Lactobacillus reuteri for promoting secretion of host beta defensin and immune globulin and relieving vaginal mucosa damage by oral administration and external application and metagen of lactobacillus reuteri

Abstract

The invention discloses lactobacillus reuteri for promoting host beta defensin and immune globulin secretion and relieving vaginal mucosa damage through oral administration and external application and a metagen thereof, and belongs to the technical field of microorganisms. The lactobacillus reuteri is separated from vaginal secretion of healthy women, has the effects of improving vaginal mucosa injury and inhibiting vagina inflammation, and is specifically reflected in that secretion of a vaginal epithelial cell host defense peptide HBD2 is promoted; the secretion of a vaginal epithelial cell host polymeric immunoglobulin receptor pIgR is promoted; the secretion of animal vaginal tissue host defense peptide mBD3 is improved; the production of secretory immunoglobulin sIgA is promoted; the generation of animal serum immune globulin IgG is promoted, so that the systemic immunity is improved; the pathological characterization of the vagina of the mouse is improved. Therefore, the lactobacillus reuteri has a huge application prospect in products for enhancing the anti-infection barrier of the host and improving the local immunity and systemic immunity of the host.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus reuteri for oral and external use, which can promote the secretion of host beta-defensins and immunoglobulins and alleviate vaginal mucosal damage, and its postbiotics, belonging to the technical field of microorganisms. Background Art

[0002] Candida albicans infection is a common vaginal infection, also known as candidal vaginitis. It is caused by the fungus Candida albicans and causes patients to experience symptoms such as itching, redness, swelling, and abnormal secretions. Clinically, the treatment of Candida albicans infection mainly includes the use of antifungal drugs. The main mechanism of antifungal treatment is to eliminate pathogens. Although it can relieve symptoms in the short term, it may lead to the development of drug resistance, high recurrence rate of the disease, and interference with the host microecological balance.

[0003] The host's own anti-infection effect in the vagina mainly depends on its unique immune system and microecological environment. The body resists the colonization and invasion of pathogens by producing antibacterial proteins such as immunoglobulins (such as sIgA, IgG) and defensins. Immunoglobulins, especially sIgA, can effectively reduce the colonization of Candida albicans in the vagina and reduce the risk of infection by enhancing the function of the vaginal mucosal immune barrier. IgG not only participates in acute immune responses, but also plays an important role in the formation of immune memory. When B cells are stimulated by pathogens, some B cells will be transformed into memory B cells. These memory B cells can rapidly proliferate and secrete IgG antibodies when they are exposed to the same pathogen again in the future. This "memory" enables the body to quickly produce a large number of IgG antibodies during the second infection, greatly improving the immune defense against the same pathogen. Therefore, IgG is an important marker of immune memory and can quickly activate the immune response during the second infection. Defensins are natural immune factors of the host with broad-spectrum antibacterial activity, which can directly inhibit the growth and colonization of Candida albicans. Under the premise of not destroying the vaginal microecology, enhancing host immunity and improving host anti-infection ability are of great significance for preventing vaginal infection and reducing the occurrence of diseases.

[0004] Existing patents mainly emphasize that vaginal commensal bacteria Lactobacillus reuteri can maintain vaginal pH and have good antibacterial effects (CN117683691A, CN116751705A), but fail to start from enhancing host immunity, and the therapeutic effect cannot always be converted into a preventive effect, because the treatment is mainly aimed at the infection that has occurred, while prevention requires strengthening the host's defense capabilities. "Staphylococcus epidermidis Contributes to Healthy Maturation of the Nasal Microbiome by Stimulating Antimicrobial Peptide Production" discloses that commensal Staphylococcus epidermidis can stimulate the host's nasal epithelial cells to highly express antimicrobial peptides and inhibit the colonization of pathogenic bacteria such as Staphylococcus aureus and Moraxella catarrhalis; "Homeostatic Control of Sebaceous Glands by Innate Lymphoid Cells Regulates Commensal Bacteria Equilibrium" reveals that commensal bacteria are necessary for skin immune adaptability, and they play a role by enhancing IL-1 signal transduction. Symbiotic bacteria are very important for maintaining human health, but there are very few studies on the role of symbiotic bacteria in regulating the body's immunity and enhancing the host's anti-infection effect in the vagina. Therefore, it is particularly important to link symbiotic bacteria with activating the host's vaginal anti-infection, which is particularly important for preventing vaginal infections, enhancing the host's mucosal immunity when invaded by pathogenic bacteria, and reducing mucosal damage. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Lactobacillus reuteri and its bacterial suspension that promotes the secretion of host defense peptides and immunoglobulins to enhance anti-infection ability, aiming to solve the technical problem that the prior art lacks research on improving host defense ability and reducing the incidence of vaginal infection symptoms, and can regulate the host's immune ability.

[0006] The first technical solution provided by the present invention is the use of a strain of Lactobacillus reuteri CCFM1432 and a bacterial suspension thereof in the preparation of a product for enhancing the host's ability to defend against pathogenic bacteria infection and reducing damage to the vaginal mucosa.

[0007] The present invention provides a strain of Limosilactobacillus reuteri CCFM1432, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65158.

[0008] The Lactobacillus reuteri CCFM1432 was isolated from the vaginal secretions of healthy women. The strain was sequenced and analyzed, and the obtained sequence was compared with the nucleic acid sequence in NCBI. The result showed that the strain was Lactobacillus reuteri of the genus Lactobacillus, and was named Lactobacillus reuteri CCFM1432.

[0009] The colonies of Lactobacillus reuteri CCFM1432 on the MRS solid culture medium are round and convex, white, and relatively moist.

[0010] The second technical solution provided by the present invention is a bacterial suspension-related microbial preparation prepared using the Lactobacillus reuteri CCFM1432.

[0011] In one embodiment, the content of Lactobacillus reuteri CCFM1432 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0012] The second technical solution provided by the present invention is the postbiotics prepared by the Lactobacillus reuteri CCFM1432.

[0013] In one embodiment, the postbiotics include killed or inactivated cells, fermentation supernatant or bacterial lysate of the Lactobacillus reuteri CCFM1432.

[0014] In one embodiment, the inactivated or inactivated cells are prepared as follows: the Lactobacillus reuteri CCFM1432 is cultured in a culture medium for a period of time, bacterial cells in the cell culture medium are collected, and inactivated bacterial cells are obtained after heat treatment or freeze-drying.

[0015] In one embodiment, the heat treatment conditions are: 60°C to 70°C, 25 to 35 min.

[0016] In one embodiment, the method for preparing the bacterial lysate is: culturing the Lactobacillus reuteri CCFM1432 in a culture medium for a period of time, collecting bacterial cells, high-pressure homogenizing, and obtaining the bacterial lysate from the supernatant of the centrifugation.

[0017] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing Lactobacillus reuteri CCFM1432 in a culture medium for a period of time and then centrifuging the culture medium.

[0018] The third technical solution provided by the present invention is a product containing Lactobacillus ijenii CCFM1432 described in the first technical solution or the microbial preparation or the postbiotic described in the second technical solution.

[0019] In certain embodiments, the product is a food, a medicine, a health product or a hygiene product.

[0020] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0021] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0022] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0023] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0024] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0025] The fourth technical solution provided by the present invention is the use of the Lactobacillus reuteri CCFM1432 or the microbial preparation in the preparation of a product for improving vaginal immunity.

[0026] The fifth technical solution provided by the present invention is the use of the Lactobacillus reuteri CCFM1432 or the microbial preparation in the preparation of a product for preventing vaginal pathogenic bacteria infection, wherein the pathogenic bacteria include but are not limited to Candida albicans.

[0027] Beneficial effects:

[0028] The present invention provides a strain of Lactobacillus reuteri CCFM1432, which is separated from the vaginal secretions of healthy women. The strain has the functions of strengthening the anti-infection barrier of the vagina, reducing mucosal damage, and improving the host's defense system by oral and external use, which is specifically embodied in: (1) increasing the secretion of vaginal epithelial cell host defense peptide (HBD2) and polymeric immunoglobulin receptor (pIgR); (2) increasing the expression of host defense peptide mBD3 in the vagina of the body; (3) promoting the production of polymeric immunoglobulin receptor (pIgR) and secretory immunoglobulin sIgA; (4) promoting the production of immunoglobulin IgG; (5) improving the vaginal pathological manifestations of the body. Therefore, the Lactobacillus reuteri has a huge application prospect in products that enhance the vaginal anti-infection barrier and reduce vaginal mucosal damage.

[0029] Biomaterial Deposit

[0030] The Limosilactobacillus reuteri CCFM1432 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65158, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Streaking results of Lactobacillus reuteri CCFM1432.

[0032] Figure 2 :The effect of Lactobacillus reuteri on the secretion of β-defensins in vaginal epithelial cells.

[0033] Figure 3 :The effect of Lactobacillus reuteri on the secretion of polymeric immunoglobulin receptor.

[0034] Figure 4 : Flowchart of animal experiments.

[0035] Figure 5 :The effect of topical application of Lactobacillus reuteri on the expression of defense peptide β-defensin in vaginal tissue of mice.

[0036] Figure 6 :The effect of topical Lactobacillus reuteri on secretory immunoglobulins pIgR and sIgA in vaginal tissue.

[0037] Figure 7 :The effect of topical Lactobacillus reuteri on the secretion of immunoglobulin IgG in animal serum.

[0038] Figure 8: Effects of topical Lactobacillus reuteri on vaginal pathological manifestations in mice.

[0039] Fig. 9 :The effect of oral administration of Lactobacillus reuteri on the expression of defense peptide β-defensin in vaginal tissue of mice.

[0040] Fig.10 :The effect of oral administration of Lactobacillus reuteri on secretory immunoglobulins pIgR and sIgA in vaginal tissue.

[0041] Fig.11 :The effect of oral administration of Lactobacillus reuteri on the secretion of immunoglobulin IgG in animal serum.

[0042] Fig.12 : Effects of oral administration of Lactobacillus reuteri on vaginal pathological manifestations in mice.

[0043] (Different lowercase letters in the figure indicate significant differences among the groups, p < 0.05). DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with specific embodiments.

[0045] The human vaginal epithelial cells (VK2 / E6E7) involved in the following examples were donated by the Department of Obstetrics and Gynecology, Wuxi People's Hospital, Jiangsu Province.

[0046] The female BALB / c mice involved in the following examples were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0047] The Lactobacillus reuteri CCFM1432 and Lactobacillus reuteri VJSWX1L3 involved in the following embodiments are from strains screened by the Food Biotechnology Center of Jiangnan University; Lactobacillus delbrueckii DM8909 is isolated from Dingjunsheng vaginal lactobacillus capsules and stored in the strain bank of the Food Biotechnology Center of Jiangnan University; Candida albicans SC5314 is purchased from the strain collection center GDMCC of Guangdong Institute of Microbiology.

[0048] The culture medium involved in the following examples is as follows:

[0049] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× mixed solution of penicillin and streptomycin (the content of penicillin in the mixed solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL).

[0050] LBS solid culture medium ( / L): 5g tryptic peptone, 5g yeast extract powder, 6g potassium dihydrogen phosphate, 0.034g potassium dihydrogen phosphate, 0.575g magnesium sulfate, 20g glucose, 25g sodium acetate, 2g ammonium citrate, 0.12g manganese sulfate, 15g agar, 1mL Tween-80, 1.3mL glacial acetic acid, pH 5.3-5.7.

[0051] 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.

[0052] 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.

[0053] SDA medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 40g, agar 15g, pH 5.6±0.2.

[0054] SDB medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 20g, pH 5.6±0.2.

[0055] The preparation methods of the reagents involved in the following embodiments are as follows:

[0056] Estradiol: 0.1 mg of β-estradiol is dissolved in 0.05 mL of sesame oil and is ready for use.

[0057] Example 1: Cell recovery and culture

[0058] First, take out the frozen human vaginal epithelial cells (VK2 / E6E7), quickly thaw them in a 37°C water bath, then centrifuge at 1000r / min for 5min, discard the supernatant, add an appropriate volume of cell culture medium to resuspend the cells, place them in a culture dish, and culture them in a 37°C incubator containing 5% CO2. When the cells regain vitality and grow for 1-2 days to reach 70%-80% fusion, cell passage is performed.

[0059] Example 2: Isolation of Lactobacillus reuteri CCFM1432 and intervention sample preparation

[0060] 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 -1Dilution, 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 LBS solid culture medium, mix gently, and after the culture medium solidifies, invert and culture at 37°C for 48 h.

[0061] Select colonies of different morphologies and streak them on MRS plates for purification. Pick the purified single colony and inoculate it into 5mL liquid culture medium and culture it at 37℃ for 48h. Take 1.5mL of 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 forward primer (10μM), 0.5μL reverse primer (10μM), 10μL 2×Taq Mixture, 0.5μL bacterial suspension, and 8.5μL double distilled water. Primer information is shown in Table 1.

[0062] Table 1: Primer information

[0063]

[0064] PCR conditions: 95℃5min; 95℃10s; 55℃30s; 72℃30s; step2-4 30×; 72℃5min; 12℃2min. The PCR product was sent to a professional sequencing company, and the sequencing results were searched and compared with similarity in GeneBank using BLAST, and it was identified as Limosilactobacillus reuteri, named Limosilactobacillus reuteri CCFM1432.

[0065] 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.

[0066] 16S sequence information (SEQ ID No.1):

[0067] TCACCTGTCCGCTTAGGCGGCTCCCTCCATAAAGGTTAGGCCACCGACTTTGGGCGTTAC

[0068] AAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCA

[0069] TGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCC

[0070] GAACTGAGAACGGCTTTAAGAGATTAGCTTACTCTCGCGAGCTTGCGACTCGTTGTACC

[0071] GTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCC

[0072] CACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAA

[0073] CTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTG

[0074] ACGACGACCATGCACCACCTGTCATTGCGTCCCCGAAGGGAACGCCTTATCTCTAAGGT

[0075] TAGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATG

[0076] CTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTC

[0077] CCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACA

[0078] CCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATG

[0079] CTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTC

[0080] CATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGT

[0081] CGCCCGGTTTCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGC

[0082] AACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTA

[0083] CCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGGTTGGATACCGTCACTGCGTG

[0084] AACAGTACTCTCACGCACGTTCTTCTCCACACAGAGCTTTACGAGCGAAACCCTTCTTC

[0085] ACTCACGCGTGTGCTCATCAGCTGCGCCCATTGTGAGATCCCTACTGCTGCCTCCCGTAG

[0086] GAGTATGACGTGTCTCAGTCATTGTGCGATCAGTCCTCTCACTCGCTATGCATCATCGCT

[0087] GCTAGCGTTACTACACATGCCTATGACCGAGTCATCCGAGTGATGCTAAGCCACTCTTCCACACCAAGCCATTT。

[0088] Preparation of the intervention sample:

[0089] Live Lactobacillus reuteri CCFM1432 (CCFM1432-L): Lactobacillus reuteri CCFM1432 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 a 4% inoculum, cultured in an incubator at 37°C for 20 h, centrifuged to obtain bacterial sludge, and the bacterial solution concentration was adjusted to 5×10 9 CFU / mL. Lactobacillus delbrueckii DM8909 was prepared as above and was recorded as DM8909-L.

[0090] Lactobacillus reuteri CCFM1432 inactivated bacterial suspension (CCFM1432-D): 5×10 9 The bacterial suspension of Lactobacillus reuteri CCFM1432 with a CFU / mL was homogenized (800-1200 MPa) 10 times in a high-pressure homogenizer and pasteurized (65°C for 30 min) to obtain an inactivated bacterial suspension, which was resuspended to the original volume with sterile saline before the experiment.

[0091] Lactobacillus reuteri CCFM1432 bacterial lysate (CCFM1432-sub): 5×10 9 After centrifugation of Lactobacillus reuteri CCFM1432 with CFU / mL, the cells were taken out and resuspended with an equal volume of sterile water. After homogenization in a high-pressure homogenizer (800-1200MPa) 10 times and pasteurization (65℃30min), the cell lysate was obtained, freeze-dried and stored in a -80℃ refrigerator, and resuspended to the original volume with sterile saline before the experiment. The preparation method of Lactobacillus delbrueckii DM8909 cell lysate and Lactobacillus reuteri VJSWX1L3 cell lysate was the same as above, and they were recorded as DM8909-sub and VJSWX1L3-sub, respectively.

[0092] Lactobacillus reuteri CCFM1432 fermentation supernatant (CCFM1432-sup): 5×10 9 CFU / mL of Lactobacillus reuteri CCFM1432 were centrifuged and the fermentation supernatant was taken and resuspended with sterile saline before the experiment to adjust the bacterial solution concentration to 5×10 9 CFU / mL was resuspended to the original volume with sterile saline. The fermentation supernatant of Lactobacillus reuteri VJSWX1L3 was prepared in the same way and was recorded as VJSWX1L3-sup.

[0093] Example 3: Cultivation of Candida albicans

[0094] Candida albicans suspension: Candida albicans 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 and shaken for expansion. The final concentration of the bacterial suspension was adjusted to 5×10 8 CFU / mL.

[0095] Example 4: Effects of postbiotic components of Lactobacillus reuteri CCFM1432 on the secretion of host defense peptide (HBD2) by vaginal epithelial cells

[0096] The specific steps are as follows:

[0097] 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:

[0098] (1) The experiment of bacterial lysate intervention on cells was divided into three groups: blank group, bacterial lysate prepared from Lactobacillus reuteri CCFM1432 group (CCFM1432-sub) and bacterial lysate prepared from Lactobacillus reuteri VJSWX1L3 group (VJSWX1L3-sub). Cell culture medium containing 5% (v / v) saline, lysate of Lactobacillus reuteri CCFM1432 (CCFM1432-sub) and lysate of Lactobacillus reuteri VJSWX1L3 (VJSWX1L3-sub) was added to the corresponding groups, respectively.

[0099] (2) The experiment of fermentation supernatant intervention on cells was divided into three groups: blank group, Lactobacillus reuteri CCFM1432 fermentation supernatant (CCFM1432-sup), Lactobacillus reuteri VJSWX1L3 fermentation supernatant (VJSWX1L3-sup) were added to the cell culture medium containing 5% (v / v) MRS liquid culture medium, Lactobacillus reuteri CCFM1432 fermentation supernatant, and Lactobacillus reuteri VJSWX1L3 fermentation supernatant, respectively.

[0100] After adding the sample, the cells were incubated under 5% CO2 and 37°C. After incubation for 24 hours, the cell culture supernatant was aspirated and the host defense peptide HBD2 was determined using an ELISA kit.

[0101] The results are as follows Figure 2As shown, the secretion of host defense peptide HBD2 by vaginal epithelial cells intervened by bacterial lysate of Lactobacillus reuteri CCFM1432 and Lactobacillus reuteri VJSWX1L3 were 105.51 pg / mL and 87.13 pg / mL, respectively, compared with the control group (74.10 pg / mL), increased by 142.3% and 117.5%, respectively; the secretion of host defense peptide HBD2 by vaginal epithelial cells intervened by fermentation supernatant of Lactobacillus reuteri CCFM1432 and Lactobacillus reuteri VJSWX1L3 were 97.26 pg / mL and 85.54 pg / mL, respectively, compared with the control group (82.52 pg / mL), increased by 117.8% and 103.6%, respectively. Therefore, Lactobacillus reuteri CCFM1432 can significantly increase the content of host defense peptide HBD2.

[0102] Example 5 Effects of postbiotic components of Lactobacillus reuteri CCFM1432 on secretion of polymeric immunoglobulin receptor (pIgR) by vaginal epithelial cells

[0103] The specific steps are as follows:

[0104] 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:

[0105] (1) The experiment of bacterial lysate intervention on cells was divided into three groups: blank group, bacterial lysate prepared from Lactobacillus reuteri CCFM1432 group (CCFM1432-sub) and bacterial lysate prepared from Lactobacillus reuteri VJSWX1L3 group (VJSWX1L3-sub). Cell culture medium containing 5% (v / v) saline, lysate of Lactobacillus reuteri CCFM1432 (CCFM1432-sub) and lysate of Lactobacillus reuteri VJSWX1L3 (VJSWX1L3-sub) was added to the corresponding groups, respectively.

[0106] (2) The experiment of fermentation supernatant intervention on cells was divided into three groups: blank group, Lactobacillus reuteri CCFM1432 fermentation supernatant (CCFM1432-sup), Lactobacillus reuteri VJSWX1L3 fermentation supernatant (VJSWX1L3-sup) were added to the cell culture medium containing 5% (v / v) MRS liquid culture medium, Lactobacillus reuteri CCFM1432 fermentation supernatant, and Lactobacillus reuteri VJSWX1L3 fermentation supernatant, respectively.

[0107] After adding the sample, the cells were incubated under 5% CO2 and 37°C. After incubation for 24 hours, the cell culture supernatant was aspirated and used for ELISA to determine the content of polymeric immunoglobulin receptor (pIgR) secreted by vaginal epithelial cells.

[0108] Experimental results:

[0109] As an important membrane protein, pIgR is responsible for transporting polymeric immunoglobulins (such as IgA) from plasma cells to the mucosal surface. Through this transport, pIgR helps guide immunoglobulins into secretions, thereby enhancing the immune defense of the mucosa. pIgR is of great significance for understanding mucosal immune mechanisms, developing new immunotherapies, and improving public health levels.

[0110] like Figure 3 As shown, the levels of pIgR secreted by vaginal epithelial cells by Lactobacillus reuteri CCFM1432 bacterial lysate and Lactobacillus reuteri VJSWX1L3 bacterial lysate were 109.02 pg / mL and 82.01 pg / mL, respectively, compared with the blank control group (85.26 pg / mL), which were increased by 127.9% and 96.2%, respectively; the levels of pIgR secreted by vaginal epithelial cells by Lactobacillus reuteri CCFM1432 fermentation supernatant and Lactobacillus reuteri VJSWX1L3 fermentation supernatant were 107.46 pg / mL and 76.95 pg / mL, respectively, compared with the control group (58.17 pg / mL), which were increased by 184.7% and 132.3%, respectively. In summary, Lactobacillus reuteri CCFM1432 can significantly increase the content of pIgR.

[0111] Example 6 Application of topical Lactobacillus reuteri CCFM1432 postbiotic components in enhancing the defense of mice against pathogenic bacterial infection

[0112] Experimental animals and strains:

[0113] Twenty-five 7-week-old SPF female BALB / c mice weighing 18-20 g were randomly divided into 8 cages, with 5 mice in each cage. They 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:

[0114] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology Culture Collection Center (GDMCC). Figure 4 Flow chart of animal experiments.

[0115] Table 2: Animal experimental plan and grouping

[0116]

[0117] The mice were randomly divided into 5 groups according to their body weight, and all the mice in the groups were kept normally during the whole experiment. The experiment adopted a preventive model, that is, the probiotics were used for intervention first, and then the model was established. The mice in the topical group were inoculated vaginally with 20 μL of 5×10 9 The intervention of Lactobacillus delbrueckii DM8909 bacterial lysate prepared with CFU / mL bacterial suspension, Lactobacillus reuteri CCFM1432 bacterial lysate, and Lactobacillus reuteri CCFM1432 fermentation supernatant, the specific operation steps are to use a pipette to draw 20μL of the corresponding sample, slowly inject it into the mouse vagina, and stand the mouse upside down for 1-2 minutes. The blank group and the model group are replaced with 20μL of saline to intervene in the vagina. On the 12th-15th day, the blank group was subcutaneously injected with 50μL of saline, and the model group and the experimental group were subcutaneously injected with 50μL of estradiol to induce estrus. On the 15th-17th day, the model group and the experimental group used a pipette to draw 20μL of a concentration 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 vaginally inoculated with normal saline.

[0118] The experimental period was 17 days. On the 18th day, all experimental mice were killed and their vaginal tissues were peeled off for the detection of mouse β-defensin 3 (homologous to human β-defensin 2), polymeric immunoglobulin receptor, and immunoglobulin content in the vaginal tissues, as well as subsequent experimental tissue pathology analysis.

[0119] Determination method: At the end of the experiment, blood was collected from the eyeball and centrifuged to obtain serum for IgG content determination. 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 12000r / min for 15min at 4°C, and the vaginal tissue supernatant was taken for mBD3, pIgR, and sIgA content determination according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in a 4% paraformaldehyde solution for histopathological examination.

[0120] 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.

[0121] Experimental results:

[0122] (1) Host defense peptide (mBD3) in mouse vagina

[0123] Mucosal immunity is one of the most important immune defense mechanisms of the body, mainly occurring on the mucosal surface of the body, and female vaginal mucosal immunity is one of them. Host defense peptides (HDPs) are a class of polypeptides with less than 50 amino acids that are positively charged and hydrophobic and can interact with microbial cell membranes. HBD2 is the most important host defense peptide in the human body, which has homology with mBD3 in mice. HDPs play an important role in mucosal immunity and can enhance immune responses by directly killing pathogens or regulating the activity of immune cells.

[0124] like Figure 5 As shown, the model group significantly reduced the level of defensins, with mBD3 secretion of 32.44ng / L and 50.65ng / L in the blank group. External application of Lactobacillus delbrueckii DM8909 bacterial lysate can increase mBD3 secretion, with secretion amounts of 48.70ng / L (48.70% higher than the model group), and external application of Lactobacillus reuteri CCFM1432 bacterial lysate and fermentation supernatant produced mBD3 of 55.29ng / L and 52.03ng / L (70.41% and 60.37% higher than the model group, respectively). Therefore, external application of Lactobacillus reuteri CCFM1432 can significantly increase the content of host defense peptide mBD3 in mice.

[0125] (2) Secretion of polymeric immunoglobulin receptor (pIgR) and secretory immunoglobulin (sIgA) in mouse vaginal tissue

[0126] Polymeric immunoglobulin receptor (pIgR) is a protein expressed on the surface of mucosal epithelial cells. It transports polymeric immunoglobulin A (pIgA) in the blood to the mucosal surface through the transcellular transport process to form secretory immunoglobulin A (sIgA). Detecting sIgA in the vagina is beneficial to enhancing local vaginal immunity, maintaining the immune protection layer on the mucosal surface, and preventing the invasion of pathogens.

[0127] like Figure 6 As shown in A, compared with the blank group (146.61pg / mL), the model group significantly reduced the level of polymeric immunoglobulin receptor (pIgR), and the pIgR secretion was 116.61pg / mL. External application of Lactobacillus delbrueckii DM8909 bacterial lysate can increase pIgR secretion, and its secretion is 179.25pg / mL (53.70% higher than the model group), and external application of Lactobacillus reuteri CCFM1432 bacterial lysate and fermentation supernatant produced pIgR of 241.85pg / mL and 212.99pg / mL (107.40% and 82.65% higher than the model group, respectively).

[0128] like Figure 6 As shown in B, compared with the blank group (0.83 μg / mL), the model group significantly reduced secretory immunoglobulin (sIgA), and the sIgA secretion was 0.54 μg / mL. External application of Lactobacillus delbrueckii DM8909 bacterial lysate can increase sIgA secretion, and its secretion amount was 0.62 μg / mL (15.46% higher than the model group), and external application of Lactobacillus reuteri CCFM1432 bacterial lysate and fermentation supernatant produced sIgA of 0.85 μg / mL and 0.73 μg / mL (57.79% and 35.22% higher than the model group, respectively). In summary, external application of Lactobacillus reuteri CCFM1432 can increase the protein expression of secretory immunoglobulin A by increasing the secretion of polymerized immunoglobulins, thereby improving the body's anti-infection ability.

[0129] (3) Secretion of immunoglobulin G (IgG) in mouse serum

[0130] IgG is the body's main antibody against bacterial and viral infections, and plays an important role by neutralizing toxins and marking pathogens for clearance by immune cells. IgG can recognize and bind to specific antigens of pathogens, preventing them from further infecting the body, and activate the complement system to promote phagocytosis by macrophages and neutrophils, thereby clearing pathogens. After the first contact with an antigen, memory B cells are produced in the body. These cells can quickly produce IgG when they are exposed to the same antigen again. This memory response helps to improve immune protection against specific pathogens.

[0131] like Figure 7 As shown in the figure, compared with the blank group (7.22μg / mL), the model group significantly reduced immunoglobulin (IgG), and the IgG secretion was 6.23μg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 and topical application of Lactobacillus delbrueckii DM8909 bacterial lysate can increase IgG secretion, with secretions of 6.53μg / mL and 6.91μg / mL, respectively (104.8% and 110.9% higher than the model group, respectively). Oral administration of live Lactobacillus reuteri CCFM1432 bacteria and bacterial suspension produced IgG of 7.42μg / mL and 7.72μg / mL (119.1% and 123.9% higher than the model group, respectively); topical application of Lactobacillus reuteri CCFM1432 bacterial lysate and fermentation supernatant produced IgG of 8.42μg / mL and 7.23μg / mL (135.2% and 116.1% higher than the model group, respectively). In summary, Lactobacillus reuteri CCFM1432 can significantly increase the content of IgG in serum and enhance systemic immunity to resist pathogenic bacteria infection.

[0132] (4) Mouse vaginal histopathological analysis

[0133] Vaginal tissue pathology studies can be used to evaluate the efficacy of different treatments for vaginal infection. For mice in the blank control group, the vaginal mucosal structure remained intact and the surface keratinized layer was normal. Mice in the model group showed the disappearance of the vaginal mucosal keratinized layer, squamous hyperplasia of epithelial cells, and infiltration of a large number of inflammatory cells in the mucosa. The surface of the vaginal tissue intervened by Lactobacillus delbrueckii DM8909 bacterial lysate was intact, but there were a small number of inflammatory infiltrating cells; after intervention by the bacterial lysate group of Lactobacillus reuteri CCFM1432 and fermentation supernatant, the surface cells of the vaginal tissue were relatively smooth, and the infiltration of inflammatory cells was significantly reduced compared with the model group. This shows that after the prevention of Lactobacillus reuteri, the damage to the surface of the vaginal tissue was greatly reduced, and it has a good effect in preventing the invasion of pathogens.

[0134] Example 7 Application of oral Lactobacillus mucilaginosus CCFM1432 postbiotic components in enhancing the defense of mice against pathogenic bacterial infection

[0135] Experimental animals and strains:

[0136] Twenty-five 7-week-old SPF female BALB / c mice weighing 18-20 g were randomly divided into 8 cages, with 5 mice in each cage. They 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:

[0137] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology, GDMCC. Figure 4 .

[0138] Table 3: Animal experimental plan and grouping

[0139]

[0140] The mice were randomly divided into 5 groups according to their body weight, and all the mice were kept normally during the whole experiment. The experiment adopted a preventive model, that is, probiotics were used for intervention first, and then the model was established. The oral group was gavaged with 200 μL of 5×10 9 CFU / mL suspension of live Lactobacillus delbrueckii DM8909, live Lactobacillus reuteri CCFM1432, and inactivated Lactobacillus reuteri CCFM1432. 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 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 vaginally inoculated with normal saline in the same way.

[0141] The experimental period was 17 days. On the 18th day, all experimental mice were killed and their vaginal tissues were peeled off for the detection of mouse β-defensin 3, polymeric immunoglobulin receptor, and immunoglobulin content in the vaginal tissues and subsequent experimental tissue pathology analysis.

[0142] Determination method: At the end of the experiment, blood was collected from the eyeball and centrifuged to obtain serum for IgG content determination. 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 12000r / min for 15min at 4°C, and the vaginal tissue supernatant was taken for mBD3, pIgR, and sIgA content determination according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in a 4% paraformaldehyde solution for histopathological examination.

[0143] 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.

[0144] Experimental results:

[0145] (1) Host defense peptide (mBD3) in mouse vagina

[0146] like Fig. 9 As shown, the model group significantly reduced the level of defensins, and the secretion of mBD3 dropped from 49.67ng / L to 33.33ng / L. Oral administration of live Lactobacillus delbrueckii DM8909 secreted mBD3 of 49.88ng / L (an increase of 49.68% compared with the model group), and oral administration of live and inactivated Lactobacillus reuteri CCFM1432 suspension produced mBD3 of 58.42ng / L and 55.99ng / L (an increase of 75.31% and 68.02% respectively compared with the model group). Therefore, Lactobacillus reuteri CCFM1432 can significantly increase the content of host defense peptide mBD3 in mice.

[0147] (2) Secretion of polymeric immunoglobulin receptor (pIgR) and secretory immunoglobulin (sIgA) in mouse vaginal tissue Fig.10As shown in A, compared with the blank group (136.02pg / mL), the model group significantly reduced the level of polymerized immunoglobulin receptor (pIgR), and the pIgR secretion was 120.92pg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 bacteria can increase pIgR secretion, and its secretion is 130.53pg / mL (7.95% higher than the model group), and oral administration of live Lactobacillus reuteri CCFM1432 bacteria and inactivated bacterial suspensions produced pIgR of 146.41pg / mL and 176.80pg / mL (21.08% and 46.21% higher than the model group, respectively).

[0148] like Fig.10 As shown in B, compared with the blank group (0.70 μg / mL), the model group significantly reduced secretory immunoglobulin (sIgA), and the sIgA secretion was 0.45 μg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 bacteria can increase sIgA secretion, and its secretion amount was 0.62 μg / mL (an increase of 38.22% compared with the model group), and oral administration of live Lactobacillus reuteri CCFM1432 bacteria and inactivated bacterial suspension produced sIgA of 1.22 μg / mL and 0.86 μg / mL (an increase of 171.15% and 91.16% respectively compared with the model group). In summary, oral administration of Lactobacillus reuteri CCFM1432 can increase the protein expression of secretory immunoglobulin A by increasing the secretion of polymerized immunoglobulins, thereby improving the body's anti-infection ability.

[0149] (3) Secretion of immunoglobulin G (IgG) in mouse serum

[0150] like Fig.11 As shown, compared with the blank group (7.17 μg / mL), the model group significantly reduced immunoglobulin (IgG), and the IgG secretion was 6.21 μg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 bacteria can increase IgG secretion, and its secretion amount increased to 6.53 μg / mL (an increase of 5.08% compared with the model group). Oral administration of live and inactivated Lactobacillus reuteri CCFM1432 bacteria suspension produced IgG of 7.42 μg / mL and 7.72 μg / mL (an increase of 19.41% and 24.25% respectively compared with the model group). In summary, oral administration of Lactobacillus reuteri CCFM1432 can significantly increase the content of IgG in serum and enhance systemic immunity to resist pathogenic bacterial infection. (4) Vaginal tissue pathological analysis of mice

[0151] Vaginal tissue pathology studies can be used to evaluate the efficacy of different treatments for vaginal infections. For mice in the blank control group, the vaginal mucosal structure remained intact, the vaginal tissue had a clear squamous epithelial cell layer and basal layer, and the immune cells were evenly distributed in the tissue. In the model group, the vaginal mucosal epithelial cell layer disappeared, and there was a large number of inflammatory cell chemotaxis on the mucosal surface. After intervention with live Lactobacillus delbrueckii DM8909, the number of inflammatory infiltrating cells in the vaginal tissue decreased, but the surface of the tissue was slightly damaged; after intervention with live Lactobacillus reuteri CCFM1432 and inactivated bacterial suspension, the surface of the tissue was intact, the infiltration of inflammatory cells was reduced, and the keratinized layer on the mucosal surface was restored, and the surface was relatively smooth.

[0152] 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 Limosilactobacillus reuteri CCFM1432, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65158.

2. A microbial preparation, characterized in that: The microbial preparation contains the Lactobacillus reuteri CCFM1432 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of Lactobacillus reuteri CCFM1432 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 reuteri CCFM1432 according to claim 1.

5. The postbiotic of Lactobacillus mucilaginosus CCFM1432 according to claim 4, characterized in that: The postbiotics include inactivated cells, fermentation supernatant or bacterial lysate of the Lactobacillus reuteri CCFM1432.

6. A product containing Lactobacillus reuteri CCFM1432 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 or 5, characterized in that: The product is a medicine or a sanitary product.

7. The product according to claim 6, characterized in that The drug can be used orally or externally.

8. The product according to claim 7, characterized in that The sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary napkins, sanitary tampons, vaginal washes, and women's antibacterial washes or antibacterial washes.

9. Use of Lactobacillus reuteri CCFM1432 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a product for improving vaginal immunity.

10. Use of Lactobacillus reuteri CCFM1432 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a product for preventing vaginal pathogenic bacteria infection, characterized in that: The pathogenic bacteria include but are not limited to Candida albicans.

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