Lactobacillus gasseri capable of improving expression of host antibacterial peptide Cathelicidin and secretory immunoglobulin sIgA and enhancing infection resistance by oral administration and external application and metagen of lactobacillus gasseri capable of improving expression of host antibacterial peptide Cathelicidin and secretory immunoglobulin sIgA

Abstract

The invention discloses lactobacillus gasseri capable of improving expression of host antibacterial peptide Cathelicidin and secretory immunoglobulin sIgA and enhancing infection resistance by oral administration and external application and a metagen thereof, and belongs to the technical field of microorganisms. The lactobacillus gasseri disclosed by the invention can be used for promoting the generation of a vaginal epithelial cell antibacterial peptide Cathelicidin; the shedding of vaginal epithelial cells is reduced, the fungal load is reduced, and the generation of vaginal tissue antibacterial peptide CRAMP is improved; the pIgR and immune globulin content of the vaginal tissue is increased; the invention also finds that the lactobacillus gasseri for external use can regulate and control TLR2 (Toll-Like Receptor 2), and further has a good balancing effect on body immunity. Therefore, the lactobacillus gasseri has a huge application prospect in a product for improving the pathogenic bacterium infection resistance of a host, enhancing the self-defense capability and inhibiting the vagina inflammation.

Description

Technical Field

[0001] The invention relates to a lactobacillus gasseri strain capable of increasing the expression of host antimicrobial peptide cathelicidin and secretory immunoglobulin sIgA and enhancing anti-infection and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] Vaginal pathogenic bacteria infection in women can cause a series of problems such as vaginal barrier destruction, oxidative damage, inflammation and excessive immune damage. Candida albicans often exists in the gastrointestinal tract and female lower reproductive tract as part of the local microbial flora and does not show any clinical symptoms. However, when the host's immunity is reduced or the use of broad-spectrum antibiotics causes local flora imbalance, Candida albicans will cause mucosal diseases such as vulvovaginal candidiasis (VVC). VVC is the most common vaginal infection in the world. 70% to 75% of women will have vulvovaginal candidiasis at least once in their lifetime, and the disease has a high recurrence rate. Clinically, the only effective means for Candida albicans infection in the female reproductive tract is to use antibiotics (fluconazole, clotrimazole, etc.) to directly inhibit bacteria. Antibiotics quickly and effectively eliminate pathogens, and may also kill beneficial flora in the vagina, especially Lactobacillus. After antibiotics eliminate these beneficial bacteria, the vaginal environment may become more suitable for the growth of pathogens, leading to secondary infection or recurrence, further endangering the host immune system. In addition, long-term or excessive use of antibiotics can promote the development of drug resistance in pathogenic bacteria, posing a threat to other parts of the body.

[0003] The vaginal microecological system with Lactobacillus as the main flora is an important part of maintaining female reproductive health and an important feature of the vaginal microecology. Lactobacillus can not only maintain the acidic environment of the vagina and inhibit the growth of harmful bacteria, but also promote and regulate host immunity, such as promoting the host to produce antimicrobial peptides and immune molecules such as immunoglobulins. The cathelicidin family of antimicrobial peptides is a small molecule peptide widely present in organisms and has a wide spectrum of antimicrobial activity. There is only one in the human body, namely LL-37. In mice, cathelicidin-related antimicrobial peptides (CRAMP) are homologous to human LL-37. They have the characteristics of high efficiency and stability. After using antimicrobial peptides, the resistance of pathogenic bacteria is greatly reduced because cathelicidin antimicrobial peptides are secreted by the host. Therefore, we propose to use vaginal commensal bacteria to stimulate the host to produce antimicrobial peptides, enhance the host's defense ability, and thus prevent vaginal infection.

[0004] In recent years, postbiotics have been widely used in the food industry, and they can be used as an alternative additive to probiotics. Postbiotics are defined as inactivated microorganisms and / or microbial cell components that are beneficial to the health of the host, including metabolites produced during their fermentation, active small molecules released by decomposition of fermentation substrates, dead cells, and cell components after lysis. The main active ingredients of postbiotics include short-chain fatty acids (SCFAs), peptides, and lipid walls. These ingredients have a variety of prebiotic functions, such as enhancing immunity. Postbiotics not only have direct biological activity, but can also act as signal molecules to affect the host's physiological processes. As a new type of health product, the research and application of postbiotics are developing rapidly, and it is expected to bring more benefits to human health in the future.

[0005] At present, the probiotics for adjuvant treatment of vaginal bacterial or mycotic vaginitis sold on the market are numerous, their mechanism of action is to treat by directly inhibiting the growth of pathogenic bacteria, the most extensive research is to utilize lactobacillus bacteriocin to inhibit pathogenic bacteria, and the bacteriocin utilizing lactobacillus secretion described in patent CN113797158A and CN113975368A plays the effect of resisting pathogenic bacteria. Because the mechanism of action of bacteriocin is mostly by combining with the specific receptor of target cell membrane, so its antimicrobial spectrum is narrow, it is difficult to cover a variety of different pathogenic microorganisms, in addition, bacteriocin belongs to protein substances, is easily degraded by the protease in the host or environment, thereby affects its stability and durability. The product of preventing vaginal infection is limited, can activate the innate immunity of the host, and simultaneously produces auxiliary and enhancing effects to adaptive immunity, and is rarely reported with the product of rapid response infection. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Lactobacillus gasseri and its postbiotics which can increase the expression of host antimicrobial peptides and secretory immunoglobulins and enhance anti-infection both orally and topically. The purpose is to solve the technical problem that there is a lack of research in the prior art on improving the host's ability to resist pathogenic bacteria infection, and to provide Lactobacillus gasseri and its postbiotics which can enhance the host's defense ability.

[0007] The first technical solution provided by the present invention is a strain of Lactobacillus gasseri CCFM1430, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65156. The Lactobacillus gasseri CCFM1430 was isolated from the vagina of a healthy woman.

[0008] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus gasseri CCFM1430.

[0009] In some embodiments, the content of Lactobacillus gasseri CCFM1430 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] The third technical solution provided by the present invention is the postbiotics prepared by the Lactobacillus gasseri CCFM1430.

[0011] In certain embodiments, the postbiotics include inactivated cells, fermentation supernatant or lysate of the Lactobacillus gasseri CCFM1430.

[0012] In certain embodiments, the preparation method of the postbiotics is any one of the following:

[0013] (a) culturing the Lactobacillus gasseri CCFM1430 to a stable phase, collecting the bacterial solution, and sterilizing it after high-pressure homogenization to obtain postbiotics;

[0014] (b) culturing the Lactobacillus gasseri CCFM1430 to a stable phase, collecting the bacterial solution, and centrifuging to obtain the supernatant to obtain postbiotics;

[0015] (c) culturing the Lactobacillus gasseri CCFM1430 to a stable phase, collecting the bacterial cells, and sterilizing them after high-pressure homogenization to obtain postbiotics.

[0016] In certain embodiments, the postbiotics are used in a liquid or solid form.

[0017] The fourth technical solution provided by the present invention is a product containing the Lactobacillus gasseri CCFM1430 or the microbial preparation or the postbiotic.

[0018] In certain embodiments, the product is a food, a medicine, or a hygiene product.

[0019] Furthermore, the medicine comprises the above-mentioned strain and / or preparation and / or postbiotic, and a pharmaceutically acceptable carrier.

[0020] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0021] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0022] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0023] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0024] The fifth technical solution provided by the present invention is a method for producing antimicrobial peptides, wherein the postbiotics are added to a culture environment of vaginal epithelial cells for culture.

[0025] In certain embodiments, the postbiotic is the lysate or fermentation supernatant of the Lactobacillus gasseri CCFM1430.

[0026] The sixth technical solution provided by the present invention is the use of the Lactobacillus gasseri CCFM1430 or the microbial preparation in the preparation of a product that enhances the host's ability to resist pathogenic bacteria infection and enhances its own defense ability.

[0027] In certain embodiments, the effect of enhancing resistance to vaginal pathogenic bacteria infection includes reducing the shedding of vaginal mucosal epithelial cells in mice, increasing the expression of antimicrobial peptide CRAMP, and promoting the secretion of vaginal polymeric immunoglobulin receptor and secretory immunoglobulin IgA.

[0028] In certain embodiments, the product is a medicine or a hygiene product.

[0029] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0030] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0031] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0032] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0033] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0034] The seventh technical solution provided by the present invention is the use of the Lactobacillus gasseri CCFM1430 or the microbial preparation or the postbiotic in the preparation of products for treating and / or preventing vaginal inflammation.

[0035] In certain embodiments, the effect of inhibiting inflammation comprises reducing the secretion of TNF-α and IL-6 in the spleen tissue of mice.

[0036] In certain embodiments, the product is a medicine or a hygiene product.

[0037] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0038] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0039] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0040] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0041] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0042] Beneficial effects:

[0043] The invention provides a strain of Lactobacillus gasseri CCFM1430, which is separated from the vagina of a healthy woman. The Lactobacillus gasseri CCFM1430 has the function of promoting vaginal epithelial cells to secrete antimicrobial peptides.

[0044] The Lactobacillus gasseri CCFM1430 of the present invention has the effects of improving the host's ability to resist pathogenic bacteria infection, enhancing its own defense ability and inhibiting vaginal inflammation, which is specifically embodied in: (1) reducing the shedding of vaginal mucosal epithelial cells; (2) increasing the expression of antimicrobial peptide CRAMP; (3) promoting the secretion of vaginal polymeric immunoglobulin receptor and secretory immunoglobulin sIgA; (4) reducing the secretion of TNF-α and IL-6 in spleen tissue; (5) improving vaginal pathological manifestations. Therefore, the Lactobacillus gasseri CCFM1430 has great application prospects in products that improve vaginal mucosal damage and inhibit vaginal inflammation.

[0045] Biomaterial Deposit

[0046] The Lactobacillus gasseri CCFM1430 provided by the present invention was isolated from the vagina of a healthy woman and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65156, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1: Streaking results of Lactobacillus gasseri CCFM1430.

[0048] Figure 2 :Lactobacillus gasseri CCFM1430 promotes the secretion of antimicrobial peptide LL-37 in vaginal epithelial cells.

[0049] Figure 3 :Flowchart of animal experiment design plan.

[0050] Figure 4 :The effect of topical Lactobacillus gasseri CCFM1430 on vaginal epithelial cell shedding and fungal load in vaginal lavage fluid of mice.

[0051] Figure 5 :The effect of topical Lactobacillus gasseri CCFM1430 on the expression of antimicrobial peptide CRAMP in vaginal tissue.

[0052] Figure 6 :The effect of topical Lactobacillus gasseri CCFM1430 on polymeric immunoglobulin receptors and secretory immunoglobulins in vaginal tissue.

[0053] Figure 7 :Effects of topical Lactobacillus gasseri CCFM1430 on TLR2 expression in vaginal tissue.

[0054] Figure 8 :The effect of topical Lactobacillus gasseri CCFM1430 on the expression of cytokines TNF-α and IL-6 in spleen suspension.

[0055] Fig. 9 : Effects of topical Lactobacillus gasseri CCFM1430 on vaginal tissue pathological sections.

[0056] Fig.10 :The effect of oral administration of Lactobacillus gasseri CCFM1430 on vaginal epithelial cell shedding and fungal load in vaginal lavage fluid of mice.

[0057] Fig.11 :The effect of oral administration of Lactobacillus gasseri CCFM1430 on the expression of antimicrobial peptide CRAMP in vaginal tissue.

[0058] Fig.12 :Effects of oral administration of Lactobacillus gasseri CCFM1430 on polymeric immunoglobulin receptors and secretory immunoglobulins in vaginal tissue.

[0059] Fig.13 :The effect of oral administration of Lactobacillus gasseri CCFM1430 on the expression of cytokines TNF-α and IL-6 in spleen suspension.

[0060] Fig.14 : Effects of oral administration of Lactobacillus gasseri CCFM1430 on vaginal tissue pathological sections.

[0061] (Different lowercase letters in the figure indicate significant differences between the groups, p < 0.05) DETAILED DESCRIPTION

[0062] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0063] The culture medium involved in the following examples is as follows:

[0064] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the penicillin content in the mixed solution is 10000 U / mL, and the streptomycin concentration is 10 mg / mL).

[0065] MRS solid medium ( / L): 5g peptone, 5g yeast extract powder, 15g glucose, 3g disodium hydrogen phosphate, 1mL Tween-80, 3g dipotassium hydrogen phosphate, 3g diammonium hydrogen citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 20g agar, pH 6.2-6.4.

[0066] MRS liquid medium ( / L): 5g peptone, 5g yeast extract powder, 15g glucose, 3g disodium hydrogen phosphate, 1mL Tween-80, 3g dipotassium hydrogen phosphate, 3g diammonium hydrogen citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, pH 6.2-6.4.

[0067] SDA medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 40g, agar 15g, pH 5.6±0.2.

[0068] SDB medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 20g, pH 5.6±0.2.

[0069] SDA medium (containing chloramphenicol) ( / L): 10 g of equal mixture of animal tissue pepsin hydrolysate and trypticase, 40 g of glucose, 15 g of agar, 0.1 g of chloramphenicol, pH 5.6±0.2.

[0070] The strains, cells and animals involved in the following examples are as follows:

[0071] Vaginal epithelial cells (VK2 / E6E7) were kindly donated by the Department of Obstetrics and Gynecology, Wuxi People's Hospital, Jiangsu Province.

[0072] Lactobacillus delbrueckii DM8909 was isolated from Dingjunsheng vaginal lactobacillus capsules and stored in the strain bank of Food Biotechnology Center of Jiangnan University.

[0073] Lactobacillus gasseri CCFM1430 and Lactobacillus gasseri VJSWX136L1 were isolated from female vagina and stored in the strain bank of Food Biotechnology Center of Jiangnan University.

[0074] 7-week-old SPF female BALB / c mice, weighing 18-20 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0075] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology Culture Collection Center (GDMCC).

[0076] The preparation methods of samples and reagents involved in the following examples are as follows:

[0077] Lactobacillus gasseri CCFM1430 live bacteria (CCFM1430-L): Lactobacillus gasseri CCFM1430 was streaked and isolated in MRS solid medium, cultured in an anaerobic incubator at 37°C for 36 h, single colonies were picked and placed in 5 mL of MRS liquid medium, cultured in an anaerobic incubator at 37°C for 20 h, inoculated into 1 L of culture system at 4% inoculum, cultured in an incubator at 37°C for 20 h, and centrifuged to obtain the bacterial cells, and the bacterial solution concentration was adjusted to 5×10 9 CFU / mL, as the live bacterial suspension. Lactobacillus delbrueckii DM8909 was prepared as above and was recorded as DM8909-L.

[0078] Lactobacillus gasseri CCFM1430 inactivated bacterial suspension (CCFM1430-D): 5×10 9 The live bacterial suspension of Lactobacillus gasseri CCFM1430 at CFU / mL was homogenized 10 times (800-1200 MPa) in a high-pressure homogenizer and pasteurized (65°C for 30 min) to obtain the inactivated bacterial suspension, which was freeze-dried and stored in a -80°C refrigerator and resuspended to the original volume with sterile saline before the experiment.

[0079] Lactobacillus gasseri CCFM1430 cell lysate (CCFM1430-sub): 5×10 9 After centrifugation of the live bacterial suspension of Lactobacillus gasseri CCFM1430 at CFU / mL, the cells were taken out and resuspended with an equal volume of sterile water. After homogenization (800-1200 MPa) 10 times in a high-pressure homogenizer and pasteurization (65°C 30 min), the cell lysate was obtained, freeze-dried and stored in a -80°C refrigerator, and resuspended to the original volume with sterile saline before the experiment. The preparation method of the cell lysate of Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri VJSWX136L1 was the same as above, and they were recorded as DM8909-sub and VJSWX136L1-sub, respectively.

[0080] Lactobacillus gasseri CCFM1430 fermentation supernatant (CCFM1430-sup): 5×10 9 After centrifugation of the CFU / mL live bacterial suspension of Lactobacillus gasseri CCFM1430, the fermentation supernatant was obtained, freeze-dried and stored in a -80°C refrigerator, and resuspended to the original volume with sterile saline before the experiment. The fermentation supernatant of Lactobacillus gasseri VJSWX136L1 was prepared in the same manner as above and was recorded as VJSWX136L1-sup.

[0081] Candida albicans suspension: SC5314 was streaked on SDA medium and cultured in a 28°C incubator for 48 h. A single colony was picked and inoculated into SDB medium for expansion. The final concentration of the bacterial suspension was adjusted to 5×10 8 CFU / mL.

[0082] Estradiol: 0.1 mg of β-estradiol is dissolved in 0.05 mL of sesame oil and is prepared for immediate use.

[0083] Example 1 Isolation and Identification of Lactobacillus gasseri CCFM1430

[0084] Collect vaginal swab samples from healthy women and place them in an EP tube containing 1 mL of sterile saline. Pipette 0.2 mL into 1.8 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.5mL 10 -1 Dilute in 4.5 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution. Take 10 -4 , 10 -5 , 10 -6 Place 1 mL of each dilution in a dish, pour into MRS solid culture medium, mix gently, and after the culture medium solidifies, invert and culture at 37°C for 48 h.

[0085] Select colonies of different morphologies and perform streak purification on MRS plates ( Figure 1 ), pick the purified single colony and inoculate it into 5mL liquid culture medium, and culture it at 37℃ for 48h. Take 1.5mL of the cultured bacterial solution, centrifuge it at 6000r / min for 3min, discard the supernatant, add 1.5mL of sterile water to wash 3 times, resuspend it in 1.5mL of sterile water, and use it as a template for bacterial identification. Set up a PCR system with a volume of 20μL, add 0.5μL of forward primer (10μM), 0.5μL of reverse primer (10μM, 10μL2×Taq Mixture, 0.5μL of bacterial suspension, and 8.5μL of double distilled water. Primer information is shown in Table 1.

[0086] Table 1: Primer information

[0087]

[0088] For the correctly identified strains, take 1.5 mL of bacterial solution into a 2 mL strain storage tube, centrifuge at 6000 r / min for 3 min, remove the supernatant in a clean bench, add 1 mL of 30% sterile glycerol, mix thoroughly with a vortex oscillator, and store in a -80°C refrigerator.

[0089] 16S sequence information of Lactobacillus gasseri CCFM1430 (SEQ ID No.1):

[0090] CCATTGGTCTACTTAGACGGCTGACTCCTATAAAGGTTATCCCACCGGCTTTGGGTGTTA

[0091] CAGACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGC

[0092] GTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGTAGGCGAGTTGCAGCCTACAGTC

[0093] CGAACTGAGAACGGCTTTCAGAGATCCGCTTGCCTTCGCAGGTTCGCTTCTCGTTGTAC

[0094] CGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCC

[0095] CCACCTTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGATGGCAA

[0096] CTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTG

[0097] ACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGGAACACCTAATCTCTTAGGT

[0098] TTGCACTGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGC

[0099] TCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCC

[0100] CCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACAC

[0101] TTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGC

[0102] TTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCC

[0103] ATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTT

[0104] CAACAGTTTCTGATGCAATTCTCCGGTTGAGCCAAAGGCTTTCACATCAGACTTATTGAA

[0105] CCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACC

[0106] GCGCTGCTGGCACGTAGTAGCCGTGACTTTCTAAGTAATACGTCAAATAAAGGCAGTACT

[0107] ACTCTATCTTTCTCACTACACAGAGCTTACGAGCGAAACCCTTCTCACTCACGCGCGTGC

[0108] TCATCAGACTGCGTCATTGTGAGATCCCTACTGCTGCCTCCGGTGAGAGTGGCAGTTCTC

[0109] AGTTCCAATGTGGCCGAATCAGCTCTCTCACCTCGCGTAATAGCACTATGCCTTGAAGCGATACCTTACCACATGCTATGGAACCGCAAGGTCTCATCTCTACC.

[0110] The screening and identification methods of Lactobacillus gasseri VJSWX136L1 used in the cell experiment were the same as above.

[0111] Example 2 Effect of Lactobacillus gasseri CCFM1430 on the secretion of antimicrobial peptide LL-37 by vaginal epithelial cells

[0112] The specific steps are as follows:

[0113] Vaginal epithelial cells (VK2 / E6E7) were cultured at 2×10 5 Inoculate cells / well in a 6-well cell plate and culture the cells overnight until the cells adhere to the wall. Discard the old culture medium, rinse with PBS buffer 3 times, and add samples according to the following steps:

[0114] (1) The experiment of intervening cells with bacterial lysate was divided into three groups: blank group, Lactobacillus gasseri CCFM1430 lysate (CCFM1430-sub) group, and Lactobacillus gasseri VJSWX136L1 lysate (VJSWX136L1-sub) group. Cell culture medium containing 5% (v / v) normal saline, Lactobacillus gasseri CCFM1430 lysate, and Lactobacillus gasseri VJSWX136L1 lysate was added, respectively.

[0115] (2) The experiment of fermentation supernatant intervention on cells was divided into three groups: blank group, Lactobacillus gasseri CCFM1430 fermentation supernatant (CCFM1430-sup), Lactobacillus gasseri VJSWX136L1 fermentation supernatant (VJSWX136L1-sup) were added to the cell culture medium containing 5% (v / v) MRS liquid culture medium, Lactobacillus gasseri CCFM1430 fermentation supernatant, and Lactobacillus gasseri VJSWX136L1 fermentation supernatant, respectively.

[0116] After addition, the cells were incubated in 5% CO 2 , carried out at 37°C. After incubation for 24 h, the cell culture supernatant was aspirated and the antimicrobial peptide LL-37 was determined using an ELISA kit.

[0117] The increase in the content of host antimicrobial peptide LL-37 can effectively resist pathogenic bacteria infection, reduce pathogenic bacteria adhesion, and enhance host immunity. Figure 2As shown, there was no significant difference between the Lactobacillus gasseri VJSWX136L1 bacterial lysate group and the blank group. After being treated with the bacterial lysate of Lactobacillus gasseri CCFM1430, the content of LL-37 secreted by vaginal epithelial cells was significantly increased, reaching 21.39pg / mL (p < 0.05). In the fermentation supernatant group, there was no significant difference between the secretion of LL-37 by the fermentation supernatant of Lactobacillus gasseri VJSWX136L1 and the blank group. The secretion of LL-37 by the fermentation supernatant of Lactobacillus gasseri CCFM1430 was significantly increased by 26% compared with the blank group (p < 0.05). In summary, the bacterial lysate and fermentation supernatant of Lactobacillus gasseri CCFM1430 can stimulate vaginal epithelial cells, promote the secretion of antimicrobial peptide LL-37 and prevent vaginal infection, and have great application potential for enhancing host resistance to pathogenic bacteria infection.

[0118] Example 3 Application of topical Lactobacillus gasseri CCFM1430 in improving mice's resistance to pathogenic bacteria infection

[0119] Experimental animals and strains:

[0120] Twenty-five 7-week-old SPF female BALB / c mice weighing 18-20 g were randomly divided into 5 cages, with 5 mice in each cage.

[0121] Table 2: Animal experimental plan and grouping

[0122]

[0123] The animals were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001). The animal experiment scheme and grouping are shown in Table 2. The specific steps are as follows:

[0124] The vaginal infection prevention experiment adopted the method of intervention first and then modeling. The blank group and the model group were required to be inoculated with 20 μL of normal saline in the vagina for 17 consecutive days, and the experimental group was required to use a pipette to draw 20 μL of 5×10 9 The bacterial lysate of Lactobacillus delbrueckii DM8909, the bacterial lysate of Lactobacillus gasseri CCFM1430, and the fermentation supernatant of Lactobacillus gasseri CCFM1430 prepared with CFU / mL bacterial suspension were slowly injected into the vagina of mice, and the mice were inverted for 1-2 minutes. On days 12-15, the blank group was subcutaneously injected with 50 μL of normal saline, and the model group and the experimental group were subcutaneously injected with 50 μL of estradiol to induce estrus (subcutaneous injection on days 12 and 15). On days 15-17, the model group and the experimental group used a pipette to draw 20 μL of a concentration of 5×10 8CFU / mL of Candida albicans suspension was slowly injected into the vagina of mice, and the mice were inverted for 1-2 minutes. The blank group was inoculated with normal saline in the vagina once a day. The experimental period was 17 days. At the end of the intervention (day 18), 50 μL of normal saline was sucked out of the mouse vagina with a gun tip each time to sample, and 300 μL of vaginal lavage fluid was finally collected for subsequent analysis of vaginal epithelial cell shedding in the mouse vagina. At the same time, on day 18, all experimental mice were killed and vaginal tissues were peeled off to detect the content of antimicrobial peptide CRAMP (human antimicrobial peptide LL-37 is homologous to mouse antimicrobial peptide CRAMP), inflammatory factor IL-6, and α tumor necrosis factor (TNF-α) in vaginal tissues, as well as subsequent experimental tissue pathology analysis.

[0125] Determination method: At the end of the experiment, the mice were killed and the vaginal tissue was dissected. A portion of the tissue was homogenized with pre-cooled RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd.) and a mixture of protease inhibitors. The sample was centrifuged at 2000r / min for 15min at 4°C, and the supernatant of the vaginal tissue was taken for determination of the antimicrobial peptide CRAMP and polymeric immunoglobulin receptor content according to the instructions of the kit; the vaginal tissue RNA was extracted by Trizol method and reverse transcribed into cDNA to detect pathway-related genes including TLR2 in the process of antimicrobial peptide production. The primers are shown in Table 3 below. In addition, the spleen tissue of the mouse was homogenized, and the spleen supernatant was taken for determination of IL-6 and TNF-α content (Nanjing Senbeijia Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in 4% paraformaldehyde solution for histopathological examination.

[0126] Table 3 Animal experiment primer information

[0127]

[0128] 10 μL of sample solution was taken from the vaginal lavage fluid of mice on day 18, transferred to a glass slide, gently smeared with the outer wall of the pipette tip, dried with an alcohol burner flame, and stained with Diff-Quik staining (a rapid staining method improved from Wright's staining). Five fields of view were captured from each sample (1 mouse) under a microscope, and epithelial cells were counted from each image to determine the average value.

[0129] Histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced ​​into 5 mm thick slices, and stained with hematoxylin and eosin (H&E). Vaginal tissue samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0130] The results are as follows:

[0131] (1) Vaginal epithelial cell shedding and Candida albicans load in mouse vaginal lavage fluid

[0132] Vaginal epithelial cell shedding refers to the natural shedding of epithelial cells in the vagina from the tissue surface. This is a normal part of the female physiological process and is usually related to the vaginal self-cleaning mechanism. In the case of vaginal infection or other problems that endanger vaginal health, vaginal epithelial cells will shed in large quantities. This excessive shedding of vaginal epithelial cells can cause damage to the vaginal barrier, increase the invasion of multiple pathogens, and further increase the risk of vaginal infection. By evaluating the shedding of vaginal epithelial cells in the vagina of mice (such as Figure 4 A) It can be seen that the epithelial cells in the model group fall off seriously, which is 27.13 / visual field, while the blank group has about 4.43 cells under each visual field, indicating that the intervention of Candida albicans damages the vaginal epithelial cells, causing them to fall off in large quantities. The number of mouse vaginal epithelial cells falling off of the external group Lactobacillus delbrueckii DM8909 bacterial lysate (DM8909-sub) is significant compared with the model group, which is 9.2% lower than the model group, and the number of mouse vaginal epithelial cells falling off of the intervention of Lactobacillus gasseri CCFM1430 bacterial lysate is significant compared with the model group, which is 78.7% lower than the model group, and the number of mouse vaginal epithelial cells falling off of the intervention of Lactobacillus gasseri CCFM1430 fermentation supernatant is significant compared with the model group, which is 53.0% lower than the model group. Therefore, the external application of Lactobacillus gasseri CCFM1430 bacterial lysate and fermentation supernatant can enhance the defense ability of the mouse vagina and reduce the large-scale shedding of vaginal epithelial cells.

[0133] 0.1 mL of the collected vaginal lavage fluid was taken out and added to 0.9 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.1mL 10 -1 Dilute in 0.9 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilutions. Take 0.1 ml of each of the above gradient dilutions in a plate, pour in SDA culture medium containing chloramphenicol, mix gently, and after the culture medium solidifies, invert and culture at 28°C for 48 hours, and record the number of live bacteria. After the intervention of probiotics, the body will strengthen its inherent immunity and enhance the host's ability to resist pathogenic bacteria. When Candida albicans suddenly infects mice, the enhancement of inherent immunity will inhibit the colonization of Candida albicans. Chloramphenicol has inhibitory effects on both Gram-positive and Gram-negative bacteria and has a stronger effect on the latter. It is often used for the selective separation of fungi and the growth and development of inhibitory bacteria. We use the calculated number of Candida albicans divided by the logarithmic function to calculate the result. Figure 4As shown in B, the average load of Candida albicans in the model group was 4.18 after taking the logarithm, while the Candida albicans was reduced to 3.41 after the intervention with external Lactobacillus delbrueckii DM8909 (p < 0.001), and the Candida albicans was reduced more significantly after the intervention with external Lactobacillus gasseri slurry and supernatant, which were 2.00 and 2.69, respectively (p < 0.0001).

[0134] (2) Expression of antimicrobial peptide CRAMP in mouse vaginal tissue

[0135] CRAMP (Cathelicidin-Related Antimicrobial Peptide) is an important antimicrobial peptide in mice. It is a key component of the mouse antimicrobial defense system, similar to human LL-37. CRAMP plays an important role in a variety of immune response processes and has broad-spectrum antibacterial, antiviral and immunomodulatory functions. CRAMP can promote the chemotaxis of macrophages and neutrophils and enhance their phagocytic ability. In addition, CRAMP can regulate the secretion of cytokines, balance the inflammatory response, and prevent tissue damage caused by excessive inflammation.

[0136] like Figure 5 As shown in the figure, the secretion of CRAMP in the model group was 19.98pg / mL, while that in the blank group was 31.15pg / mL. The secretion of antimicrobial peptide CRAMP after external application of Lactobacillus delbrueckii DM8909 bacterial lysate increased by 48.0% compared with the model group, and the secretion of antimicrobial peptide CRAMP after external application of Lactobacillus gasseri CCFM1430 bacterial lysate and fermentation supernatant increased by 65.2% and 44.4% respectively compared with the model group. There were significant differences between the three and the model group (p < 0.05). The level of antimicrobial peptide CRAMP secreted by Lactobacillus gasseri CCFM1430 bacterial lysate was better than that of Lactobacillus delbrueckii DM8909 bacterial lysate group.

[0137] (3) Production of polymerized immunoglobulin receptors and immunoglobulin secretion in mouse vaginal tissue

[0138] The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane glycoprotein and an important component of the mucosal immune system. It mediates the transcytosis of polymeric immunoglobulin (pIg) through epithelial cells to the mucosal surface to form secretory immunoglobulin A (sIgA). The transport of each immunoglobulin molecule requires the synthesis of one molecule of pIgR. The lack of pIgR will affect the secretion of sIgA. sIgA can protect cells from the adhesion and invasion of pathogens and discharge pathogens into mucosal secretions. Its effective secretion is crucial for mucosal immune defense.

[0139] like Figure 6As shown in (A), the content of polymerized immunoglobulin receptor pIgR in the blank group was 140.07pg / mL, and that in the model group was 125.47pg / mL. The content of pIgR produced by the host stimulated by external application of Lactobacillus delbrueckii DM8909 bacterial lysate was 43.9% higher than that in the model group, and the content of pIgR produced by the host stimulated by the bacterial lysate of Lactobacillus gasseri CCFM1430 was 50.3% higher than that in the model group. There was no significant difference between the fermentation supernatant of Lactobacillus gasseri CCFM1430 and the model group. Therefore, the level of pIgR produced by the host stimulated by external application of bacterial lysate of Lactobacillus gasseri CCFM1430 was better than that of the bacterial lysate of Lactobacillus delbrueckii DM8909. The bacterial lysate of Lactobacillus gasseri CCFM1430 has more potential to produce more sIgA and enhance host resistance.

[0140] like Figure 6 As shown in (B), the content of secretory immunoglobulin sIgA in the blank group was 0.76μg / mL, and that in the model group was 0.50μg / mL. The content of sIgA produced by the host stimulated by external application of Lactobacillus delbrueckii DM8909 bacterial lysate was 20.6% higher than that in the model group, the content of sIgA produced by the host stimulated by Lactobacillus gasseri CCFM1430 bacterial lysate was 80.0% higher than that in the model group, and the content of sIgA produced by the host stimulated by Lactobacillus gasseri CCFM1430 fermentation supernatant was 28.0% higher than that in the model group. After probiotic intervention, the content of sIgA produced by the host was significantly different from that in the model group (p < 0.05), and the level of sIgA produced by the host stimulated by external application of Lactobacillus gasseri CCFM1430 bacterial lysate and fermentation supernatant was better than that of external application of Lactobacillus delbrueckii DM8909 bacterial lysate.

[0141] In summary, Lactobacillus gasseri CCFM1430 can significantly increase the secretion of polymeric immunoglobulin receptor pIgR and secretory immunoglobulin sIgA in mouse vaginal tissue, further enhancing the host's own ability to resist pathogenic bacteria infection.

[0142] (4) TLR2 gene expression

[0143] Lactobacillus can inhibit the growth of pathogenic bacteria by producing a large amount of lactic acid to lower pH, compete for nutrients, and occupy adhesion sites. Existing studies have shown that inactivated lactobacillus suspension can regulate host immunity, which proves that certain components on the surface of lactobacillus are at work. It is speculated that components such as lipoteichoic acid, peptidoglycan, or extracellular polysaccharides secreted by lactobacillus on the surface of lactobacillus can be recognized by host cells, triggering the Toll-like receptor (TLRs) signaling pathway, thereby promoting the expression and secretion of antimicrobial peptides. Therefore, we measured the expression of TLR2 gene in vaginal tissue.

[0144] like Figure 7As shown, the expression of TLR2 in the model group was higher than that in the blank group, indicating that the body may be mobilizing defense pathways to resist pathogens. After topical application of Lactobacillus delbrueckii DM8909 bacterial lysate, the relative expression of TLR2 increased by 14.8% compared with the model group, and after topical application of Lactobacillus gasseri CCFM1430 bacterial lysate, the relative expression of TLR2 increased by 44.3% compared with the model group. There was no significant difference between topical application of Lactobacillus gasseri CCFM1430 fermentation supernatant and the model group. Therefore, topical application of Lactobacillus gasseri CCFM1430 can bind to Toll-like receptor 2 on vaginal epithelial cells, thereby promoting immune regulation and the secretion of defense factors.

[0145] (5) Activity of immune-regulatory tumor necrosis factor-α (TNF-α) and secretion of cytokine IL-6 in mouse spleen tissue

[0146] Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine secreted by a variety of cells (including macrophages, T cells, natural killer cells, etc.), which plays a central role in host defense, inflammatory response, apoptosis and immune regulation. IL-6 is an important inflammatory cytokine. IL-6 plays a key role in the development of acute phase reactions, chronic inflammation and a variety of diseases. IL-6 can promote the production of chemokines, activate and regulate different types of immune cells, including neutrophils, macrophages and lymphocytes, which play an antimicrobial role at the site of infection.

[0147] like Figure 8 As shown in (A), the content of tumor necrosis factor-α (TNF-α) secreted by the blank group was 238.99 ng / L, and the content of TNF-α secreted by the model group was 407.83 ng / L. The content of TNF-α produced by the host stimulated by external application of bacterial lysate of Lactobacillus delbrueckii DM8909 was 29.2% lower than that of the model group, the content of TNF-α produced by the host stimulated by bacterial lysate of Lactobacillus gasseri CCFM1430 was 38.9% lower than that of the model group, and the content of TNF-α produced by the host stimulated by fermentation supernatant of Lactobacillus gasseri CCFM1430 was 29.6% lower than that of the model group. After probiotic intervention, the content of TNF-α produced by the host was significantly different from that of the model group (p < 0.05), and the external application of bacterial lysate and fermentation supernatant of Lactobacillus gasseri CCFM1430 was better than that of Lactobacillus delbrueckii DM8909 in reducing the level of TNF-α.

[0148] like Figure 8As shown in (B), the interleukin 6 (IL-6) content in the blank group was 62.98 pg / mL, and that in the model group was 96.76 pg / mL. The content of IL-6 produced by the host stimulated by external application of Lactobacillus delbrueckii DM8909 bacterial lysate was 27.6% lower than that in the model group, the content of IL-6 produced by the host stimulated by Lactobacillus gasseri CCFM1430 bacterial lysate was 31.5% lower than that in the model group, and the content of IL-6 produced by the host stimulated by the fermentation supernatant of Lactobacillus gasseri CCFM1430 was 25.5% lower than that in the model group. There were significant differences in the content of IL-6 produced by the host after probiotic intervention and that in the model group (p < 0.05), and the pro-inflammatory level produced by external application of Lactobacillus gasseri CCFM1430 bacterial lysate was lower than the inflammatory level produced by Lactobacillus delbrueckii DM8909 bacterial lysate.

[0149] In summary, Lactobacillus gasseri CCFM1430 can significantly reduce the secretion of TNF-α and IL-6 in the spleen tissue of mice, inhibit the further development of inflammation, and resist pathogenic bacteria infection.

[0150] (6) Mouse vaginal histopathological analysis

[0151] HE staining of mouse vaginal tissue can effectively evaluate the integrity of the vaginal barrier and inflammation of each group of mice. Fig. 9 As shown in the figure, the vaginal structure of the blank group mice was intact, with intact vaginal epithelial cells on the surface and moderate thickness of the cornified layer. The vaginal tissue of the model group mice was damaged, the cornified layer on the mucosal surface disappeared, the squamous epithelial cells proliferated, and a large number of inflammatory cells infiltrated in the mucosa; the vaginal tissue inflammatory cell infiltration in the group treated with external Lactobacillus delbrueckii DM8909 bacterial lysate was less than that in the model group, but there was obvious squamous epithelial hyperplasia; after external intervention with Lactobacillus gasseri CCFM1430 bacterial lysate and fermentation supernatant, the epithelial layer was gradually repaired, the inflammatory infiltration was significantly reduced, and a certain amount of cornified layer appeared on the mucosal surface, which could effectively protect the integrity of the vaginal mucosa of the mice. This shows that the degree of damage to the vaginal tissue of the mice after intervention with Lactobacillus gasseri CCFM1430 was less than that of the tissue after intervention with Lactobacillus delbrueckii DM8909.

[0152] Example 4 Application of oral Lactobacillus gasseri CCFM1430 in improving mice's resistance to pathogenic bacteria infection

[0153] Experimental animals and strains:

[0154] 25 7-week-old SPF female BALB / c mice weighing 18-20 g were randomly divided into 5 cages, 5 mice in each cage. They were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2012-0001). The animal experiment scheme and grouping are shown in Table 4. The specific steps are as follows:

[0155] Table 4: Animal experimental plan and grouping

[0156]

[0157] The vaginal infection prevention experiment adopted the method of intervention first and then modeling. The blank group and the model group were gavaged with 200 μL of normal saline for 17 consecutive days, and the experimental group was gavaged with 200 μL of 5×10 9 CFU / mL suspension of live Lactobacillus delbrueckii DM8909, live Lactobacillus gasseri CCFM1430, and inactivated Lactobacillus gasseri CCFM1430. On days 12-15, the blank group was subcutaneously injected with 50 μL of saline, and the model group and experimental group were subcutaneously injected with 50 μL of estradiol to induce estrus. On days 15-17, the model group and experimental group used a pipette to draw 20 μL of 5×10 8 CFU / mL of Candida albicans suspension was slowly injected into the vagina of mice, and the mice were inverted for 1-2 minutes. The blank group was inoculated with saline in the same way. The experimental period was 17 days. At the end of the intervention (day 18), 50 μL of saline was sucked out of the mouse vagina with a gun tip each time to sample, and 300 μL of vaginal lavage fluid was finally collected for subsequent analysis of vaginal epithelial cell shedding in the mouse vagina. At the same time, on day 18, all experimental mice were killed and vaginal tissues were peeled off to detect the content of antimicrobial peptide CRAMP (human antimicrobial peptide LL-37 is homologous to mouse antimicrobial peptide CRAMP), inflammatory factor IL-6, and α tumor necrosis factor (TNF-α) in vaginal tissues, as well as subsequent experimental tissue pathology analysis.

[0158] Determination method: At the end of the experiment, mice were killed and vaginal tissue was dissected. A portion of the tissue was homogenized with pre-cooled RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd.) and a mixture of protease inhibitors. The sample was centrifuged at 2000r / min for 15min at 4°C, and the supernatant of the vaginal tissue was taken for the determination of antimicrobial peptide CRAMP and polymeric immunoglobulin receptor content according to the instructions of the kit; in addition, the spleen tissue of the mouse was homogenized, and the spleen supernatant was taken for the determination of IL-6 and TNF-α content (Nanjing Senbeijia Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in 4% paraformaldehyde solution for histopathological examination.

[0159] 10 μL of sample solution was taken from the vaginal lavage fluid of mice on day 18, transferred to a glass slide, gently smeared with the outer wall of the pipette tip, dried with an alcohol burner flame, and stained with Diff-Quik staining (a rapid staining method improved from Wright's staining). Five fields of view were captured from each sample (1 mouse) under a microscope, and epithelial cells were counted from each image to determine the average value.

[0160] Histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced ​​into 5 mm thick slices, and stained with hematoxylin and eosin (H&E). Vaginal tissue samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0161] The results are as follows:

[0162] (1) Vaginal epithelial cell shedding and fungal load in mouse vaginal lavage fluid

[0163] Extensive shedding of vaginal epithelial cells can lead to damage to vaginal tissue, such as Fig.10 As shown in A, the model group had severe vaginal epithelial cell shedding, with about 27.33 cells in each field of view. After oral administration of live Lactobacillus delbrueckii DM8909, live Lactobacillus gasseri CCFM1430, and inactivated Lactobacillus gasseri CCFM1430 suspension, the vaginal epithelial cell shedding was significantly improved. The live Lactobacillus delbrueckii DM8909 group was reduced by 29.0% compared with the model group, the live Lactobacillus gasseri CCFM1430 group was reduced by 37.1% compared with the model group, and the inactivated Lactobacillus gasseri CCFM1430 suspension group was reduced by 71.7% compared with the model group. Overall, the vaginal epithelial cell shedding of mice after intervention with the inactivated Lactobacillus gasseri CCFM1430 suspension group was greatly reduced, and the live Lactobacillus gasseri CCFM1430 group could also significantly reduce the vaginal epithelial cell shedding. The effect of the live bacteria group of Lactobacillus gasseri CCFM1430 was significantly better than that of the live bacteria group of Lactobacillus delbrueckii DM8909. Therefore, Lactobacillus gasseri CCFM1430 can alleviate the shedding of vaginal epithelial cells and effectively prevent the adhesion and invasion of pathogenic bacteria to tissues.

[0164] The detection of Candida albicans load helps us to gain a deeper understanding of the colonization, reproduction and pathogenicity of Candida albicans in the vaginal microenvironment, such as Fig.10 As shown in B, the average load of Candida albicans in the model group was 4.21 after taking the logarithm, while the Candida albicans was reduced to 3.66 after intervention with Lactobacillus delbrueckii DM8909 (p < 0.001), and the Candida albicans was reduced more significantly after intervention with oral live Lactobacillus gasseri and inactivated bacterial solution, which were 2.80 and 3.06, respectively (p < 0.0001).

[0165] (2) Expression of antimicrobial peptide CRAMP in mouse vaginal tissue

[0166] like Fig.11As shown in the figure, the secretion of CRAMP in the model group was 15.23pg / mL, while that in the blank group was 33.91pg / mL. After oral administration of live Lactobacillus delbrueckii DM8909, the secretion of antimicrobial peptide CRAMP increased by 48.2% compared with the model group. After oral administration of live Lactobacillus gasseri CCFM1430 and inactivated bacterial suspension, the secretion of antimicrobial peptide CRAMP increased by 79.1% and 99.4% respectively compared with the model group. There were significant differences between the three groups and the model group (p < 0.05), but the secretion levels of live Lactobacillus gasseri CCFM1430 group and inactivated bacterial suspension group were better than those of live Lactobacillus delbrueckii DM8909 group, indicating that after oral administration of Lactobacillus gasseri CCFM1430, the body's defense ability is enhanced.

[0167] (3) Production of polymerized immunoglobulin receptors and immunoglobulin secretion in mouse vaginal tissue

[0168] like Fig.12 (A), the content of polymerized immunoglobulin receptor pIgR in the blank group was 142.81pg / mL, and that in the model group was 119.47pg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 bacteria was not significant compared with the model group, but oral administration of live Lactobacillus gasseri CCFM1430 bacteria and inactivated Lactobacillus gasseri CCFM1430 suspension stimulated the host to produce pIgR significantly compared with the model group, and the pIgR produced by live Lactobacillus gasseri CCFM1430 bacteria and inactivated Lactobacillus gasseri CCFM1430 suspension was 14.9% and 40.7% higher than that in the model group. Therefore, Lactobacillus gasseri CCFM1430 can well upregulate pIgR, thereby possibly transporting more IgA in the body to increase the expression of sIgA and enhance the anti-infection effect in the mouse vagina.

[0169] like Fig.12 As shown in (B), the content of secretory immunoglobulin sIgA in the blank group was 0.77μg / mL, and that in the model group was 0.49μg / mL. There was no significant difference in the content of sIgA produced by the host stimulated by oral administration of live Lactobacillus delbrueckii DM8909 compared with the model group. The content of sIgA produced by the host stimulated by oral administration of live Lactobacillus gasseri CCFM1430 was 38.8% higher than that in the model group. The content of sIgA produced by the host stimulated by the inactivated bacterial suspension of Lactobacillus gasseri CCFM1430 was 118.4% higher than that in the model group. There were significant differences in the content of sIgA produced by the host after intervention with Lactobacillus gasseri CCFM1430 and that in the model group (p<0.05).

[0170] (4) Activity of immune-regulatory tumor necrosis factor-α (TNF-α) and secretion of cytokine IL-6 in mouse spleen tissue

[0171] like Fig.13As shown in (A), the content of TNF-α produced by the spleen tissue of the blank group mice was 269.85ng / L, and that of the model group was 433.91ng / L. After oral administration of live bacteria of Lactobacillus delbrueckii DM8909, the content of TNF-α produced by the host was 22.9% lower than that of the model group, and the content of TNF-α produced by the host stimulated by oral administration of live bacteria of Lactobacillus gasseri CCFM1430 was 36.7% lower than that of the model group, and the content of TNF-α produced by the host stimulated by the inactivated bacterial suspension of Lactobacillus gasseri CCFM1430 was 35.4% lower than that of the model group. After probiotic intervention, the content of TNF-α produced by the host was significantly different from that of the model group (p<0.05), and the oral administration of live bacteria and inactivated bacterial suspension of Lactobacillus gasseri CCFM1430 was better than that of Lactobacillus delbrueckii DM8909 in reducing the level of TNF-α.

[0172] like Fig.13 As shown in (B), the interleukin 6 (IL-6) content in the blank group was 52.78 pg / mL, and that in the model group was 99.06 pg / mL. The content of IL-6 produced by the host stimulated by oral administration of live Lactobacillus delbrueckii DM8909 was 21.6% lower than that in the model group, the content of IL-6 produced by the host stimulated by live Lactobacillus gasseri CCFM1430 was 27.6% lower than that in the model group, and the content of IL-6 produced by the host stimulated by inactivated bacterial suspension of Lactobacillus gasseri CCFM1430 was 30.1% lower than that in the model group. After probiotic intervention, the content of IL-6 produced by the host was significantly different from that in the model group (p < 0.05), and the pro-inflammatory level regulated by live and inactivated bacterial suspension of Lactobacillus gasseri CCFM1430 was better than that produced by Lactobacillus delbrueckii DM8909.

[0173] In summary, Lactobacillus gasseri CCFM1430 can significantly reduce the secretion of TNF-α and IL-6 in the spleen tissue of mice, inhibit the further development of inflammation, and resist pathogenic bacteria infection.

[0174] (5) Mouse vaginal histopathological analysis

[0175] HE staining of mouse vaginal tissue can effectively evaluate the integrity of the vaginal barrier and inflammation of each group of mice. Fig.14As shown in the figure, the vaginal structure of mice in the blank group was intact, with a certain amount of keratinized layer on the surface, while the vaginal mucosal surface of mice in the model group was severely damaged, with a large number of inflammatory cells gathered in the mucosa; the vaginal tissue inflammatory cell infiltration in the oral Lactobacillus delbrueckii DM8909 live bacteria group was less than that in the model group, but there was obvious squamous epithelial hyperplasia; after oral Lactobacillus gasseri CCFM1430 live bacteria group and inactivated bacteria suspension group intervention, the epithelial layer was gradually repaired, the inflammatory infiltration was significantly reduced, and a certain amount of keratinized layer appeared on the mucosal surface, which can effectively protect the integrity of the vaginal mucosa of mice. This indicates that the degree of damage to the vaginal tissue of mice after intervention with Lactobacillus gasseri CCFM1430 was less than that of the tissue after intervention with Lactobacillus delbrueckii DM8909.

[0176] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus gasseri CCFM1430, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65156.

2. A microbial preparation containing the Lactobacillus gasseri CCFM1430 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of Lactobacillus gasseri CCFM1430 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The postbiotic prepared by Lactobacillus gasseri CCFM1430 according to claim 1, characterized in that: The postbiotics include inactivated cells, fermentation supernatant or lysate of the Lactobacillus gasseri CCFM1430.

5. The method for preparing the postbiotics according to claim 4, characterized in that: Include any of the following: (1) Cultivating the Lactobacillus gasseri CCFM1430 described in claim 1 to a stable phase, collecting the bacterial liquid, homogenizing under high pressure, and inactivating it by heat treatment to obtain postbiotics; (2) culturing the Lactobacillus gasseri CCFM1430 described in claim 1 to a stable phase, collecting the bacterial solution, and centrifuging to obtain the supernatant to obtain postbiotics; (3) Cultivating the Lactobacillus gasseri CCFM1430 described in claim 1 to a stable phase, collecting the cells, and sequentially performing high-pressure homogenization and heat treatment to obtain postbiotics.

6. A product, characterized in that The product contains the Lactobacillus gasseri CCFM1430 described in claim 1, or the microbial preparation described in claim 2 or 3, or the postbiotic described in claim 4.

7. The product according to claim 6, characterized in that The products include food, medicine or sanitary products; the usage of the medicine includes oral or external use; the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary napkins, sanitary tampons, vaginal washes, women's antibacterial washes or women's antibacterial washes.

8. A method for producing an antimicrobial peptide, characterized in that: The postbiotics according to claim 4 are added to an environment containing vaginal epithelial cells for cultivation; the postbiotics are the lysate or fermentation supernatant of the Lactobacillus gasseri CCFM1430.

9. Use of Lactobacillus gasseri CCFM1430 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in preparing a product for treating and / or preventing vaginal pathogenic bacteria infection, or in preparing a product for treating and / or preventing vaginal inflammation, characterized in that: The product has at least one of the following functions: (1) Promote the secretion of antimicrobial peptide LL-37 in vaginal epithelial cells; (2) Reduce the shedding of vaginal mucosal epithelial cells; (3) increase the expression of the antimicrobial peptide CRAMP in vaginal epithelial cells; (4) Promote the secretion of polyimmunoglobulin receptor pIgR and secretory immunoglobulin sIgA in the mouse vagina.

10. The use according to claim 9, characterized in that: The product is a medicine or a sanitary product; preferably, the sanitary product includes sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, women's antibacterial washes or women's antibacterial washes.

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