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for increasing the level of anti-inflammatory factors.

[0012] Through the above technical solution, the beneficial effects obtained by the present invention include at least: (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 Atopobium, Staphylococcus aureus, Candida albicans, Escherichia coli and Gardnerella, and can also reduce the degree of infection of pathogenic bacteria by inhibiting the colonization and reproduction of pathogenic bacteria; (2) The plant lactobacillus provided by the present invention can reduce the level of pro-inflammatory factors and increase the level of anti-inflammatory factors in tissues suffering from vaginitis, reduce the degree of tissue disease, will not cause cell hemolysis, and is sensitive to common antibiotics. Therefore, it can be used to prepare microbial preparations and has the potential to prepare drugs for preventing and / or treating vaginal diseases.

[0013] Biological Deposit The strain provided by the present invention is classified and named as Lactobacillus plantarum Lactobacillus plantarum , was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviated as CGMCC) on December 23, 2024. Its deposit number is CGMCC No.33152, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a colony morphology diagram of Lactobacillus plantarum CCNH263 provided by the present invention; Figure 2 It is a Gram microscopic image of Lactobacillus plantarum CCNH263 provided by the present invention; Figure 3 It is the cell adhesion microscopy result of the negative control group in Example 9; Figure 4 It is the cell adhesion microscopy result of Lactobacillus plantarum CCNH263 in Example 9; Figure 5 is the cell adhesion microscopy result of UREX-1 in Example 9; Figure 6 It is the microscopic result of the negative control group in Example 10 inhibiting the cell adhesion of Candida albicans; Figure 7The microscopic result of the inhibition of cell adhesion of Candida albicans by Lactobacillus plantarum CCNH263 in Example 10; Figure 8 It is the microscopic result of UREX-1 inhibiting the adhesion of Candida albicans cells in Example 10; Fig. 9 This is the score result of vaginal swelling in mice in Example 12; Fig.10 Pathological sections of vaginal tissues of the model group, control group and UREX-1 group in Example 12; Fig.11 These are the vaginal tissue pathological sections of the CCNH263L group, CCNH263M group and CCNH263H group in Example 12. DETAILED DESCRIPTION

[0015] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0016] The first aspect of the present invention provides a plant lactobacillus ( Lactobacillus plantarum ), the preservation number of the plant lactobacillus is CGMCC No.33152.

[0017] The plant lactobacillus with the deposit number of CGMCC No.33152 provided by the present invention is isolated from Xinjiang sour mare's milk, and a single colony with milky white, smooth, round or oval shape is obtained after culture. The colony morphology and Gram microscopic examination image on the culture medium plate are as follows: Figure 1 and Figure 2 As shown. Further experiments have shown that the plant lactobacillus with the deposit number CGMCC No. 33152 provided by the present invention has excellent growth performance, gastric acid resistance, bile salt resistance, and acid production performance, and can inhibit pathogenic bacteria from colonizing the gastrointestinal tract through high self-aggregation ability and hydrophobicity. In addition, the strain does not cause hemolysis.

[0018] "CCNH263" is the number given to the strain by the inventor of the present invention during the strain screening process, and "CGMCC No.33152" is the preservation number of the strain. The two represent the same strain, and their numbers can be used interchangeably in the following text.

[0019] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the aforementioned Lactobacillus plantarum.

[0020] The present invention has no particular limitation on the type of the bacterial agent, which can be any type of bacterial agent currently available in the art. According to a preferred embodiment of the present invention, the bacterial agent is selected from at least one of a liquid bacterial agent, a concentrated bacterial agent and a solid bacterial agent.

[0021] In the present invention, the bacterial agent may further contain auxiliary materials, and the auxiliary materials are preferably selected from protective agents (e.g., freeze-dried protective agents, such as skim milk powder, maltodextrin, trehalose, dextran and glycerol, etc.) and / or buffers (e.g., buffers remaining in the process of preparing solid bacterial agents, etc.).

[0022] According to the present invention, the auxiliary material can be selected from glycerol and / or corn oil.

[0023] Preferably, relative to 1 mL of auxiliary material, the viable count of the plant lactobacillus is 10 4 -10 12 CFU.

[0024] The plant lactobacillus provided by the present invention can produce a large number of live cells of plant lactobacillus through liquid culture. There is no special requirement for the culture method, as long as the plant lactobacillus can be proliferated. For example, the live cells of plant lactobacillus can be inoculated into a culture medium at an inoculum amount of 1-5 vol%, and cultured at a temperature of 30-40° C. for 9-50 hours to obtain a culture solution. The culture medium can be any culture medium suitable for culturing plant lactobacillus known in the art, for example, an MRS culture medium.

[0025] The present invention can further separate the live cells of Lactobacillus plantarum in the above-mentioned culture solution. The separation method is not particularly limited as long as the cells can be enriched from the culture solution. For example, it can be achieved by centrifugation and / or filtration. The conditions of the centrifugation and the filtration can be well-known conditions, and the present invention will not be repeated here.

[0026] The third aspect of the present invention provides the use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting the colonization of pathogenic bacteria.

[0027] A fourth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria.

[0028] In the present invention, the pathogenic bacteria may include Escherichia coli ( Escherichia coli ), Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella )、Candida albicans( Candida albicans )、Gardnerella ( Gardnerella vaginalis ) and Atopobium ( Atopobium minutum ), preferably Atopobium.

[0029] According to some embodiments of the present invention, the Salmonella preferably includes Salmonella typhimurium ( Salmonella typhimurium ) and Salmonella Enteritidis ( Salmonella enteritidis ).

[0030] The fermentation supernatant prepared by the Lactobacillus plantarum provided by the present invention can inhibit the reproduction of Atopobium. According to a most preferred embodiment of the present invention, the inhibition rate of the supernatant of the Lactobacillus plantarum provided by the present invention on Atopobium can reach 99%, so the Lactobacillus plantarum provided by the present invention is particularly suitable for inhibiting Atopobium.

[0031] A fifth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for preventing and / or treating vaginitis.

[0032] A sixth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for reducing the level of pro-inflammatory factors.

[0033] According to the present invention, the Lactobacillus plantarum provided by the present invention can inhibit the increase in the levels of proinflammatory factors (TNF-α, IL-6, IL-1β and IL-8) caused by Candida albicans infection and proinflammatory factors (TNF-α and IL-1β) caused by Gardnerella infection.

[0034] A seventh aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for increasing the level of anti-inflammatory factors.

[0035] According to the present invention, the Lactobacillus plantarum provided by the present invention can inhibit the reduction of the levels of anti-inflammatory factors IL-4 and IL-10 caused by Candida albicans infection.

[0036] According to the present invention, the drug may include pharmaceutically acceptable excipients.

[0037] According to the present invention, the medicine preferably includes the fermentation supernatant of Lactobacillus plantarum and / or the bacterial solution containing live bacteria as described above.

[0038] The present invention will be described in detail below through examples.

[0039] In the following examples, unless otherwise specified, the chemical / biological reagents used were purchased from regular chemical / biological reagent suppliers.

[0040] Escherichia coli ( Escherichia coli ), numbered (1) ATCC 25922, purchased from the American Type Culture Collection, (2) CICC 10421, purchased from the China Industrial Microbiological Culture Collection; Staphylococcus aureus ( Staphylococcus aureus), numbered CMCC(B)26001 and CMCC(B)26003, both purchased from the Chinese Medical Bacteria Collection Center; Salmonella, (1) Salmonella typhimurium ( Salmonella typhimurium ), numbered ATCC 14028, purchased from the American Type Culture Collection; (2) Salmonella Enteritidis ( Salmonella enteritidis ), numbered CVCC 3378, purchased from the National Veterinary Microbiological Bacteria (Virus) Collection Center; Candida albicans ( Candida albicans ), numbered (1) CICC 1965, purchased from China Industrial Microbiological Culture Collection, (2) ATCC 10231, purchased from American Type Culture Collection; Gardnerella Gardnerella vaginalis ), numbered ATCC 14018, purchased from the American Type Culture Collection; Atopobium Atopobium minutum ), numbered ATCC 33267, purchased from the American Type Culture Collection; MRS liquid culture medium: peptone 10g, beef extract powder 10g, yeast extract powder 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween-80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, deionized water 1L, pH 6.5, high pressure sterilization; add 18g agar before constant volume to obtain MRS solid culture medium.

[0041] Human vaginal epithelial cells (VK2 / E6E7) were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number BFN60808401; Double-antibody cell culture medium: VK2 / E6E7 cell-specific culture medium, purchased from Shanghai Jinyuan Biotechnology Co., Ltd., catalog number JY-Y1196; Penicillin-streptomycin: purchased from Thermo Fisher Scientific, catalog number 15140-122; K-SFM complete medium: purchased from Guangzhou Keluojie Biotechnology Co., Ltd., catalog number M1017; PBS buffer was purchased from Solebol, catalog number P1003; Reference strains: (1) Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), numbered ATCC 53103, purchased from the American Type Culture Collection, hereinafter referred to as LGG; (2) Lactobacillus reuteri ( Lactobacillus reuteri) and Lactobacillus rhamnosus, wherein the ratio of the number of live bacteria of Lactobacillus reuteri to Lactobacillus rhamnosus is 1:1, and both strains are isolated from UREX® bacterial powder produced by Chr. Hansen of Denmark (purchased from Shaanxi Mufan Biotechnology Co., Ltd., product number MF-00395). The compound bacterial agent is hereinafter referred to as UREX-1.

[0042] Example 1 The frozen glycerol tubes of strain CCNH263 and LGG were inoculated into fresh MRS liquid medium at 2% and cultured at 37°C overnight for 18 h. 600 Normalization was performed to obtain an activated bacterial solution, which was inoculated into a fresh MRS liquid medium at a 2% inoculation rate and cultured at 37°C, 200 rpm for 18 h to obtain the CCNH263 culture solution and the LGG culture solution. The OD values ​​of the CCNH263 culture solution and the LGG culture solution were measured. 600 They were 8.27±0.05 and 8.13±0.02, respectively, indicating that the growth performance of CCNH263 was better than that of LGG.

[0043] Example 2 Take the cultured bacterial liquid of Lactobacillus plantarum CCNH263 and LGG, and refer to T / CNHFA435-2024 "Test Method for Gastric Juice Tolerance of Probiotics" to evaluate the strains' resistance to gastric acid and bile salts.

[0044] In the gastric acid resistance test, the pH was 3 and the incubation time was 2 h; in the bile salt resistance test, the bile salt content was 0.2 wt% and the incubation time was 4 h. 600 Measure the absorbance of the bacterial solution before and after the experiment.

[0045] Survival rate: (OD before experiment 600 Value - OD after experiment 600 value) / OD before the experiment 600 value × 100%.

[0046] The gastric acid resistance survival rates of CCNH263 and LGG were 94.51% and 93.33%, and the bile salt resistance survival rates were 62.4% and 53.13%, respectively. It can be found that the gastric acid resistance and bile salt resistance of CCNH263 are better than those of LGG.

[0047] Example 3 The activated bacterial liquid CCNH263 and LGG were inoculated at a 2% inoculation rate into 30 mL of fresh MRS liquid culture medium, cultured at 37°C for 18 h, and then centrifuged to obtain the supernatant, which was passed through a membrane (membrane pore size of 0.22 μm), and the lactic acid production was detected by high performance liquid chromatography (HPLC).

[0048] The lactic acid production of strain CCNH263 was 16.52 g / L, and the lactic acid production of LGG was 15.1 g / L, indicating that the strain CCNH263 activated under the same conditions had a higher lactic acid production performance, indicating that the strain CCNH263 could metabolize more sugars during the cultivation process and reduce the sugar content in the environment.

[0049] Example 4 Take the cultured bacteria, wash twice with saline, and resuspend in 0.1M PBS buffer to make the OD of the bacterial suspension 600 =1, draw 100 μL of bacterial suspension into the ELISA plate and detect the initial OD 600 The bacterial suspension was allowed to stand for 4 h, 100 μL of the upper bacterial suspension was taken to the ELISA plate, and the OD value after standing was detected. 600 The value was recorded as A1, and the self-aggregation rate of the strain was calculated; 900 μL of the above bacterial suspension was taken, 180 μL of dodecane was added, vortexed until mixed evenly, and the mixture was allowed to stand for 20 minutes to separate, and the lower aqueous phase was taken to measure its OD 600 value, recorded as A2, to calculate the hydrophobicity of the strain; The self-aggregation rate or hydrophobicity rate of the strain = (A0-A1 or A2) / A0×100%.

[0050] The self-aggregation rates of strains CCNH263 and LGG were 53.21% and 54.55%, and the hydrophobicity rates were 46.74% and 43.85%, respectively. The two data combined showed that CCNH263 had a good adhesion effect, could bind to intestinal epithelial cells and colonize in the intestine, thereby inhibiting the colonization of pathogens in the gastrointestinal tract.

[0051] Example 5 Weigh 44 g of Columbia blood agar medium powder (purchased from Qingdao Haibo Biological, catalog number HB9295), add 1000 mL of water, sterilize at 121°C for 15 min, and then add 10 vol% sterile defibrinated sheep blood to obtain a Columbia blood agar plate.

[0052] 10 μL of the culture solution was inoculated on the Columbia blood agar plate. In addition, Escherichia coli ATCC25922 and Staphylococcus aureus CMCC(B)26001 with the same number of viable cells were used as control strains.

[0053] The results showed that CCNH263 had no hemolysis, and Escherichia coli showed no hemolysis, while the white colonies of Staphylococcus aureus were surrounded by β-hemolysis zones, indicating that the strain CCNH263 provided by the present invention would not cause hemolysis when applied to an organism, and thus has the possibility of in vivo application.

[0054] Example 6 Prepare H2O2 solutions with concentrations of 0, 0.5, 1, 1.5, 2, 5, 8, 10, 25, and 50 μg / mL, and detect the OD at different concentrations. 505 The absorbance was measured to establish a standard curve of absorbance-concentration.

[0055] Take 30 mL of the culture solution, centrifuge it, take the supernatant, measure its absorbance, and calculate the H2O2 production based on the absorbance-concentration standard curve.

[0056] The H2O2 production of Lactobacillus plantarum CCNH263 and LGG were 4.38μg / mL and 4.33μg / mL, respectively, indicating that both strains can non-specifically inhibit the growth of pathogenic bacteria by producing H2O2.

[0057] Example 7 Escherichia coli (CICC10421 and ATCC25922), Staphylococcus aureus (CMCC(B)26001 and CMCC(B)26003), Salmonella (ATCC14028) and CVCC3378) were inoculated in LB liquid medium, Candida albicans (CICC1965 and ATCC10231) were inoculated in YPD solid medium, Gardnerella (ATCC14018) and Atopobium (ATCC33267) were planted in blood agar plates. Except for Gardnerella, all other indicator strains were cultured aerobically, cultured and activated at 37°C for 18 hours, transferred to the corresponding fresh culture medium, and cultured until the viable count was 10 5 -10 7 CFU / mL, dilute to 10 5 CFU / mL was used as the indicator bacterial solution.

[0058] The activated bacterial liquid of Lactobacillus plantarum CCNH263 and LGG was inoculated into 30 mL of fresh MRS liquid culture medium at a 2% inoculation rate, and after static culture at 37°C for 18 hours, the supernatant was centrifuged and passed through a membrane (the membrane pore size was 0.22 μm) to obtain the fermentation supernatant.

[0059] Three experimental groups were set up: S1: 100 μL fermentation supernatant, 100 μL indicator bacteria solution; S2: 50 μL fermentation supernatant, 150 μL indicator bacteria solution; S3: 25 μL fermentation supernatant, 175 μL indicator bacteria solution; the fermentation supernatant was replaced with an equal volume of sterile MRS liquid culture medium as the negative control group, and the fermentation supernatant was replaced with 10 mL of kanamycin (content 100 ug / mL) as the positive control group. Measure the OD of the experimental group and the negative control group 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 %: Inhibition rate (%) = (A0-A) / A0×100%.

[0060] Table 1

[0061] As can be seen from Table 1 above, strain CCNH263 has a certain inhibition rate against Escherichia coli, Staphylococcus aureus, Salmonella, and common vaginal pathogens Candida albicans, Gardnerella and Atopobium, especially against Gardnerella and Candida albicans. The results indicate that strain CCNH263 has the potential to prevent or alleviate BV and VVC diseases caused by Candida albicans, Gardnerella and Atopobium.

[0062] Example 8 Take the culture solution and dilute it with MRS medium until the viable count is 1.5×10 5 CFU / mL.

[0063] Prepare antibiotic samples with different initial concentrations (INC) according to Table 2, dilute the antibiotics by double dilution method and measure the growth of the strains in them until the strains grow. At this time, the antibiotic concentration is the minimum inhibitory concentration (MIC). Compare the MIC with the breakpoint value in the "General Rules for Probiotics for Food" (T / CIFST 009-2022) to determine its antibiotic sensitivity. If the MIC is less than the judgment concentration, it means that the strain is highly sensitive to the antibiotic, and a low concentration of antibiotics can inhibit the growth of the strain; if the MIC is greater than the breakpoint value, it means that the strain is highly resistant to the antibiotic, and a higher concentration of antibiotics is required to inhibit the growth of the strain. Among them, nr means not required, and the test results and standard values ​​are shown in Table 2. MRS liquid culture medium is used as a blank control.

[0064] Table 2

[0065] Previous studies have shown that vancomycin and streptomycin have poor inhibitory effects on Lactobacillus plantarum, and their MICs are usually greater than 64. However, it can be seen from Table 2 that, compared with other conventional Lactobacillus plantarum, the sensitivity of Lactobacillus plantarum provided by the present invention to streptomycin is significantly improved, and lower levels of vancomycin and streptomycin can also inhibit the growth of Lactobacillus plantarum CCNH263. In addition, the sensitivity of Lactobacillus plantarum CCNH263 to other antibiotics except erythromycin is stronger than or not lower than the standard. The results show that strain CCNH263 is safe and has the potential to be used in the development of probiotic additives or as a food fermentation strain.

[0066] Example 9 VK2 / E6E7 cells were activated with cell culture medium containing dual antibodies. The activation method was to quickly thaw the frozen VK2 / E6E7 cells at 37°C, centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend the cells with VK2 / E6E7 culture medium containing 10vol% fetal bovine serum and 1vol% dual antibodies, adjust the cell density, inoculate a 6cm culture dish, and culture at 5vol% CO2 and 37°C. The medium was changed every 36h, and the cells were subcultured (subcultured once every 3 days) when they covered 90% of the surface area of ​​the cell culture dish.

[0067] Human vaginal epithelial cells were cultured at a cell number of 10 6 Cells / mL were inoculated into a six-well plate with a sterile cover glass. After culturing for 24 h, the cells adhered to the wall and grew. The cell culture medium containing the double antibody was removed, and the bacterial solution was diluted with K-SFM complete culture medium. 2 mL of 10 8 CFU / mL of cultured bacterial solution, and UREX-1 was used as the positive control group, and physiological saline was used instead of bacterial solution as the negative control group. Co-culture with cells for 2 hours under 5vol%CO2 and 37℃. After the co-culture, the non-adherent strains were washed with PBS buffer and fixed with methanol at room temperature for 20 minutes. After the fixative was washed with double deionized water and dried, the cover slip was removed and placed on a slide after staining with a Gram staining kit, and the cover slip was fixed with neutral resin. The adhesion of strains to cells in different groups was observed under a microscope (such as Figure 3 , Figure 4 and Figure 5 20 cells were randomly selected and the number of bacterial strains adhering to each cell was counted.

[0068] The cell adhesion number of Lactobacillus plantarum CCNH263 was 13.2±3.8 CFU / cell, and the cell adhesion number of the positive control group was 9±2.7 CFU / cell. The results showed that the strain Lactobacillus plantarum CCNH263 can better colonize in the body, thereby exerting a probiotic effect.

[0069] Example 10 The Candida albicans suspension was prepared according to the methods of Examples 1 and 4, and the cell adhesion experiment was performed according to the method of Example 9. When the culture solution was added to the six-well plate, 2 mL of 10% 7 CFU / mL of Candida albicans suspension. Observe the adhesion of Candida albicans to cells under a microscope (e.g. Figure 6 , Figure 7 and Figure 8 The number of C. albicans adherent to each cell was counted.

[0070] The cell adhesion number of Candida albicans in the Lactobacillus plantarum CCNH263 group was 14.4±3.9 CFU / cell, while the cell adhesion numbers of 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 of tissues, thereby treating or alleviating vaginitis.

[0071] Embodiment 11 VK2 / E6E7 cells were cultured in dual-antibody medium at a concentration of 1×10 5 Cells / mL were inoculated in 12-well plates. After the cells were cultured for 24 h and adhered to the wall, the dual-antibody culture medium was replaced with dual-antibody-free culture medium. The dual-antibody-free culture medium containing VK2 / E6E7 cells was added with a final concentration of 1×10 5 CFU / mL of Candida albicans, 1×10 6 CFU / mL of Gardnerella and 1×10 6 CFU / mL of Atopobium was cultured at 5 vol% CO2 and 37°C for 2 h.

[0072] Dilute the bacterial solution to 1×10 9 CFU / mL, add bacterial solution to each well to make the final concentration of 10 7 CFU / mL. In addition, a model group (no strain), a negative control group (no pathogenic bacteria and strains), and a positive control group (with pathogenic bacteria, and the strain CCNH263 was replaced by UREX-1) were set up in a similar manner. After incubation for 6 hours under 5% CO2 and 37°C, the cell culture medium was removed, and the cells were washed three times with PBS buffer. After the cell RNA samples of each group were extracted using a cell RNA extraction kit, the RNA detection kit was used to detect 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 infection group, and TNF-α, IL-1β, IL-6 and IL-8 in the Atopobium infection group relative to the control group (the control group was 1). The results are shown in Table 3.

[0073] Table 3

[0074] As shown in Table 3, both Lactobacillus plantarum CCNH263 and UREX-1 inhibit the expression of cellular inflammatory factors caused by Candida albicans, Gardnerella and Atopobium infection, that is, the Lactobacillus plantarum CCNH263 provided by the present invention can significantly reduce the inflammatory factors after Candida albicans, Gardnerella and Atopobium infection, thereby reducing cellular inflammation, and has the potential to improve vaginal inflammation caused by Candida albicans, Gardnerella and Atopobium.

[0075] Example 12 7-week-old BALB / c female mice (purchased from Vital River) were selected and adaptively fed for 4 days. Then they were kept in a constant temperature (22°C) and constant humidity (relative humidity 50%) environment with a 12-h light-dark cycle and free access to standard feed and sterile water. 90 mice were randomly divided into six groups, one of which was the control group, one was the model group, and four were the experimental groups.

[0076] In addition to 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 inoculated with 10 live bacteria in the vagina. 9 20 μL of CFU / mL Candida albicans suspension was inoculated for 5 consecutive days. The control group of mice was inoculated intravaginally with an equal amount of PBS buffer.

[0077] The experimental groups were divided into low-dose CCNH263 group (CCNH263L, 1×10 8 CFU / mL), medium-dose CCNH263 group (CCNH263M, 1×10 9 CFU / mL), high-dose CCNH263 group (CCNH263H, 1×10 10 CFU / mL and UREX-1 group (1×10 9 CFU / mL), 200 μL bacterial solution was gavaged daily; the control group and model group were gavaged with equal volume of PBS; the gavage lasted for 12 consecutive days, and the vaginal redness and swelling of the six groups of mice were scored every day (full score is 10 points, the higher the score, the more severe the redness and swelling). The specific scoring conditions are as follows Fig. 9 shown.

[0078] On the day before strain intervention, except for the control group, the vaginal swelling scores of mice in each group were relatively high, and the vaginal scores of mice in the model group without strain intervention treatment remained at a high level. After strain CCNH263 and UREX-1 intervention treatment, the vaginal swelling scores showed a downward trend. The results show that strain CCNH263 can effectively alleviate vaginal swelling caused by Candida albicans infection.

[0079] The vaginal tissue of mice was obtained and stained using the Diff-Quik staining kit. The slides of the stained tissue were placed under a 400x optical microscope for observation. Five microscope fields were randomly selected, and the number of epithelial cells shed in each field was counted. The number of epithelial cells shed in the control group, model group, and UREX-1 group was 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 vaginal epithelial cells of mice in the model group shed severely, and the number of cell shed decreased significantly after CCNH263 intervention treatment ( p<0.0001), among which there was no significant difference in the number of cell shedding between the medium-dose and high-dose CCNH263 groups and the control group ( p >0.05). The results showed that Lactobacillus plantarum CCNH263 had significant efficacy in treating vaginal epithelial cell damage caused by Candida albicans infection and could reduce vaginal epithelial cell damage caused by bacterial infection.

[0080] The vaginal tissue of mice was taken, sectioned, and stained with HE to observe the epithelial morphology. Fig.10 and Fig.11 As shown. The vaginal mucosal epithelium of the control group was smooth and continuous, with intact tissue structure and no obvious inflammatory cell infiltration; the vaginal mucosal epithelium of the model group had poor continuity, accompanied by superficial erosion, holes, congestion of the submucosal interstitial layer, and a large number of inflammatory cells infiltrated into the epithelial layer and interstitium. After strain intervention, the inflammatory cell infiltration of the vaginal tissue of mice in the CCNH263H group and the UREX-1 group was reduced to varying degrees, and the shedding of the mucosal layer was improved. The results show that the appropriate dose of strain CCNH263 can effectively promote the recovery of the vaginal mucosa of mice, alleviate the inflammatory symptoms of the vaginal tissue of mice, and improve the damaged vaginal structure of mice.

[0081] The mouse vaginal tissue samples were placed in 200 μL of radioimmunoprecipitation lysis buffer (RIPA lysis buffer) containing 2 vol% protease inhibitor mixture and 2 vol% phosphatase inhibitor mixture, and broken with a high-throughput tissue grinder to obtain a homogenate. The supernatant was obtained by centrifugation at 12000×g for 15 min at 4°C, and the concentrations of pro-inflammatory factors (TNF-ɑ, IL-6 and IL-1β) and anti-inflammatory factors (IL-4 and IL-10) in the supernatant were determined using the kit (unit: ng / g supernatant), as shown in Table 4.

[0082] Table 4

[0083] The levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α in the vaginal tissue of the model group mice infected with Candida albicans were significantly increased compared with those of the control group mice ( p <0.05), while the levels of pro-inflammatory factors in the vaginal tissue of mice treated with strain CCNH263 were significantly decreased compared with the model group ( p <0.05). The content of anti-inflammatory factors IL-4 and IL-10 in the vaginal tissue of mice in the model group was significantly decreased compared with that in the control group ( p <0.05), while its content increased significantly after the intervention of strain CCNH263 compared with the model group ( p<0.05). From the results of inflammatory factor detection in vaginal tissue, it can be seen that, except for the low-dose group of strain CCNH263, the anti-inflammatory factors and pro-inflammatory factors of mice in the CCNH263 intervention treatment group were significantly improved, and it has a strong potential to improve vaginal inflammation caused by Candida albicans.

[0084] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

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

2. A bacterial agent, characterized in that The bacterial agent contains the Lactobacillus plantarum according to claim 1.

3. The bacterial agent according to claim 2, wherein The bacterial agent contains auxiliary materials, and the auxiliary materials are selected from glycerol and / or corn oil.

4. Use of the plant lactobacillus according to claim 1 in the preparation of a medicament for inhibiting colonization of pathogenic bacteria.

5. Use of the plant lactobacillus according to claim 1 in preparing a medicine for inhibiting the reproduction of pathogenic bacteria.

6. The use according to claim 4 or 5, wherein: The pathogenic bacteria include Atopobium.

7. Use of the plant lactobacillus according to claim 1 in the preparation of a medicament for preventing and / or treating vaginitis.

8. Use of the Lactobacillus plantarum according to claim 1 in the preparation of a medicament for reducing the level of pro-inflammatory factors.

9. Use of the plant lactobacillus according to claim 1 in the preparation of a medicament for increasing the level of anti-inflammatory factors.

10. The use according to claim 8 or 9, wherein: The pro-inflammatory factor includes at least one of TNF-α, IL-6, IL-1β and IL-8; And / or, the anti-inflammatory factors include IL-4 and / or IL-10.

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