A strain of Lactobacillus rhamnosus BB520 capable of regulating reproductive tract microecology and repairing reproductive tract mucosal barrier damage and its application

Abstract

The present invention provides a strain of Lactobacillus rhamnosus BB520 that can regulate genital microecology and repair genital mucosal barrier damage and its application. The Lactobacillus rhamnosus BB520 is preserved in the China Center for Type Culture Collection, with a preservation date of December 28, 2023, and a preservation number of CCTCC NO: M20232713. The Lactobacillus rhamnosus BB520 is non-irritating to the vaginal mucosa, has good safety, can quickly regulate and intervene in the imbalance of genital flora caused by bacterial vaginitis, HPV infection, and candidal vaginitis, and effectively treat genital tract infections; in addition, BB520 can also repair genital mucosal barrier damage caused by Gardnerella, especially when used in combination with Lactobacillus reuteri MeetU, the two have a synergistic effect, which can significantly improve the repair effect. Finally, the BB520 can also effectively inhibit Streptococcus pyogenes, Cryptococcus neoformans and / or Candida albicans, and has a certain intervention effect on atrophic vaginitis.

Description

Technical Field

[0001] The invention belongs to the technical field of Lactobacillus rhamnosus, and particularly relates to a Lactobacillus rhamnosus BB520 capable of regulating reproductive tract microecology and repairing reproductive tract mucosal barrier damage and an application thereof. Background Art

[0002] Under normal circumstances, lactobacilli coexist with other organisms in the female reproductive tract in a state of microecological balance. Lactobacilli inhibit the adhesion of pathogenic microorganisms by producing lactic acid, and the metabolites they produce have a direct bactericidal effect. Inhibiting the excessive growth of pathogenic bacteria, their large number forms a competitive space occupation, which can inhibit the growth of pathogenic bacteria. Once the former loses its advantage and the latter overgrows, this balance of normal flora is broken, manifested as an imbalance of the female reproductive tract microecology. The imbalance of the female reproductive tract microecology can also cause pathogens. When the relative number of lactic acid bacteria decreases, some pathogenic bacteria will invade vigorously, such as Escherichia coli, Peptococcus, Staphylococcus aureus, Group B Streptococcus, Candida, Trichomonas, Mycoplasma, etc. Leading to various types of vaginitis. The ultimate treatment goal of vaginal infection caused by imbalance of female reproductive tract microecology is to restore the balance of vaginal microecology. At present, the treatment of vaginal infection usually uses antibiotics, etc., but the side effects are large.

[0003] As mentioned above, the female reproductive tract has a stable flora environment, with beneficial bacteria and harmful bacteria in a dynamic balance. At the same time, it has a strong barrier function to prevent the invasion of external pathogens and microorganisms, and effectively prevent the occurrence of gynecological inflammation. However, the perfect barrier function of the reproductive tract is easily destroyed, such as failure to pay attention to local hygiene, changes in vaginal pH, and uterine cavity operations. Clinically, the occurrence, development and outcome of many diseases are related to reproductive tract mucosal barrier damage. Therefore, how to repair mucosal barrier damage safely, effectively, and without toxic side effects requires further research. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a strain of Lactobacillus rhamnosus BB520 and its application which can regulate reproductive tract microecology and repair reproductive tract mucosal barrier damage.

[0005] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0006] In the first aspect, the present invention provides a strain of Lactobacillus rhamnosus that can repair genital mucosal barrier damage. The Lactobacillus rhamnosus is Lactobacillus rhamnosus BB520 (Lacticaseibacillus rhamnosus BB520), which is deposited in the China Center for Type Culture Collection on December 28, 2023, and the deposit number is CCTCC NO: M20232713.

[0007] In a second aspect, the present invention provides a fermentation and culture method of the Lactobacillus rhamnosus BB520, wherein the fermentation and culture method uses a culture medium containing the following components for culture:

[0008] Soy peptone 8-20g / L, yeast extract powder 8-20g / L, anhydrous glucose 15-30g / L, lactose 3-5g / L, dipotassium hydrogen phosphate 1.0-2.5g / L, diammonium hydrogen citrate 1-3g / L, sodium acetate 2-8g / L, magnesium sulfate 0.05-0.2g / L, manganese sulfate 0.3-0.7g / L, L-cysteine ​​hydrochloride 0.5-1.5g / L, Tween 80 0.5-1.5ml / L, soybean lecithin 1-5g / L;

[0009] Preferably, the culture is carried out using a culture medium comprising the following components:

[0010] Soy peptone 13.5g / L, yeast extract powder 15g / L, anhydrous glucose 20g / L, lactose 5g / L, dipotassium hydrogen phosphate 1.5g / L, diammonium hydrogen citrate 1.5g / L, sodium acetate 5g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.5g / L, L-cysteine ​​hydrochloride 1g / L, Tween 80 1ml / L, soybean lecithin 2.5g / L.

[0011] As a specific implementation scheme, the fermentation culture method comprises: adjusting the pH value of the culture medium to 6.0-7.0 after preparation, sterilizing, inoculating the fermenter at a 3%-10% inoculation amount, preferably 8% inoculation amount, and continuously culturing at 35°C-37°C for 8-16 hours.

[0012] In a third aspect, the present invention provides a composition comprising the Lactobacillus rhamnosus BB520

[0013] In a fourth aspect, the present invention provides the use of the Lactobacillus rhamnosus BB520 and the composition in the preparation of a product for treating reproductive tract infection; preferably, the reproductive tract infection is bacterial vaginitis, HPV infection or candidal vaginitis.

[0014] In a fifth aspect, the present invention provides the use of the rhamnosus Lactobacillus BB520 and the composition in the preparation of a product for regulating reproductive tract microecology; preferably, the regulation of reproductive tract microecology is the regulation of reproductive tract flora imbalance caused by bacterial vaginitis, HPV infection or candidal vaginitis.

[0015] In a sixth aspect, the present invention provides the use of the Lactobacillus rhamnosus BB520 and the composition in the preparation of a product for repairing genital mucosal barrier damage; preferably, the genital mucosal barrier damage is genital mucosal barrier damage caused by Gardnerella.

[0016] In a seventh aspect, the present invention provides the use of the Lactobacillus rhamnosus BB520 and the composition in the preparation of a product for treating atrophic vaginitis.

[0017] In an eighth aspect, the present invention provides use of the Lactobacillus rhamnosus BB520 and the composition in the preparation of an antibacterial agent; preferably, the antibacterial agent can inhibit Streptococcus pyogenes, Cryptococcus neoformans and / or Candida albicans.

[0018] In a ninth aspect, the present invention provides a composition, comprising the rhamnosus Lactobacillus BB520 and Lactobacillus reuteri MeetU, wherein the Lactobacillus reuteri MeetU is deposited in the China Center for Type Culture Collection on December 11, 2023, and the deposit number is CCTCC NO: M 20232531. Preferably, in the composition, the ratio of the amount of live bacteria of Lactobacillus rhamnosus BB520 and Lactobacillus reuteri MeetU is (1-5): (1-5), and more preferably 1:1.

[0019] In a tenth aspect, the present invention provides the use of the above-mentioned composition comprising Lactobacillus rhamnosus BB520 and Lactobacillus reuteri MeetU in the preparation of a product for repairing genital mucosal barrier damage; preferably, the genital mucosal barrier damage is genital mucosal barrier damage caused by Gardnerella.

[0020] The products described in the present invention include food, antibacterial agents or medicines.

[0021] Beneficial effects: Compared with the prior art, the present invention provides a new strain of Lactobacillus rhamnosus BB520, which can effectively regulate the genital microecology and repair genital mucosal barrier damage. Experimental results show that Lactobacillus rhamnosus BB520 is non-irritating to the vaginal mucosa and has good safety. It can quickly regulate and intervene in the imbalance of genital flora caused by bacterial vaginitis (BV), HPV infection (HPV), and candidal vaginitis (VVC), and effectively treat genital tract infections; in addition, BB520 can also repair genital mucosal barrier damage caused by Gardnerella, especially when used in combination with Lactobacillus reuteri MeetU, the two have a synergistic effect, which can significantly improve the repair effect. Finally, BB520 can also effectively inhibit Streptococcus pyogenes, Cryptococcus neoformans and / or Candida albicans, and has a certain intervention effect on atrophic vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Gram stain of BB520 strain.

[0023] Figure 2 This is the biological evolution tree of BB520.

[0024] Figure 3 This is the abundance structure at the microbial genus classification level for each group of samples in Example 7.

[0025] Figure 4 It is the Pearson correlation coefficient of the microbial structure of the disease group and the microbial structure of the healthy group after 7 and 14 days of intervention in Example 7.

[0026] Figure 5 The effect of BB520 on the gene expression of genital mucosal tight junction proteins ZO-1 and Occludin.

[0027] Figure 6 The effect of BB520 on the gene expression of reproductive tract mucins MUC-1 and MUC-2. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0029] Example 1 Strain Isolation, Screening and Preservation

[0030] According to clinical medical standards, samples of reproductive tract flora of healthy women were collected. The health conditions must meet the following conditions: no oral or vaginal topical application of antibacterial drugs within 1 month, no menstrual disorders, avoiding menstrual period, no intrauterine contraceptive device, sexual life and vaginal lavage within 7 days, vaginal pH value ≤ 4.5, cleanliness I-II degree. Each sample was diluted gradiently and spread on MRS agar medium for culture. The same sample was spread twice in parallel and cultured in anaerobic and 5% oxygen environments for 48 hours, and the culture temperature was 35-37℃. Single colonies were selected from MRS agar medium and placed in 15ml MRS liquid culture medium. A total of 125 single colonies were selected and placed in a 35-37℃ shaker for 12 hours. Samples with poor liquid fermentation effect or no obvious bacterial amplification were discarded. 0.5ml of each of the 38 strains with better growth was placed in a 1.5ml cryopreservation tube, and an equal volume of 30% glycerol was added and mixed evenly for cryopreservation.

[0031] In order to further screen strains that are acid-resistant, have weak self-inhibition and strong survival ability, the remaining fermentation liquid was placed in a 35-37℃ shaker for fermentation and the pH value was measured every 8 hours for 3 consecutive times. After another 24 hours of fermentation, the lowest pH of the fermentation liquid could reach 3.5. 600 The samples with higher final fermentation pH values ​​of 3.5-4.0 were selected as candidate strains, with a total of 3 strains, correspondingly numbered BB418, BB473 and BB520.

[0032] Example 2

[0033] The three strains screened in Example 1 were inoculated in 50 ml of MRS liquid culture medium, cultured at 37°C for 16 h, and then the fermented liquid was centrifuged at 10,000 r / min for 10 min, and the supernatant and bacterial cells were collected respectively. The bacterial cells were washed twice with 0.9% sterile saline, and then prepared into a bacterial suspension with an OD600 of 0.5-0.8 with 0.9% sterile saline, and stored at 4°C for later use.

[0034] Streptococcus pyogenes ATCC19615, Cryptococcus neoformans ATCC 32609 and Candida albicans ATCC10231 were selected as test strains for in vitro antibacterial experiments, and Lactobacillus rhamnosus GR-1 was selected as the control test strain. The fermentation supernatant and bacterial suspension of each strain were tested by agar punching method for inhibition zone test. The MRS culture medium was adjusted with lactic acid to have the same pH value as the supernatant as a blank control, and amphotericin B with a concentration of 0.05 mg / mL was used as a positive control for inhibiting each pathogenic bacteria. The size of the inhibition zone was measured, and the antibacterial zone radius produced by the sample to be tested was greater than that of the MRS control solution, which was considered to have antibacterial activity. The evaluation results of strains with significant antibacterial effects are shown in Table 1.

[0035] The results showed that the bacterial suspension and fermentation supernatant of strain BB520 had strong inhibitory effects on Streptococcus pyogenes, Cryptococcus neoformans and Candida albicans, which were significantly better than strains BB418 and BB473. The antibacterial ability against Cryptococcus neoformans and Candida albicans was significantly better than the control strain Lactobacillus rhamnosus GR-1. Strain BB520 was selected as the preferred strain.

[0036] Table 1 Evaluation of antibacterial effect

[0037]

[0038] Example 3 Identification of strains and collection of strains

[0039] Based on the evaluation results during the comprehensive screening of strains, strain BB520, which has both high acid production / acid resistance and the ability to inhibit pathogenic bacteria, was selected as the target strain.

[0040] (1) Morphological identification

[0041] Observation of the purified bacterial liquid under a microscope showed that the cells were short, thick, rod-shaped, and without bifurcations. The colonies of the strain on MRS agar medium were milky white, semicircular protrusions, with a smooth, shiny surface and neat edges.

[0042] (2) Gram staining

[0043] Gram staining results are Gram-positive, such as Figure 1 shown.

[0044] (3) 16S rRNA molecular biological identification

[0045] The 16S rDNA gene sequence of the BB520 strain was amplified and sequenced using the published 16S universal primers (primer sequences: 8F: 5'-AGAGTTTGATCCTGGCTCA-3'; 1510R: 5'-GGTTACCTTGTTACGACTT-3'), and the PCR amplification product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing and identification. The nucleotide sequence of the 16SrDNA of the BB520 strain is sequence 1 in the sequence table; after 16SrDNA gene alignment, the similarity rate with Lacticaseibacillus rhamnosus in Genebank reached 99%; combined with microbial system identification, the BB520 strain was a rhamnosus Lactobacillus, named rhamnosus Lactobacillus BB520 (Lacticaseibacillus rhamnosusBB520), its 16S rDNA is shown in SEQ ID NO.1, and the biological evolution tree is shown in Figure 2 shown.

[0046] The biologically identified Lactobacillus rhamnosus BB520 was deposited in the China Center for Type Culture Collection (CCTCC) on December 28, 2023, with the deposit number CCTCC NO: M20232713. The deposit address is Wuhan University, Wuhan City, Hubei Province, People's Republic of China.

[0047] Example 4 Industrial scale fermentation of Lactobacillus rhamnosus BB520 and preparation of freeze-dried powder

[0048] In order to facilitate industrial expansion, the culture medium fermentation test and freeze-dried powder preparation of Lactobacillus rhamnosus BB520 were carried out.

[0049] (1) Culture medium configuration.

[0050] Rhamnosus lactobacillus casei BB520 culture medium scheme 1: soy peptone 13.5g / L, yeast extract powder 15g / L, anhydrous glucose 20g / L, lactose 5g / L, dipotassium hydrogen phosphate 1.5g / L, diammonium hydrogen citrate 1.5g / L, sodium acetate 5g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.5g / L, L-cysteine ​​hydrochloride 1g / L, Tween 80 1ml / L, soybean lecithin 2.5g / L. All raw materials are food grade raw materials.

[0051] Rhamnosus lactobacillus casei BB520 culture medium preparation scheme 2: soy peptone 8g / L, yeast extract powder 8g / L, anhydrous glucose 15g / L, lactose 3g / L, dipotassium hydrogen phosphate 1g / L, diammonium hydrogen citrate 1g / L, sodium acetate 2g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.5g / L, L-cysteine ​​hydrochloride 1g / L, Tween 80 1ml / L, soybean lecithin 1g / L. All raw materials are food grade raw materials.

[0052] Rhamnosus Lactobacillus casei BB520 culture medium preparation scheme 3: soy peptone 20g / L, yeast extract powder 20g / L, anhydrous glucose 30g / L, lactose 5g / L, dipotassium hydrogen phosphate 2.5g / L, diammonium hydrogen citrate 3g / L, sodium acetate 8g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.5g / L, L-cysteine ​​hydrochloride 1g / L, Tween 80 1ml / L, soybean lecithin 5g / L. All raw materials are food grade raw materials.

[0053] (2) Fermentation culture

[0054] After the medium was prepared, the pH value was adjusted to 6.5, sterilized at 115°C for 30 minutes, and the fermenter was inoculated at a 3%-10% inoculation amount, preferably 8%, and cultured continuously at 35°C-37°C for 8-16 hours. At 12 hours, the live bacterial counts of the fermentation broth under the three culture medium schemes were 6.5×10 9 CFU / mL, 3.7×10 8 CFU / mL, 2.2×10 9 The viable bacteria in the fermentation broth were 2.3×10 10 CFU / mL, 1.8×10 9 CFU / mL, 1.4×10 10 The CFU / mL level of live fermentation bacteria is already high, meeting the requirements of industrial high-density fermentation production. The preferred culture medium configuration scheme 1 has more advantages in fermentation bacterial growth rate and production ratio, and is used as the preferred culture medium scheme for Lactobacillus rhamnosus.

[0055] (3) Centrifugation and freeze-drying

[0056] After 12 hours of culture, the OD 600After the growth curve enters the plateau phase, the fermentation liquid is quickly cooled to below 15°C, centrifuged at 5000rpm using a tubular centrifuge, and the sludge is harvested. The harvested sludge is diluted to a water content of 60%-80%, and emulsified with a lyophilization protective agent of equal weight; the sludge protective agent comprises: 36% skim milk powder, 15% stachyose, 15% mannitol, 3% Tween-80, 15% betaine, 1% sodium glutamate, and 15% soluble starch. The emulsified sludge is placed in a freeze dryer for freeze drying, and the final material moisture content is less than 5%, and the water activity is 0.11-0.22aw.

[0057] The activity of freeze-dried bacterial powder can reach 9.2×10 11 CFU / g, which is convenient for storage. It is also convenient for adding lactic acid bacteria powder to food or for preparing microecological intervention preparations.

[0058] Example 5 Stimulation experiment of reproductive tract mucosal cells

[0059] According to the vaginal mucosal irritation test standard in the Disinfection Technical Specification, healthy female New Zealand rabbits in their early adulthood and in the non-estrus period were selected. Before the test, the vaginal opening of the animals was checked for secretions, congestion, edema, and other injuries. 0.5 g (9.2 × 10 11 CFU / g) was dissolved in 100ml sterile saline as the test solution. Three female New Zealand rabbits were randomly selected for exposure, and 2ml of the test solution was injected each time. The control group of animals was treated with saline in the same way. Each time was 24h apart for 5 consecutive days. 24h after the last exposure, the animals were killed by air embolism, the vagina was completely cut off and then cut longitudinally, and the mucosa was observed with the naked eye for edema, congestion, etc., and then fixed in 10% formaldehyde solution for histopathological examination.

[0060] According to the vaginal mucosal profile scoring standard and irritation intensity classification in the Technical Specifications for Disinfection, the vaginal mucosal irritation index of Lactobacillus rhamnosus BB520 freeze-dried powder is 0.89 < 1, which is considered to be non-irritating to the vaginal mucosa. It can be used as a biological agent directly for vaginal microecological intervention.

[0061] Example 6 Therapeutic effect of Lactobacillus rhamnosus BB520 on reproductive tract infection

[0062] According to clinical criteria, 6 adult women were diagnosed with bacterial vaginosis (BV), HPV infection (HPV), and candidal vaginitis (VVC) and had not used antibiotics within 1 month before diagnosis. The lyophilized powder of Lactobacillus rhamnosus BB520 was prepared into tablets, including 1 portion of lyophilized powder of Lactobacillus rhamnosus BB520 (viable count of 9.2×10 11CFU / ), 8.1 parts of sorbitol, 0.2 parts of magnesium stearate, and 0.7 parts of microcrystalline cellulose were mixed evenly to prepare 0.3-1.0g / tablet of lyophilized powder of Lactobacillus rhamnosus BB520, which disintegrated in water at room temperature for 10-60min. As a preparation for intervention of reproductive tract flora, one tablet was applied to the posterior fornix of the vagina every night. The collected healthy reproductive tract samples were selected as controls, and the reproductive tract samples of patients with gynecological inflammation were collected after the preliminary clinical diagnosis and selection into the intervention group. The reproductive tract secretion samples were collected again at 7d and 14d after using the reproductive tract flora preparation.

[0063] Among patients who used only the lyophilized powder tablets of Lactobacillus rhamnosus BB520 for intervention of gynecological diseases, no serious or worsening of the disease occurred. According to clinical standards, 12 patients with bacterial vaginitis (BV) and candidal vaginitis (VVC) showed good improvement, and 83.3% of the patients were clinically cured (10 / 12). The HPV infection (HPV) group showed good changes in the cleanliness of the reproductive tract, and the conversion rate was 50% (3 / 6). Therefore, Lactobacillus rhamnosus BB520 can intervene in the treatment of pathogenic bacteria and viruses in reproductive tract infections.

[0064] Example 7 Effect of Lactobacillus rhamnosus BB520 on microecological regulation after genital tract infection

[0065] For the samples collected in Example 6, 16S rRNA sequencing was performed using an Illumina Miseq gene sequencer to obtain sequencing data for each group of samples. At the microbial phylum classification level, Firmicutes accounted for 78.98%-97.17% of the reproductive tract samples in the healthy group, followed by Actinobacteria accounting for 0.74%-8.70%, Bacteroidetes accounting for 0.53%-2.95%, Tenericutes accounting for 0.03%-1.91%, and Proteobacteria accounting for 0.46%-1.5%.

[0066] The genus classification level of each group of samples is as follows Figure 3 As shown, Figure 3The figure is the mean graph of the genus level for the six groups. The top 19 genera with the highest proportion are selected in the figure. These 19 genera contain more than 95% of the microorganisms in all samples. It can be clearly seen that the content of Lactobacillus in the healthy group exceeds 90%. The content of Lactobacillus in the bacterial vaginitis (BV), HPV infection (HPV), and candidal vaginitis (VVC) groups was low when no intervention (0d), but the proportion of Gardnerella, which was mainly pathogenic bacteria, exceeded 15%. The abundance structure of other genera was disordered, and the proportion of various pathogenic bacteria was high, that is, the genital flora of various diseases was significantly different from that of healthy genital flora. When Lactobacillus rhamnosus BB520 was used for intervention for 7d and 14d, the flora structure was adjusted, and the flora structure dominated by Lactobacillus was restored.

[0067] Taking the healthy group as the standard, the Pearson correlation coefficients of the bacterial flora structures of each disease group and after 7 and 14 days of intervention with those of the healthy group are as follows Figure 4 As shown in the figure, it can be seen that the flora structure of each group of samples in the early stage of the disease has no correlation with the healthy group. After 7 and 14 days of intervention, the correlation coefficient is higher than 0.95, indicating that through the intervention of Lactobacillus rhamnosus BB520, the genital flora structure is highly similar to that of the healthy genital flora structure, that is, Lactobacillus rhamnosus BB520 can quickly regulate and intervene in the genital flora imbalance caused by bacterial vaginitis (BV), HPV infection (HPV), and candidal vaginitis (VVC).

[0068] Example 8 Repair effect of Lactobacillus rhamnosus BB520 on genital mucosal barrier damage caused by Gardnerella

[0069] Two-month-old female rats were selected, and according to the grouping and intervention scheme listed in Table 2, the infected model mice were genital tract infection Gardnerella infection model mice, and they were randomly divided into 5 groups. The Limosilactobacillus reuteri MeetU used was deposited in the China Center for Type Culture Collection (CCTCC) on December 11, 2023, with the deposit number CCTCC NO:M 20232531, and the deposit address is Wuhan University, Wuhan, Hubei Province, People's Republic of China. In addition to normal diet, the blank control group and the positive control group were given 20 μL of normal saline daily to irrigate the reproductive tract, the metronidazole group was given 20 μL of vitamin suspension daily to irrigate the reproductive tract (containing metronidazole 0.3 mg), and the other groups were given 20 μL of the corresponding bacterial suspension daily to irrigate the reproductive tract, and the amount of live bacteria in 20 μL was about 1.0×10 7CFU. In the TE3 and TE4 groups, the two bacteria were mixed and resuspended in a 1:1 ratio, and the concentration of live bacteria was the same as that in the TE1 and TE2 groups. After 14 days of intervention, the gene expression of the genital mucosal tight junction proteins ZO-1 and Occludin was used as an indicator of the physical barrier function of the genital mucosa by real-time fluorescence quantitative PCR technology, and the gene expression of the genital mucins MUC-1 and MUC-2 was detected as an indicator of the chemical barrier of the genital mucosa. The results were as follows: Figure 5 , Figure 6 shown.

[0070] Table 2 Experimental groups

[0071]

[0072]

[0073] The results showed that compared with the blank control group, Gardnerella infection of the reproductive mucosa led to a significant decrease in the gene expression of ZO-1, Occludin, MUC-1, and MUC-2, that is, the expression of genes related to tight junction proteins and mucins was reduced, and the mechanical and chemical barriers of the mucosa were destroyed. The intervention treatment with the antibiotic metronidazole increased and restored the gene expression of ZO-1, Occludin, MUC-1, and MUC-2 in the reproductive mucosa, but the effect was not significant. After intervention in model mice, Lactobacillus rhamnosus BB520 and Lactobacillus reuteri MeetU significantly increased the gene expression of ZO-1, Occludin, MUC-1, and MUC-2, indicating that the two strains can repair the damaged mechanical and chemical barriers of the reproductive mucosa, alleviate the normal absorption and secretion activities of the reproductive tract mucosa, increase the expression of mucins, thereby significantly increasing the thickness of the mucosal mucus layer, and strengthening the chemical intestinal barrier function of the colon. After Lactobacillus rhamnosus BB520 and Lactobacillus mucosa MeetU were compounded, the expression of genes related to tight junction proteins and mucins in the reproductive mucosa was significantly improved compared with other groups, indicating that the composite bacteria of Lactobacillus mucosa MeetU and Lactobacillus rhamnosus BB520 can significantly repair the genital mucosal barrier damage caused by Gardnerella, the strains can cooperate with each other, and this compounding method is better than other compounding methods. Therefore, Lactobacillus rhamnosus BB520, more preferably Lactobacillus rhamnosus BB520 and Lactobacillus mucosa MeetU can be compounded in a 1:1 ratio to repair the genital mucosal barrier damage caused by Gardnerella.

[0074] Example 9: Interventional effect of topical application of Lactobacillus rhamnosus BB520 on atrophic vaginitis

[0075] According to the clinical diagnostic criteria, patients diagnosed with atrophic vaginitis for the first time in the gynecological clinic aged 35-60 years old, who had not used any treatment drugs within 1 month, and who were able to cooperate with the test, were randomly divided into a treatment group and a control group of 18 patients in each group. The treatment group was given a tablet of lyophilized powder of Lactobacillus rhamnosus BB520, and the control group was given a tablet without Lactobacillus rhamnosus BB520, 1 tablet / day, soaked in clean water and placed in the posterior fornix of the vagina for 14 consecutive days. The tablet ingredients are: 1 portion of lyophilized powder of Lactobacillus rhamnosus BB520 (the number of viable bacteria is 9.2×10 11 CFU / g), 8.1 parts of sorbitol, 0.2 parts of magnesium stearate, 0.6 parts of microcrystalline cellulose, and 0.1 parts of sodium hyaluronate were mixed evenly and directly pressed into tablets of 0.5g / tablet by a tablet press. The relief of atrophic vaginitis before and after treatment was observed and compared. The results showed that the treatment group had a significant improvement in atrophic vaginitis, with a total effective rate of 94.4% for cure and marked effect, while the total effective rate of the control group was 11.1%. Therefore, Lactobacillus rhamnosus BB520 can be used as an intervention preparation for atrophic vaginitis.

[0076] The implementation modes of the present invention have been described in detail above in conjunction with specific embodiments, but the present invention is not limited to the above-mentioned implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus that can repair genital mucosal barrier damage, characterized in that: The Lactobacillus rhamnosus is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )BB520, deposited in the China Center for Type Culture Collection, the deposit date is December 28, 2023, and the deposit number is CCTCCNO:M 20232713.

2. The fermentation and culturing method of Lactobacillus rhamnosus BB520 according to claim 1, characterized in that: The fermentation culture method uses a culture medium containing the following components for culture: Soy peptone 8-20g / L, yeast extract powder 8-20g / L, anhydrous glucose 15-30g / L, lactose 3-5g / L, dipotassium hydrogen phosphate 1.0-2.5g / L, diammonium hydrogen citrate 1-3g / L, sodium acetate 2-8g / L, magnesium sulfate 0.05-0.2g / L, manganese sulfate 0.3-0.7g / L, L-cysteine ​​hydrochloride 0.5-1.5g / L, Tween 80 0.5-1.5ml / L, soybean lecithin 1-5g / L.

3. The fermentation and culture method of Lactobacillus rhamnosus BB520 according to claim 2, characterized in that: The fermentation culture method uses a culture medium containing the following components for culture: Soy peptone 13.5g / L, yeast extract powder 15g / L, anhydrous glucose 20g / L, lactose 5g / L, dipotassium hydrogen phosphate 1.5g / L, diammonium hydrogen citrate 1.5g / L, sodium acetate 5g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.5g / L, L-cysteine ​​hydrochloride 1g / L, Tween 80 1ml / L, soybean lecithin 2.5g / L; The fermentation culture method comprises: adjusting the pH value of the culture medium to 6.0-7.0 after preparation, sterilizing, inoculating and culturing in a fermenter at an inoculation amount of 3%-10%, and continuously culturing at 35°C-37°C for 8-16 hours.

4. The fermentation and culture method of Lactobacillus rhamnosus BB520 according to claim 3, characterized in that: In the fermentation culture method, the fermentation tank is inoculated and cultured at an inoculation rate of 8%.

5. A composition, characterized in that The composition comprises the Lactobacillus rhamnosus BB520 according to claim 1.

6. Use of the Lactobacillus rhamnosus BB520 according to claim 1 and the composition according to claim 5 in preparing a product for treating genital tract infection, characterized in that: The reproductive tract infection is bacterial vaginitis, HPV infection or candidal vaginitis.

7. Use of the Lactobacillus rhamnosus BB520 according to claim 1 and the composition according to claim 5 in preparing a product for regulating reproductive tract microecology, characterized in that: The regulation of reproductive tract microecology is to regulate the imbalance of reproductive tract flora caused by bacterial vaginitis, HPV infection or candidal vaginitis.

8. Use of the Lactobacillus rhamnosus BB520 according to claim 1 and the composition according to claim 5 in preparing a product for repairing genital mucosal barrier damage, characterized in that: The genital tract mucosal barrier damage is genital tract mucosal barrier damage caused by Gardnerella.

9. Use of the Lactobacillus rhamnosus BB520 according to claim 1 and the composition according to claim 5 in preparing a product for treating atrophic vaginitis.

10. Use of the Lactobacillus rhamnosus BB520 according to claim 1 and the composition according to claim 5 in preparing an antibacterial agent, characterized in that: The antibacterial agent can inhibit Streptococcus pyogenes, Cryptococcus neoformans and / or Candida albicans.

11. A composition, characterized in that The composition comprises the Lactobacillus rhamnosus BB520 and Lactobacillus reuteri MeetU according to claim 1, wherein the Lactobacillus reuteri ( Limosilactobacillus reuteri )MeetU, deposited in China Center for Type Culture Collection, the deposit date is December 11, 2023, and the deposit number is CCTCCNO:M 20232531.

12. Use of the composition according to claim 11 in preparing a product for repairing reproductive tract mucosal barrier damage, characterized in that: The genital tract mucosal barrier damage is genital tract mucosal barrier damage caused by Gardnerella.

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