Lactobacillus gasseri strain targeting vaginal barrier repair and postbiotic for alleviating vaginal infections

Abstract

The application discloses a lactobacillus gasseri for repairing a vaginal barrier and relieving a vaginal infection, and a postbiotic thereof, and belongs to the technical field of microorganisms.The lactobacillus gasseri and the postbiotic thereof can repair a vaginal barrier and relieve a vaginal infection when being used externally.The lactobacillus gasseri is separated from vaginal secretion of a healthy female, has the functions of repairing a vaginal mucosa barrier and relieving a vaginal infection, and specifically has the following effects: improving a cell monolayer barrier transendothelial electrical resistance value, increasing integrity, reducing FITC-labeled dextran permeability of the cell monolayer barrier, and reducing permeability;adjusting a mouse vaginal mucin level;improving a mouse vaginal tissue protein expression level;reducing mouse vaginal tissue IL-1 beta secretion and myeloperoxidase content;and improving a mouse vaginal tissue pathological condition.Therefore, the lactobacillus gasseri has great application prospects in products for repairing a vaginal mucosa barrier and relieving a vaginal infection caused by pathogenic bacteria.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Lactobacillus gasseri targeting repair of vaginal barrier to assist in alleviating vaginal infection and a postbiotic thereof, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Reproductive tract infection is a common disease caused by microorganisms worldwide. Due to the unique physiological structure of the female reproductive tract and the cycle of the menstrual cycle, it is particularly susceptible to infection by external pathogenic bacteria, causing patients to suffer greatly and seriously affecting the quality of life of women.

[0003] The female reproductive tract mucosa is one of the sites of interaction between the body and pathogens. Since the vaginal mucosa is the main interface connecting the internal environment of the body and the external environment of the cavity, the immune defense on the surface of the vaginal mucosa is particularly important for protecting the deep tissues and organs. A healthy cervical vaginal microbiota maintains the integrity of the cervical epithelial barrier and regulates the mucosal immune system. The mucosal barrier is composed of a mechanical barrier (mucus and epithelial cells), factors related to the innate immune system (such as antibacterial peptides and enzymes), and adaptive immune responses. Cervical mucus is mainly composed of mucin, including MUC5B and MUC16, etc., and these proteins can act on pathogens by physically capturing microorganisms and promoting antibody binding. Vaginal epithelial cells constitute an interface that separates the individual from the environment. An intact epithelial cell layer, with the help of cell-to-cell tight junctions, an important structure that maintains the mechanical barrier and permeability of the mucosal epithelium, can effectively inhibit the invasion of pathogens into the local vagina. By enhancing the integrity of the epithelial barrier, accelerating the healing of vaginal mucosal damage, and reducing the inflammatory state, women can be protected from infection by sexually transmitted disease pathogens and other pathogens.

[0004] At present, the products selected for vaginal mucosal damage are mainly plant medicine hydrogel (CN111773259A) and protein peptides (CN110151981A), which have complex components and mainly function as antibacterial and anti-inflammatory. For vaginal infections, antibiotics are commonly used for treatment in clinical practice, such as metronidazole and clindamycin, which can effectively eliminate pathogenic microorganisms and improve the clinical symptoms of patients, but have high recurrence rates and drug resistance rates, and poor prognosis.

[0005] Currently, there are very limited probiotic products on the market for targeting the repair of the vaginal mucosal barrier. Lactobacillus rhamnosus GR-1 (ATCC 55826) is isolated from the distal urethra of a healthy woman, which improves the cervical vaginal epithelial barrier function by producing L-lactic acid, hydrogen peroxide and bacteriocin-like compounds (Selection of lactobacillus strains for urogenital probiotic applications). Vaginal lactobacillus live capsule (Dingjunsheng) is the only lactobacillus live preparation approved for marketing so far in China for the prevention and treatment of bacterial vaginosis, and is the first vaginal microecological live preparation in China. The effective component of the capsule is Lactobacillus delbrueckii DM8909, which is isolated from the vagina of a healthy woman and is the original strain, does not carry plasmid, is non-toxic and has low immunogenicity. DM8909 can repair the mucosal barrier function to a certain extent, which is beneficial to the recovery of normal cell function. However, both of the two strains do not belong to the vaginal dominant lactobacillus, and the colonization ability in the vagina is weak, and the main function is not related to the targeted vaginal barrier repair. There are many probiotics on the market for relieving vaginal infections, and the patents involved mainly include the relief and treatment of vaginitis (CN114250186A, CN112313325A), but the repair of vaginal mucosal barrier damage is not studied. Therefore, there is no research on vaginal source dominant lactobacillus which can target vaginal mucosal barrier and assist in relieving vaginal infections caused by pathogenic bacteria. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a Lactobacillus gasseri and its postbiotic which can repair the vaginal mucosal barrier and relieve vaginal infections caused by pathogenic bacteria, aiming to solve the technical problems of the prior art that lack research on the repair of vaginal mucosal barrier damage and can relieve infections caused by pathogenic bacteria.

[0007] The present application provides a Lactobacillus gasseri CCFM1336, which was deposited in the Guangdong Microbial Culture Collection Center on September 12, 2023, and the deposit number is GDMCC No:63792.

[0008] The Lactobacillus gasseri CCFM1336 is isolated from the vaginal secretion of a healthy woman.

[0009] The present application further provides a microbial preparation containing the above-mentioned Lactobacillus gasseri CCFM1336 or its fermentation broth, or its lysate, or its freeze-dried powder, or its postbiotic.

[0010] In an embodiment of the present application, the content of Lactobacillus gasseri CCFM1336 in the microbial preparation is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

[0011] The present application also provides a product containing the above-mentioned Lactobacillus gasseri CCFM1336 or the above-mentioned microbial preparation and / or its postbiotic.

[0012] In an embodiment of the present application, the preparation method of the postbiotic is: culturing Lactobacillus gasseri CCFM1336 for a period of time to obtain a bacterial solution, heat-treating and inactivating the obtained bacterial solution, centrifuging, collecting the precipitate, and obtaining the postbiotic.

[0013] In an embodiment of the present application, the preparation method of the postbiotic is: culturing Lactobacillus gasseri CCFM1336 for 24 h, calculating the total number of viable bacteria by pouring method, and diluting to be consistent with the number of viable bacteria for external use before preparation, heat-treating (105℃, 10 min) and centrifuging to remove the supernatant, obtaining the postbiotic and freeze-drying for standby.

[0014] In an embodiment of the present application, the product is a food, a medicine or a hygiene product.

[0015] In an embodiment of the present application, the medicine comprises Lactobacillus gasseri CCFM1336 and a pharmaceutically acceptable carrier.

[0016] In an embodiment of the present application, the carrier comprises one or more of fillers, binders, humectants, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0017] In an embodiment of the present application, the dosage form of the medicine comprises granules, capsules, tablets, pills, suppositories or oral liquids.

[0018] In an embodiment of the present application, the medicine comprises enteric-coated tablets and capsules for oral administration, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, gels.

[0019] In an embodiment of the present application, the hygiene product comprises sanitary wet wipes, sanitary napkins, sanitary pads, sanitary plugs, sanitary cotton, vaginal lotions, and women's antibacterial / antibacterial lotions.

[0020] In an embodiment of the present application, the content of Lactobacillus gasseri CCFM1336 in the product is not less than 1 x 106 CFU / mL or 1 x 10 6 CFU / g.

[0021] The present application also provides the use of Lactobacillus gasseri CCFM1336 or the microbial preparation and / or its postbiotic described above in the preparation of a medicine or a hygiene product for repairing the vaginal barrier and / or relieving vaginal infection.

[0022] In an embodiment of the present application, the repairing of the vaginal barrier includes enhancing the vaginal epithelial barrier by increasing the cell monolayer barrier integrity, reducing the cell monolayer barrier permeability, regulating the levels of MUC5B and MUC16 of the mouse vaginal barrier, increasing the expression levels of OCLN and CLDN1 proteins of the mouse vaginal barrier, and / or repairing the barrier damage caused by pathogenic bacteria infection.

[0023] In an embodiment of the present application, the medicine comprises the strain and / or preparation described above, and a pharmaceutically acceptable carrier.

[0024] In an embodiment of the present application, the carrier includes one or more of the fillers, binders, humectants, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0025] In an embodiment of the present application, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0026] In an embodiment of the present application, the medicine includes enteric-coated tablets and capsules for oral administration, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, gels.

[0027] In an embodiment of the present application, the hygiene product includes sanitary wet wipes, sanitary napkins, sanitary pads, sanitary suppositories, sanitary cotton, vaginal lotions, and women's antibacterial / antifungal lotions.

[0028] The present application also provides the use of Lactobacillus gasseri CCFM1336 or the microbial preparation and / or its postbiotic described above in the preparation of a medicine or a hygiene product for relieving vaginal infection.

[0029] In an embodiment of the present application, the relieving of the vaginal infection includes reducing the secretion of IL-1β and myeloperoxidase (MPO) in the mouse vaginal tissue.

[0030] In an embodiment of the present application, the medicine comprises the strain and / or preparation described above, and a pharmaceutically acceptable carrier.

[0031] In an embodiment of the present application, the carrier comprises one or more of a filler, a binder, a humectant, a disintegrant, a lubricant, a flavoring agent commonly used in medicine.

[0032] In an embodiment of the present application, the dosage form of the medicine comprises granules, capsules, tablets, pills, suppositories or oral liquids.

[0033] In an embodiment of the present application, the medicine comprises enteric-coated tablets and capsules for oral use, oral liquids, suppositories, tablets, gelatin capsules, sprays, creams, gels for vaginal use.

[0034] In an embodiment of the present application, the hygiene product comprises sanitary wet wipes, sanitary napkins, sanitary pads, tampons, sanitary cotton, vaginal washes, women's antibacterial / antifungal lotions.

[0035] Beneficial effects

[0036] The present application provides a Lactobacillus gasseri CCFM1336 isolated from the vaginal secretions of healthy women, which has the effects of repairing the vaginal mucosal barrier and relieving vaginal infections caused by pathogenic bacteria, and is specifically embodied in:

[0037] (1) increasing the transendothelial electrical resistance value of the cell monolayer barrier, increasing the integrity, reducing the FITC-labeled dextran permeability of the cell monolayer barrier, and reducing the permeability;

[0038] (2) regulating the levels of vaginal mucin MUC5B and MUC16 of the individual;

[0039] (3) increasing the protein expression levels of OCLN and CLDN1 in the vaginal tissue of the individual;

[0040] (4) reducing the secretion of IL-1β and the content of myeloperoxidase in the vaginal tissue of the individual;

[0041] (5) improving the pathological condition of the vaginal tissue of the individual.

[0042] Therefore, the Lactobacillus gasseri has great application prospects in products for repairing the vaginal mucosal barrier and relieving vaginal infections caused by pathogenic bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Figure 1: Effects of different bacterial lysates on the integrity and permeability of the vaginal epithelial barrier; A is the transendothelial electrical resistance value (TEER); B is the FITC-labeled dextran (FD-4) permeability graph.

[0044] Figure 2Figure 1: Flow chart of animal experiment design; Control: blank control group; Model: model group; Metronidazole: metronidazole intervention group; h-DM8909: DM8909 live bacteria external intervention group; s-DM8909: DM8909 dead bacteria external intervention group; h-CCFM1336: CCFM1336 live bacteria external intervention group; s-CCFM1336: CCFM1336 dead bacteria external intervention group; h-FHNXY73M2: FHNXY73M2 live bacteria external intervention group.

[0045] Figure 3 Figure 6: Effect of Lactobacillus gasseri on the expression of MUC5B and MUC16 in the vaginal barrier of mice.

[0046] Figure 4 Figure 7: Effect of Lactobacillus gasseri on the expression of CLDN1 and OCLN proteins in the vaginal barrier of mice.

[0047] Figure 5 Figure 8: Effect of Lactobacillus gasseri on the secretion of IL-1β and myeloperoxidase (MPO) in the vaginal barrier of mice.

[0048] Figure 6 Figure 9: Histopathological evaluation of mouse vaginal tissue.

[0049] (Different lowercase letters in the figure indicate significant differences between groups, p<0.05).

[0050] Biological material preservation

[0051] A strain of Lactobacillus gasseri CCFM1336, taxonomically named Lactobacillus gasseri, has been preserved in the Guangdong Microbial Culture Collection Center on September 12, 2023, with the preservation number GDMCC No: 63792 and the preservation address being the 5th floor of Building 59, 100 Middle Martyrs Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. DETAILED DESCRIPTION

[0052] To make the purpose, technical solutions and advantages of the present application clearer, the following will be further described in detail in combination with specific examples and with reference to the accompanying drawings.

[0053] The strains, cells and animals involved in the following examples are as follows: SPF grade BALB / c mice, female, 7 weeks old, weighing 18-20 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001).

[0054] Lactobacillus gasseri FJPY37L3 and Lactobacillus crispatus FHNXY73M2 were from the Jiangnan University Biotechnology Center Culture Collection. Lactobacillus delbrueckii DM8909 was isolated from "Dingjunsheng" vaginal lactobacillus live capsule. Gardnerella vaginalis ATCC 14018 was purchased from Guangdong Microbial Institute Culture Collection Center (GDMCC). Human vaginal epithelial cells VK2 / E6E7 were kindly gifted by the Department of Gynecology and Obstetrics of Renmin Hospital.

[0055] The culture media involved in the following examples are as follows:

[0056] MRS culture medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, sodium acetate anhydrous 2.0 g / L, citric acid hydrogen diamine 2.0 g / L, potassium phosphate dibasic 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, pH 6.2-6.4.

[0057] BHI culture medium: tryptone 10.0 g / L, beef heart infusion powder 17.5 g / L, sodium chloride 5.0 g / L, glucose 3.0 g / L, sodium phosphate dibasic dodecahydrate 2.5 g / L, yeast powder 10.0 g / L, maltose 1.0 g / L, pH 7.2-7.4; when the temperature cools to about 55°C, add 10% sterile fetal bovine serum.

[0058] Cell complete culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100×penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution).

[0059] Serum-free cell culture medium: 99% (v / v) DMEM medium + 1% (v / v) 100×penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution).

[0060] 5% MRS serum-free cell culture medium: 94% (v / v) DMEM medium + 1% (v / v) 100×penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution) + 5% (v / v) MRS.

[0061] The bacterial liquid involved in the following examples is as follows:

[0062] Preparation of lactobacillus liquid:

[0063] (1) Seed liquid preparation: Lactobacillus gasseri CCFM1336, FJPY37L3 and Lactobacillus delbrueckii DM8909 were inoculated into MRS medium respectively, and cultured at 37℃ for 24h to prepare seed liquid;

[0064] (2) The prepared seed liquid was inoculated into MRS liquid medium at an inoculation amount of 2%, and cultured at 37℃ for 24h to obtain culture liquid for experiment. For animal experiment, the lactobacillus was cultured at 37℃ for 24h until the bacterial concentration reached 1×10 9 CFU / mL, then the bacterial cells were collected by centrifugation and freeze-dried, and resuspended with PBS to 1% of the volume of the culture liquid before centrifugation.

[0065] Bacterial cell lysate:

[0066] (1) The lactobacillus liquid was prepared, and the bacterial liquid was obtained by homogenizing (800-1200MPa) 10 times in a high-pressure homogenizer and filtering with a 0.22μm filter membrane.

[0067] (2) Preparation of serum-free cell culture medium containing 5% (v / v) lactobacillus bacterial cell lysate:

[0068] The bacterial cell lysate obtained above was added to the serum-free cell culture medium at a ratio of 5% (v / v), including 94% (v / v) DMEM medium + 1% (v / v) 100×penicillin and streptomycin mixed solution (penicillin content 10000U / mL, streptomycin concentration 10mg / mL) + 5% (v / v) lactobacillus bacterial cell lysate.

[0069] Gardnerella vaginalis suspension:

[0070] (1) Seed liquid preparation: Gardnerella vaginalis was inoculated into BHI medium and cultured at 37℃ for 24h to prepare seed liquid.

[0071] (2) The seed liquid of Gardnerella vaginalis strain ATCC 14018 was inoculated into BHI medium at an inoculation amount of 2%, and cultured at 37℃ for 24h, then the bacterial cells were collected by centrifugation and resuspended with PBS to a concentration of 1×10 10 CFU / mL.

[0072] The detection methods involved in the following examples are as follows:

[0073] Cell transmembrane resistance determination:

[0074] The trans-epithelial electrical resistance (TEER) of the cells was measured, and it was considered that a stable state was reached when a dense monolayer was formed, which can be used to characterize the integrity of the monolayer barrier of the vaginal epithelial cells. The resistance was measured using an epithelial trans-epithelial electrical resistance meter, with the cell resistance meter electrodes perpendicular to the monolayer of cells, and the two electrodes on the apical (AP) and basolateral side (BL) of the cells, respectively, with the long electrode outside and the short electrode inside (gently place and take, without scratching the cells). The measured TEER was recorded as the average of three measurements per well. The resistance value measured for the chamber without inoculated cells was the blank TEER, and then the TEER of the cell monolayer was (measured TEER-blank TEER) x effective membrane area, with the unit of Ω·cm 2 is represented.

[0075] Detection of cell permeability and FD-4 permeability:

[0076] The monolayer permeability of the VK2 / E6E7 cells was evaluated by a fluorescent yellow (FITC-labeled dextran, FD-4) marker leakage test.

[0077] After measuring the TEER value, the culture solution was discarded, and the AP and BL chambers were washed with 3 times of PBS preheated to 37°C. Then, 0.6 mL of PBS preheated to 37°C was added to the BL, and 100 μL of FD-4 solution with a concentration of 100 μg / mL was added to the AP, and the mixture was incubated in a 37°C, 5% CO2 incubator for 1 hour (the above steps involving FD-4 were performed in the dark). The AP and BL liquids were taken, and the fluorescence intensity was measured by a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. A standard curve was prepared using different concentrations of FD-4, and the concentration of fluorescent yellow in the sample was calculated according to the standard curve.

[0078] FD-4 permeability (%) = BL side fluorescent yellow concentration / initial AP side fluorescent yellow concentration (x 100%).

[0079] Example 1: Isolation and identification of Lactobacillus gasseri CCFM1336

[0080] The specific steps are as follows:

[0081] Healthy female vaginal swab samples were collected and placed in EP tubes containing 1 mL of sterile normal saline. 0.2 mL was taken in 1.8 mL of sterile normal saline to obtain a 10 -1 dilution solution, and then 0.5 mL of the 10 -1 dilution solution was taken in 4.5 mL of normal saline to obtain a 10 -2 dilution solution. This operation was repeated to obtain 10 -3 , 10 -4 , 10 -5 , and 10 -6 gradient dilution solutions. 10 -4、10 -5 、10 -6 Each 1 mL of the diluent was poured into MRS solid medium, mixed gently, and then incubated at 37°C for 48 h after solidification.

[0082] The different morphological colonies were selected for streaking and purification on MRS plates, and then the purified single colonies were inoculated into 10 mL liquid medium and incubated at 37°C for 48 h. 1.5 mL of the cultured bacterial solution was centrifuged at 6000 r / min for 3 min, and the supernatant was discarded. After washing with 1.5 mL of sterile water for 3 times, the bacterial solution was resuspended in 1.5 mL of sterile water and used as a template for strain identification. The PCR system with a volume of 20 μL was set, in which 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 10 μL of 2×Taq Mixture, 0.5 μL of bacterial suspension, and 8.5 μL of double-distilled water were added. The primer information is shown in Table 1.

[0083] Table 1: Primer information table

[0084]

[0085] The correct strain was identified, 1.5 mL of the bacterial solution was taken into a 2 mL strain preservation tube, centrifuged at 6000 r / min for 3 min, and then the supernatant was removed in an ultra-clean bench. 1 mL of 30% sterile glycerol was added, mixed well with a vortex shaker, and then stored in a -80°C refrigerator.

[0086] The 16S sequence information is as follows (SEQ ID NO. 1):

[0087]

[0088] The obtained 16S sequence was subjected to species confirmation by BLAST of NCBI (http: / / www.ncbi.nlm.nih.gov / BLAST). The closer Query Cover and Identification to 100%, the more feasible. If there are multiple species in the alignment results, the Complete genome species is preferred in consideration of the numerical value. The results show that the above strain is Lactobacillus gasseri, named Lactobacillus gasseri CCFM1336.

[0089] Example 2: Ability of Lactobacillus to regulate cell monolayer barrier integrity and permeability in vitro

[0090] The specific steps are as follows:

[0091] 1. Construction of monolayer barrier model:

[0092] To establish a human vaginal epithelial cell (VK2 / E6E7) monolayer barrier model, the upper chamber of the Transwell cell culture plate was moistened with pre-cooled PBS solution, and then the recovered cells VK2 / E6E7 were inoculated into the upper chamber of the Transwell plate at a density of 1 x 10 4 cells per well, and cultured in serum-free medium at 37°C, 5% CO2 for 24 h, then replaced with cell complete medium, and continued to culture at 37°C, 5% CO2 for 18-21 days. After the culture ended, the transmembrane resistance of the cells was measured, and if it reached a stable state, it was considered to form a dense monolayer, which could be used to characterize the integrity of the vaginal epithelial cell monolayer barrier. The monolayer permeability of VK2 / E6E7 cells was evaluated by fluorescence yellow (FITC-labeled dextran, FD-4) marker leakage test.

[0093] 2. Verification of the ability of Lactobacillus to regulate cell monolayer barrier integrity and permeability in vitro

[0094] The whole experiment included control group, model group and treatment group, and the grouping information is shown in Table 2.

[0095] Table 2: Grouping information table

[0096]

[0097] Control group: To the well plate of the monolayer barrier model obtained in step 1, add serum-free cell culture medium containing 5% MRS, respectively, and culture at 37°C, 5% CO2 for 48 h;

[0098] Model group: to the hole plate of single layer barrier model obtained in step 1, add lipopolysaccharide (LPS) with a final concentration of 25 μg / mL respectively, after incubation for 24 h at 37℃, 5% CO2, then add serum-free culture medium containing 5% MRS, continue to incubate for 24 h at 37℃, 5% CO2;

[0099] Lactobacillus delbrueckii DM8909 group: to the hole plate of single layer barrier model obtained in step 1, add lipopolysaccharide (LPS) with a final concentration of 25 μg / mL respectively, after incubation for 24 h at 37℃, 5% CO2, then add serum-free culture medium containing 5% (v / v) Lactobacillus delbrueckii DM8909 cell lysate, continue to incubate for 24 h at 37℃, 5% CO2;

[0100] Lactobacillus gasseri CCFM1336 group: to the hole plate of single layer barrier model obtained in step 1, add lipopolysaccharide (LPS) with a final concentration of 25 μg / mL respectively, after incubation for 24 h at 37℃, 5% CO2, then add serum-free culture medium containing 5% (v / v) Lactobacillus gasseri CCFM1336 cell lysate, continue to incubate for 24 h at 37℃, 5% CO2;

[0101] Lactobacillus gasseri FJXPY37L3 group: to the hole plate of single layer barrier model obtained in step 1, add lipopolysaccharide (LPS) with a final concentration of 25 μg / mL respectively, after incubation for 24 h at 37℃, 5% CO2, then add serum-free culture medium containing 5% (v / v) Lactobacillus gasseri FJXPY37L3 cell lysate, continue to incubate for 24 h at 37℃, 5% CO2;

[0102] All groups of intervention, add to Transwell chamber AP side, give 1.5 mL with AP side same cell culture medium on BL side. After the end of the experiment, carry out cell transmembrane resistance value detection and cell permeability determination.

[0103] 3、Experimental results:

[0104] (1) The evaluation results of cell monolayer barrier integrity are shown as A in Figure 1 .

[0105] The detection results of cell transmembrane resistance value show that: compared with the control group (475.33 Ω·cm 2 ), the cell monolayer barrier resistance value of the model group is 323.33 Ω·cm 2 , the barrier resistance value decreases significantly (p<0.05), which indicates that the LPS model is successfully constructed.

[0106] Compared with the LPS treatment group (model group), the cell transmembrane resistance value of the Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri CCFM1336 cell lysate treatment groups was significantly increased (p<0.05), which was 455.33 Ω·cm 2 and 469 Ω·cm 2 respectively. The cell transmembrane resistance value of the Lactobacillus gasseri FJPY37L3 treatment group was 331 Ω·cm 2 , which had no significant difference compared with the model group, indicating that it had no effect on repairing the integrity of the vaginal epithelial cell monolayer barrier.

[0107] (2) Figure 1 The results in Table B show that the FD-4 permeability of the control group was 1.22%, and that of the model group was 4.57%, indicating that the permeability of the VK2 / E6E7 cells was significantly increased (p<0.05). The permeability of the Lactobacillus delbrueckii DM8909, Lactobacillus gasseri CCFM1336, and Lactobacillus gasseri FJPY37L3 treatment groups was 3.09%, 2.87%, and 4.14%, respectively. The permeability of the Lactobacillus gasseri CCFM1336 treatment group was lower than that of the Lactobacillus delbrueckii DM8909, but the difference was not significant. The permeability of the Lactobacillus gasseri FJPY37L3 treatment group was significantly higher than that of the Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri CCFM1336 groups, indicating that it could not reduce the permeability of the VK2 / E6E7 cell monolayer.

[0108] From the determination of the TEER value and FD-4 permeability of the VK2 / E6E7 cell monolayer, it can be seen that Lactobacillus gasseri CCFM1336 has the ability to improve the barrier integrity and reduce the permeability of the cell monolayer, and the effect is better than that of Lactobacillus delbrueckii DM8909.

[0109] Example 3: Application of Lactobacillus gasseri CCFM1336 in vaginal epithelial barrier damaged mice

[0110] 1. Preparation of Lactobacillus suspension

[0111] Lactobacillus delbrueckii DM8909 live bacteria group: Lactobacillus delbrueckii DM8909 was inoculated into MRS medium to prepare a seed solution at 37°C. The prepared seed solution was inoculated into MRS liquid medium at a 2% (v / v) inoculation amount, and cultured in a 37°C incubator for 24 h until the bacterial concentration reached 1×10 9 CFU / mL, then the bacteria were collected by centrifugation and freeze-dried, and resuspended with PBS to 1% of the volume of the culture before centrifugation.

[0112] Lactobacillus delbrueckii DM8909 dead bacteria group: the preparation method and concentration of the bacteria liquid of the dead bacteria group are the same as those of the live bacteria, except that high-pressure steam inactivation at 105°C for 10 minutes is used, the inactivation effect is checked by plate coating, and after heat treatment, the supernatant is removed by centrifugation to obtain the postbiotic and freeze-dried for standby, and resuspended with sterile PBS before intervention.

[0113] Lactobacillus gasseri CCFM1336 live bacteria group: Lactobacillus gasseri CCFM1336 was inoculated into MRS medium to prepare a seed liquid at 37°C; the prepared seed liquid was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and cultured in a 37°C incubator for 24 h until the bacterial concentration reached 1×10 9 CFU / mL, the bacterial cells were collected by centrifugation and freeze-dried, and resuspended with PBS to 1% of the volume of the culture before centrifugation.

[0114] Lactobacillus gasseri CCFM1336 dead bacteria group: the preparation method and concentration of the bacteria liquid of the dead bacteria group are the same as those of the live bacteria, except that high-pressure steam inactivation at 105°C for 10 minutes is used, the inactivation effect is checked by plate coating, and after heat treatment, the supernatant is removed by centrifugation to obtain the postbiotic and freeze-dried for standby, and resuspended with sterile PBS before intervention.

[0115] Lactobacillus crispatus FHNXY73M2 live bacteria group: Lactobacillus crispatus FHNXY73M2 was inoculated into MRS medium to prepare a seed liquid at 37°C; the prepared seed liquid was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and cultured in a 37°C incubator for 24 h until the bacterial concentration reached 1×10 9 CFU / mL, the bacterial cells were collected by centrifugation and freeze-dried, and resuspended with PBS to 1% of the volume of the culture before centrifugation.

[0116] 2. Experimental animals and strains:

[0117] SPF BALB / c mice, female, 7 weeks old, weighing 18-20 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2012-0001). Lactobacillus gasseri CCFM1336 and Lactobacillus crispatus FHNXY73M2 were from the Jiangnan University Biotechnology Center Culture Collection. Lactobacillus delbrueckii DM8909 was isolated from “Dingjunsheng” vaginal lactobacillus capsule. Lactobacillus gasseri was purchased from Guangdong Microbial Institute Culture Collection Center GDMCC. Figure 2 and Table 3 is the animal experiment process.

[0118] Table 3: Animal experiment scheme and grouping

[0119]

[0120] The mice were randomly divided into 8 groups according to the body weight, and all the mice were normally fed during the whole experiment according to Table 3. The vaginal barrier destruction model group and the intervention group both experienced continuous 5-day (3-7 days) vaginal Gardnerella infection (the specific operation method of the infection was that 20 μL of the bacterial suspension of Gardnerella was slowly injected into the vagina of the mouse by using a gun head, the mouse was inverted, and stayed for 1-2 minutes, and then was put into the cage). The probiotic intervention group was intervened with Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri CCFM1336 for 12 consecutive days (8-19 days) respectively. The specific operation method of all the interventions was that 20 μL of the corresponding bacterial suspension (the bacterial solution concentration of the dead bacteria group was the same as that of the live bacteria, which was inactivated by high-pressure steam at 105 ℃ for 10 minutes, and the inactivation effect was checked by plate coating) was slowly injected into the vagina of the mouse by using a gun head, the mouse was inverted, and stayed for 1-2 minutes, and then was put into the cage.

[0121] The specific steps are as follows:

[0122] (1) Inducing estrus: Except for the blank group, all the mice were subcutaneously injected with 100 μL of estradiol valerate (0.5 mg of estradiol valerate was dissolved in 100 μL of sesame oil) for three consecutive days (0-2 days) before being inoculated with Gardnerella vaginalis, to induce the estrus of the mice, and then once every 7 days.

[0123] (2) Infection experiment (modeling period): the mice in the blank control group were inoculated with 20 μL of PBS in the vagina on the 3rd day, and the mice in the other groups were inoculated with 20 μL of Gardnerella vaginalis bacterial suspension with a viable bacterial count of 1×10 10 CFU / mL in the vagina, which was slowly injected into the vagina of the mouse, the mouse was inverted for 1-2 minutes after inoculation to prevent the bacteria from flowing out, and the Gardnerella vaginalis inoculation was continuously performed for 5 days until the end of the 7th day.

[0124] (3) Intervention experiment: starting from the 8th day, the specific operations are as follows:

[0125] Blank control group: the mice were inoculated with 20 μL of PBS in the vagina once a day.

[0126] Model group of mice: the mice were inoculated with 20 μL of PBS in the vagina once a day.

[0127] Metronidazole treatment group: 1.62 mg of metronidazole per 20 g of mouse body weight was dissolved in PBS, and 20 μL of the solution was slowly injected into the vagina of the mouse by using a gun head.

[0128] Lactobacillus delbrueckii DM8909 live bacteria group: 20 μL of Lactobacillus delbrueckii DM8909 live bacteria bacterial suspension was inoculated in the vagina, which was slowly injected into the vagina of the mouse, the mouse was inverted for 1-2 minutes after inoculation to prevent the bacteria from flowing out, and then was put into the cage;

[0129] Lactobacillus delbrueckii subsp. bulgaricus DM8909 dead bacteria group: 20 μL of Lactobacillus delbrueckii subsp. bulgaricus DM8909 dead bacteria suspension was inoculated into the vagina of the mouse, and the mouse was placed in the cage after being inverted for 1-2 minutes (to prevent the bacteria from flowing out).

[0130] Lactobacillus gasseri CCFM1336 live bacteria group: 20 μL of Lactobacillus gasseri CCFM1336 live bacteria suspension was inoculated into the vagina of the mouse, and the mouse was placed in the cage after being inverted for 1-2 minutes (to prevent the bacteria from flowing out).

[0131] Lactobacillus gasseri CCFM1336 dead bacteria group: 20 μL of Lactobacillus gasseri CCFM1336 dead bacteria suspension was inoculated into the vagina of the mouse, and the mouse was placed in the cage after being inverted for 1-2 minutes (to prevent the bacteria from flowing out).

[0132] Lactobacillus crispatus FHNXY73M2 live bacteria group: 20 μL of Lactobacillus crispatus FHNXY73M2 live bacteria suspension was inoculated into the vagina of the mouse, and the mouse was placed in the cage after being inverted for 1-2 minutes (to prevent the bacteria from flowing out).

[0133] The experimental period was 19 days (0-18 days). On the 20th day, all experimental mice were sacrificed and the vaginal tissue was stripped for subsequent histopathological analysis, detection of mucin (MUC5B, MUC16) content, tight junction protein (OCLN, CLDN1) expression, and secretion of inflammatory factors IL-1β and myeloperoxidase in the vaginal tissue.

[0134] Determination method: at the end of the experiment, the mice were sacrificed and the vagina was removed. A part of the vaginal tissue was used for histopathological examination. Another part of the tissue was homogenized with pre-cooled RIPA lysis buffer (Bi Yun Tian Biotechnology Co., Ltd.) and protease inhibitor cocktail (Bi Yun Tian Biotechnology Co., Ltd.). The samples were centrifuged at 12000 r / min for 15 min at 4℃, and the vaginal tissue supernatant was used for determination of MUC5B, MUC16, CLDN1, OCLN, myeloperoxidase and IL-1β content according to the kit instructions.

[0135] Histopathological observation: the vaginal tissue was fixed with 4% paraformaldehyde, paraffin-embedded, and sliced into 5 mm thick sections, which were stained with hematoxylin and eosin (H&E). The vaginal tissue samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at 100 times magnification.

[0136] 3. Experimental results:

[0137] (1) Mucin expression analysis of mouse vaginal tissue

[0138] Mucus is essential for the protection of the mucosal site from molecules and microorganisms, and the cervical vaginal epithelial cells are the main source of mucus in the reproductive tract. Mucin is a large O-linked glycosylated protein, which is the main structural protein of mucus. MUC5B belongs to the secreted mucin, and MUC16 belongs to the gel-forming protein. Mucin has the functions of hydration, lubrication, protection of vaginal epithelial cells, regulation of signal transduction and transcription of vaginal epithelial cells, resistance to pathogenic microorganisms, and participation in immune functions such as lymph circulation and lymphocyte activation.

[0139] 1) The determination results of the important component mucin MUC5B of vaginal mucus are as shown in A of Figure 3

[0140] The secretion amount of the control group was 20.54 ng / mL, the model group increased compensatorily after being stimulated by pathogenic bacteria, reaching 28.20 ng / mL, and the secretion amount of MUC5B decreased to 25.67 ng / mL after metronidazole intervention. After intervention of the live Lactobacillus delbrueckii DM8909 group and the dead bacteria group, the secretion amounts were 21.86 ng / mL and 22.44 ng / mL, respectively. After intervention of the live Lactobacillus gasseri CCFM1336 group and the dead bacteria group, the secretion amounts were 20.42 ng / mL and 19.87 ng / mL, respectively, which were significantly lower than those of the model group and the metronidazole group (p<0.05), and the effects of the live bacteria group and the dead bacteria group were better than those of the Lactobacillus delbrueckii DM8909. The secretion amount of MUC5B after external use of the live Lactobacillus crispatus FHNXY73M2 was 23.77 ng / mL, which had a certain regulating effect on the secretion of MUC5B, but the effect was not as good as that of the live Lactobacillus gasseri CCFM1336 group and the dead bacteria group.

[0141] 2) The expression of vaginal tissue mucin MUC16 was detected at the same time, and the results are shown in B of Figure 3

[0142] The expression amount of the control group was 1.72 ng / mL, that of the model group was 2.75 ng / mL, and the expression amount of MUC16 increased significantly after intervention of Gardnerella vaginalis (p<0.05). After intervention, the expression amount of MUC16 of the metronidazole group was 2.14 ng / mL, that of the live Lactobacillus delbrueckii DM8909 group and the dead bacteria group was 1.85 ng / mL and 1.80 ng / mL, respectively, that of the live Lactobacillus gasseri CCFM1336 group and the dead bacteria group was 1.84 ng / mL and 1.80 ng / mL, respectively, which was better than that of the live Lactobacillus delbrueckii DM8909 group and the dead bacteria group, and was significantly different from that of the model group (p<0.05). The expression amount of MUC16 after intervention of the live Lactobacillus crispatus FHNXY73M2 was 2.18 ng / mL, which had a smaller effect on the secretion of MUC16 of the vaginal mucosal barrier caused by Gardnerella vaginalis.​​

[0143] In conclusion, both the live and dead *Lactobacillus gasseri* CCFM1336 groups can regulate the expression of vaginal epithelial mucins MUC5B and MUC16, reduce barrier inflammation, and restore the vaginal mucosal barrier mucus balance.

[0144] (2) Expression levels of tight junction proteins CLDN1 and OCLN in mouse vaginal tissue

[0145] The mechanical barrier of the cervical vaginal epithelium is mainly composed of the connections between epithelial cells and adjacent cells. These connections include tight junctions, gap junctions, adhesive junctions, and desmosomes. Tight junctions surrounding the top of the epithelium are the structural basis for maintaining the mechanical barrier and regulate the transepithelial transport of small molecules such as water and solutes. They are key factors that determine the integrity and permeability of the barrier. Figure 4 The protein expression levels of CLDN1 and OCLN in vaginal tissue.

[0146] 1) Expression level of CLDN1 protein in vaginal tissue, such as Figure 4 As shown in Figure A, the expression level of tight junction protein CLDN1 in the control group was 442.33 pg / mL, while that in the model group was 278.83 pg / mL, showing a significant difference compared to the control group (p < 0.05). The CLDN1 protein expression level in the metronidazole intervention group was 397.67 pg / mL, while the levels in the live and dead *Lactobacillus delbrueckii* DM8909 group were 385.08 pg / mL and 374.25 pg / mL, respectively. The levels in the live and dead *Lactobacillus gasseri* CCFM1336 group were 397.25 pg / mL and 438.75 pg / mL, respectively. The level in the topical intervention group with live *Lactobacillus curvatureii* FHNXY73M2 was 333.33 pg / mL. The dead *Lactobacillus gasseri* CCFM1336 group showed significantly better results than the live and dead *Lactobacillus delbrueckii* DM8909 group and the topical intervention group with live *Lactobacillus curvatureii* FHNXY73M2.

[0147] 2) In addition to CLDN1 protein expression, the expression level of OCLN protein was also detected. Figure 4The results of B in Table 2 show that the expression amount of occludin OCLN protein in the control group is 3.12 ng / mL, and the expression amount of tight junction protein in the model group is 1.74 ng / mL, indicating that vaginal Gardnerella causes damage to the basis of the mechanical barrier structure of the vagina. After metronidazole intervention, the expression amount of OCLN protein is 2.84 ng / mL. The expression amounts of OCLN protein in the live Lactobacillus delbrueckii DM8909 group and the dead group are 2.94 ng / mL and 2.30 ng / mL respectively, and the expression amounts of OCLN protein in the live Lactobacillus gasseri CCFM1336 group and the dead group are 3.03 ng / mL and 3.53 ng / mL respectively. The expression amount of OCLN protein in the dead Lactobacillus gasseri CCFM1336 group is higher than that in the live group, and is better than that in the live and dead Lactobacillus delbrueckii DM8909 groups. The expression amount of OCLN protein in the live Lactobacillus crispatus FHNXY73M2 group is 2.79 ng / mL, which is not as good as that in the live and dead Lactobacillus gasseri CCFM1336 groups.

[0148] In summary, the live and dead Lactobacillus gasseri CCFM1336 groups can effectively increase the expression of tight junction protein and adhesion junction protein of the cervical and vaginal mucosal barrier, enhance the integrity of the epithelial barrier, reduce the intercellular permeability, and maintain the function of the mechanical barrier.

[0149] (3) Mice vaginal tissue myeloperoxidase (MPO) content and IL-1β secretion

[0150] Myeloperoxidase (MPO) is mainly expressed in neutrophils in inflammatory tissues, and can reflect the aggregation of neutrophils in the vaginal tissue. Pro-inflammatory factor IL-1β plays an enhancing role in the inflammatory process, and can enhance the immune response of the host barrier and destroy the integrity of the barrier.

[0151] 1) The myeloperoxidase content in the vaginal tissue of mice was evaluated, and the results are shown in A of Table 2. Figure 5 A of Table 2 shows that, compared with the myeloperoxidase content (15.51 ng / L) of the control group, the myeloperoxidase content of the model group is significantly increased to 33.19 ng / L (p<0.05), indicating that vaginal Gardnerella causes accumulation of neutrophils in the vaginal tissue. After metronidazole intervention, the myeloperoxidase content is 22.22 ng / L, indicating that metronidazole can effectively relieve vaginal infection. The myeloperoxidase contents in the live and dead Lactobacillus delbrueckii DM8909 groups are 23.68 ng / L and 16.07 ng / L respectively, and the myeloperoxidase contents in the live and dead Lactobacillus gasseri CCFM1336 groups are 19.92 ng / L and 19.54 ng / L respectively. The myeloperoxidase content in the live Lactobacillus crispatus FHNXY73M2 group is 30.89 ng / L. Lactobacillus gasseri CCFM1336 can effectively reduce the myeloperoxidase content in the vaginal tissue, and the effect is better than that of the live Lactobacillus delbrueckii DM8909 group and the live Lactobacillus crispatus FHNXY73M2 group.

[0152] 2) Simultaneously, the inflammatory factor IL-1β in mouse vaginal tissue was detected, and the results were as follows: Figure 5 As shown in Figure B, the IL-1β secretion level in the control group was 77.94 ng / L. After infection with Gardnerella vaginalis, the IL-1β secretion level in the model group was 121.11 ng / L, significantly increased (p < 0.05). The metronidazole intervention group had a level of 85.63 ng / L, while the live and dead Lactobacillus delbrueckii DM8909 groups had levels of 87.36 ng / L and 87.73 ng / L, respectively. The live and dead Lactobacillus gasseri CCFM1336 groups had levels of 97.51 ng / L and 91.70 ng / L, respectively, all significantly lower than the model group (p < 0.05). The topical application of live Lactobacillus curvatureis FHNXY73M2 showed the worst effect, with a level of 105.62 ng / L.

[0153] In conclusion, both live and dead Lactobacillus gasseri CCFM1336 groups can effectively reduce the levels of vaginal inflammatory factors IL-1β and myeloperoxidase, and alleviate vaginal infections caused by Gardnerella vaginalis.

[0154] (4) Histopathological analysis of mouse vaginal tissue

[0155] HE staining of mouse vaginal tissue can effectively assess the integrity of the vaginal barrier and the degree of inflammation in each group of mice.

[0156] like Figure 6 As shown, the vaginal mucosa of mice in the blank control group was intact, with a certain amount of keratinized layer on the surface. In the model group, the keratinized layer on the vaginal mucosa disappeared, squamous epithelial cells proliferated, and a large number of inflammatory cells infiltrated the mucosa. After intervention with live and dead *Lactobacillus delbrueckii* DM8909 and live and dead *Lactobacillus gasseri* CCFM1336, the epithelial layer gradually repaired, inflammatory infiltration significantly decreased, and a certain amount of keratinized layer appeared on the mucosal surface. *Lactobacillus curvatureii* FHNXY73M2 showed weaker ability to restore the damaged vaginal barrier; compared with the model group, inflammatory cells infiltrated the mucosal epithelial layer to varying degrees, and the lamina propria structure was looser with inflammatory cell infiltration. *Lactobacillus gasseri* CCFM1336 can effectively promote the recovery of the vaginal mucosa in mice, alleviate inflammatory symptoms in the vaginal tissue, and improve the damaged vaginal structure.

[0157] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus gasseri ( Lactobacillus gasseri CCFM1336 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 12, 2023, with accession number GDMCC No:63792.

2. A microbial preparation, characterized in that, The microbial preparation contains the Lactobacillus gasseri CCFM1336 of claim 1 or a fermentation broth thereof, or a lysate thereof, or a freeze-dried powder thereof, or a postbiotic thereof.

3. The microbial preparation according to claim 2, characterized in that, Lactobacillus gasseri CCFM1336 is present in the microbial preparation in an amount of not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

4. A product containing the Lactobacillus gasseri CCFM1336 of claim 1 or the microbial preparation of claim 2 or 3.

5. The product of claim 4, wherein, The product is a food product, a pharmaceutical product or a hygiene product.

6. The product according to claim 4 or 5, characterized in that, Lactobacillus gasseri CCFM1336 is present in the product in an amount of not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

7. The product of claim 5, wherein, The hygiene product includes a sanitary wet tissue, a sanitary napkin, a sanitary pad, a sanitary tampon, a sanitary cotton, a vaginal wash, a lady's antibacterial / bacteriostatic lotion.

8. Use of the Lactobacillus gasseri CCFM1336 of claim 1 or the microbial preparation of claim 2 or 3 in the manufacture of a product for repairing the vaginal barrier to assist in alleviating vaginal infection or in the manufacture of a product for repairing the vaginal barrier and / or alleviating vaginal infection.

9. Use according to claim 8, characterized in that, The repairing of the vaginal barrier includes enhancing the vaginal epithelial barrier by increasing the cell monolayer barrier integrity, reducing the cell monolayer barrier permeability, modulating the levels of MUC5B and MUC16 of the individual vaginal barrier, and increasing the expression levels of OCLN and CLDN1 proteins of the individual vaginal barrier to repair the barrier damage caused by pathogenic bacteria infection.

10. Use according to claim 8 or 9, characterized in that, The product is a pharmaceutical product or a hygiene product.

11. Use according to claim 10, characterized in that, The hygiene product includes a sanitary wet tissue, a sanitary napkin, a sanitary pad, a sanitary tampon, a sanitary cotton, a vaginal wash, a lady's antibacterial / bacteriostatic lotion.

Images