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] This invention relates to a strain of *Lactobacillus reuteri* and its postbiotic that can be taken orally and applied topically to promote the secretion of host β-defensins and immunoglobulins and reduce vaginal mucosal damage, belonging to the field of microbial technology. Background Technology

[0002] Candida albicans infection is a common vaginal infection, also known as candidal vaginitis, caused by the fungus Candida albicans. Symptoms include itching, redness, swelling, and abnormal discharge. Clinically, treatment for Candida albicans infection primarily involves the use of antifungal medications. The main mechanism of antifungal treatment is to eliminate the pathogen. While it can relieve symptoms in the short term, it may lead to drug resistance, a high recurrence rate, and disruption of the host's microecological balance.

[0003] The host's own anti-infective response in the vagina relies primarily on its unique immune system and microecological environment. The body defends against pathogen colonization and invasion by producing antimicrobial proteins such as immunoglobulins (sIgA and IgG) and defensins. Immunoglobulins, especially sIgA, can effectively reduce Candida albicans colonization in the vagina and lower 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 a crucial role in the formation of immune memory. When B cells are stimulated by pathogens, some B cells transform into memory B cells. These memory B cells can rapidly proliferate and secrete IgG antibodies upon re-exposure to the same pathogen. This "memory" allows the body to rapidly produce large amounts of IgG antibodies during a second infection, greatly enhancing the immune defense against the same pathogen. Therefore, IgG is an important marker of immune memory, capable of rapidly activating the immune response during secondary infection. Defensins are the host's natural immune factors with broad-spectrum antimicrobial activity, directly inhibiting the growth and colonization of Candida albicans. Enhancing host immunity and improving the host's anti-infective capacity without disrupting the vaginal microecology is of great significance for preventing vaginal infections and reducing disease incidence.

[0004] Existing patents primarily emphasize that the vaginal symbiotic *Lactobacillus reuteri* can maintain vaginal pH and has good antibacterial effects (CN117683691A, CN116751705A), failing to address the issue of enhancing host immunity. Furthermore, therapeutic effects do not always translate into preventative effects, as treatment primarily targets existing infections, while prevention requires strengthening the host's defenses. The patent "Staphylococcus epidermidis Contributes to Healthy Maturation of the Nasal Microbiome by Stimulating Antimicrobial Peptide Production" discloses that symbiotic *Staphylococcus epidermidis* can stimulate high expression of antimicrobial peptides in host nasal epithelial cells, inhibiting the colonization of pathogenic bacteria such as *Staphylococcus aureus* and *Moraxella catarrhalis*. The patent "Homeostatic Control of Sebaceous Glands by Innate LymphoidCells Regulates Commensal Bacteria Equilibrium" reveals that symbiotic bacteria are essential for skin immune adaptation, exerting their effects by enhancing IL-1 signaling. Symbiotic bacteria are crucial for maintaining human health; however, research on their role in regulating the body's immunity and enhancing the host's resistance to infection is extremely limited in the area of ​​vaginal health. Therefore, linking symbiotic bacteria to activating the host's vaginal resistance to infection is particularly important for preventing vaginal infections, enhancing the host's mucosal immunity during pathogenic invasion, and reducing mucosal damage. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a strain of *Lactobacillus reuteri* and its suspension that promotes the secretion of host defense peptides and immunoglobulins, thereby enhancing anti-infection capabilities. The aim is to solve the technical problem of the lack of research in the existing technologies on enhancing host defense capabilities, reducing the incidence of vaginal infections, and regulating host immunity with *Lactobacillus reuteri* and its suspension.

[0006] The first technical solution provided by this invention is the application of a strain of Limosilactobacillus reuteri CCFM1432 and its bacterial suspension in the preparation of products that enhance the host's ability to defend against pathogenic bacterial infections and reduce damage to the vaginal mucosa.

[0007] This invention provides a strain of *Limosilactobacillus reuteri* CCFM1432, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 20, 2024, with accession number GDMCC No:65158.

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

[0009] The colonies of *Lactobacillus reuteri* CCFM1432 on MRS solid medium were round, raised, white, and relatively moist.

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

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

[0012] The second technical solution provided by this invention is a metabiotic prepared from the *Lactobacillus reuteri* CCFM1432.

[0013] In one embodiment, the metabiotic includes inactivated or dead cells, fermentation supernatant, or cell lysate of the *Lactobacillus reuteri* CCFM1432.

[0014] In one embodiment, the inactivated or dead cells are prepared by culturing the *Lactobacillus reuteri* CCFM1432 in a culture medium for a period of time, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells by heat treatment or freeze-drying.

[0015] In one embodiment, the heat treatment conditions are: 60℃~70℃, 25~35min.

[0016] In one embodiment, the method for preparing the bacterial lysate is as follows: the *Lactobacillus reuteri* CCFM1432 is cultured in a culture medium for a period of time, bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

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

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

[0019] In some embodiments, the product is food, medicine, or hygiene product.

[0020] Furthermore, the drug comprises the aforementioned strains and / or preparations, as well as pharmaceutically permissible carriers.

[0021] Furthermore, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, humectants, disintegrants, lubricants, and flavoring agents.

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

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

[0024] Furthermore, the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic washes for women.

[0025] The fourth technical solution provided by this invention is the application of the aforementioned *Lactobacillus reuteri* CCFM1432 or the aforementioned microbial preparation in the preparation of products that enhance vaginal immunity.

[0026] The fifth technical solution provided by the present invention is the application of the aforementioned *Lactobacillus reuteri* CCFM1432 or the aforementioned microbial preparation in the preparation of products for preventing vaginal pathogenic bacterial infections, wherein the pathogenic bacteria include, but are not limited to, *Candida albicans*.

[0027] Beneficial effects:

[0028] This invention provides a strain of *Limosilactobacillus reuteri* CCFM1432, isolated from vaginal secretions of healthy women. It can strengthen the vaginal anti-infection barrier, reduce mucosal damage, and enhance the host's defense system function through both external and oral application. Specifically, it can: (1) increase the secretion of host defense peptide (HBD2) and polymerized immunoglobulin receptor (pIgR) in vaginal epithelial cells; (2) increase the expression of host defense peptide mBD3 in the vagina; (3) promote the production of polymerized immunoglobulin receptor (pIgR) and secretory immunoglobulin sIgA; (4) promote the production of immunoglobulin IgG; and (5) improve vaginal pathological features. Therefore, this *Limosilactobacillus reuteri* has great application potential in products that enhance the vaginal anti-infection barrier and reduce vaginal mucosal damage.

[0029] Preservation of biological materials

[0030] The Limosilactobacillus reuteri CCFM1432 provided by this invention was deposited on September 20, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:65158, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

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

[0032] Figure 2 Figure: Effect of *Lactobacillus reuteri* on β-defensin secretion in vaginal epithelial cells.

[0033] Figure 3 Figure: Effect of *Lactobacillus reuteri* on the secretion of polymerase receptors.

[0034] Figure 4 Animal experiment flowchart.

[0035] Figure 5 Figure: Effect of topical application of *Lactobacillus reuteri* on the expression of the defensive peptide β-defensin in mouse vaginal tissue.

[0036] Figure 6 Effects of topical application of *Lactobacillus reuteri* on vaginal secretory immunoglobulins pIgR and sIgA.

[0037] Figure 7 Effect of topical application of *Lactobacillus reuteri* on pathological features of the mouse vagina.

[0038] Figure 8:Effect of orally administered Lactobacillus reuteri on the expression of defensin β-defensin in mouse vaginal tissue.

[0039] Figure 9 :Effect of orally administered Lactobacillus reuteri on the secretory immunoglobulins pIgR and sIgA in vaginal tissue.

[0040] Figure 10 :Effect of orally administered Lactobacillus reuteri on the secretion of immunoglobulin IgG in animal serum.

[0041] Figure 11 :Effect of orally administered Lactobacillus reuteri on the pathological manifestations of the mouse vagina.

[0042] (Different lowercase letters in the figure indicate significant differences between groups, p < 0.05). Detailed implementation manners

[0043] The present invention will be further described below in conjunction with specific embodiments.

[0044] The human vaginal epithelial cells (VK2 / E6E7) involved in the following embodiments were generously donated by the Department of Obstetrics and Gynecology of Wuxi People's Hospital, Jiangsu Province.

[0045] The female BALB / c mice involved in the following embodiments were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2012-0001).

[0046] The Lactobacillus reuteri CCFM1432 and Lactobacillus reuteri VJSWX1L3 involved in the following embodiments are self-screened strains from the Food Biotechnology Center of Jiangnan University; Lactobacillus delbrueckii DM8909 was isolated from the vaginal lactobacillus live bacteria capsule Dingjun Sheng and stored in the strain collection of the Food Biotechnology Center of Jiangnan University; Candida albicans SC5314 was purchased from the Guangdong Provincial Institute of Microbiology GDMCC.

[0047] The culture media involved in the following embodiments are as follows:

[0048] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content in the mixed solution is 10000 U / mL, streptomycin concentration is 10 mg / mL).

[0049] LBS solid medium ( / L): 5g casein peptone, 5g yeast extract, 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.

[0050] MRS solid medium ( / L): 5g peptone, 5g yeast extract, 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.

[0051] MRS liquid medium ( / L): 5g peptone, 5g yeast extract, 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.

[0052] SDA medium ( / L): animal tissue pepsin hydrolysate and trypsin 10g, glucose 40g, agar 15g, pH 5.6±0.2.

[0053] SDB medium ( / L): animal tissue pepsin hydrolysate and tryptone 10g, glucose 20g, pH 5.6±0.2.

[0054] The reagent preparation methods involved in the following examples are as follows:

[0055] Estradiol: Dissolve 0.1 mg of β-estradiol in 0.05 mL of sesame oil and prepare immediately before use.

[0056] Example 1: Cell resuscitation and culture

[0057] First, retrieve the frozen human vaginal epithelial cells (VK2 / E6E7), quickly thaw them in a 37°C water bath, then centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in an appropriate volume of cell culture medium, place them in a culture dish, and incubate them in a 37°C incubator containing 5% CO2. When the cells regain viability and reach 70%–80% confluence after 1–2 days, passage the cells.

[0058] Example 2: Isolation and Intervention Sample Preparation of Lactobacillus reuteri CCFM1432

[0059] 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... -1Diluent, then take another 0.5 mL of 10 -1 The diluent was added to 4.5 mL of physiological saline to obtain 10. -2 Diluent, follow these steps to obtain 10 -3 10 -4 10 -5 10 -6 Serial dilution buffer. Take 10... -4 10 -5 10 -6 Add 1 mL of each diluent to a Petri dish, pour in LBS solid medium, mix gently, and incubate at 37°C upside down for 48 hours after the medium has solidified.

[0060] Different morphological colonies were streaked and purified on MRS plates. A single purified colony was picked and inoculated into 5 mL of liquid culture medium and incubated at 37°C for 48 h. 1.5 mL of the cultured bacterial suspension was centrifuged at 6000 rpm for 3 min, the supernatant was discarded, and the suspension was washed three times with 1.5 mL of sterile water and resuspended in 1.5 mL of sterile water for use as a template for bacterial identification. A 20 μL PCR system was prepared, containing 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 10 μL of 2×Taq Mixture, 0.5 μL of bacterial suspension, and 8.5 μL of double-distilled water. Primer information is shown in Table 1.

[0061] Table 1: Primer Information Table

[0062]

[0063] PCR conditions: 95℃ for 5 min; 95℃ for 10 s; 55℃ for 30 s; 72℃ for 30 s; step 2-4 30×; 72℃ for 5 min; 12℃ for 2 min. The PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained using BLAST in GeneBank, and the product was identified as *Limosilactobacillus reuteri*, named *Limosilactobacillus reuteri* CCFM1432.

[0064] For correctly identified bacterial strains, transfer 1.5 mL of bacterial culture to a 2 mL bacterial culture preservation 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 mixer, and store at -80℃.

[0065] 16S sequence information (SEQ ID No. 1):

[0066]

[0067] Preparation of intervention samples:

[0068] Lactobacillus reuteri CCFM1432 cell lysate (CCFM1432-sub): Lactobacillus reuteri CCFM1432 was streaked onto MRS solid medium and cultured anaerobicly at 37°C for 36 h. Single colonies were picked and cultured in 5 mL of MRS liquid medium at 37°C for 20 h. A 4% inoculum was then added to a 1 L culture system and cultured at 37°C for 20 h. The culture was then centrifuged to collect the bacterial sludge, and the bacterial concentration was adjusted to 5 × 10⁻⁶ with physiological saline. 9 CFU / mL. 5×10 9 After centrifuging the CFU / mL *Lactobacillus reuteri* CCFM1432 bacterial suspension, the bacterial cells were collected and resuspended in an equal volume of sterile water. The suspension was then homogenized 10 times at 800–1200 MPa in an autoclave and pasteurized (65°C for 30 min) to obtain bacterial lysates. These lysates were lyophilized and stored at -80°C. Before experiments, the lysates were resuspended in sterile physiological saline to the original volume. The preparation methods for *Lactobacillus delbrueckii* DM8909 and *Lactobacillus reuteri* VJSWX1L3 bacterial lysates were the same, and they were designated DM8909-sub and VJSWX1L3-sub, respectively.

[0069] Fermentation supernatant of *Lactobacillus reuteri* CCFM1432 (CCFM1432-sup): 5 × 10 9 After centrifuging the CFU / mL *Lactobacillus reuteri* CCFM1432 suspension, the fermentation supernatant was collected, lyophilized, and stored at -80°C. Before the experiment, it was resuspended in sterile physiological saline to the original volume. The fermentation supernatant of *Lactobacillus reuteri* VJSWX1L3 was prepared in the same manner and is designated as VJSWX1L3-sup.

[0070] Lactobacillus reuteri CCFM1432 live bacterial suspension (CCFM1432-L): 5×10 9 After centrifuging the CFU / mL *Lactobacillus reuteri* CCFM1432 bacterial suspension, the bacterial sludge was collected and resuspended in an equal volume of physiological saline, and designated as *Lactobacillus reuteri* CCFM1432 live bacteria. The preparation method for *Lactobacillus delbrueckii* DM8909 live bacterial suspension is as described above, designated as DM8909-L.

[0071] Lactobacillus reuteri CCFM1432 postbiotic (CCFM1432-D): 5 × 10 9The CFU / mL suspension of *Lactobacillus reuteri* CCFM1432 was homogenized 10 times in a high-pressure homogenizer (800–1200 MPa) and then pasteurized (65°C for 30 min) to obtain an inactivated bacterial suspension. Before the experiment, the suspension was resuspended to the original volume with sterile physiological saline and denoted as *Lactobacillus reuteri* CCFM1432 postbiotic.

[0072] Example 3: Culture of Candida albicans

[0073] Candida albicans suspension: Candida albicans SC5314 was streaked on SDA medium and incubated at 28℃ for 48 h. Single colonies were picked and inoculated into SDB medium for shaker expansion. The final suspension concentration was adjusted to 5 × 10⁻⁶. 8 CFU / mL.

[0074] Example 4: Effect of postbiotic components of *Lactobacillus reuteri* CCFM1432 on the secretion of host defense peptide (HBD2) by vaginal epithelial cells

[0075] The specific steps are as follows:

[0076] Vaginal epithelial cells (VK2 / E6E7) were used at a rate of 2×10⁻⁶. 5 Seed cells per well in 6-well cell culture plates and culture overnight until cells adhere. Discard the old culture medium, wash three times with PBS buffer, and then add cells as follows:

[0077] (1) The experiment of cell intervention with bacterial lysate was divided into three groups: blank group, bacterial lysate group prepared by Lactobacillus reuteri CCFM1432 (CCFM1432-sub) and bacterial lysate group prepared by Lactobacillus reuteri VJSWX1L3 (VJSWX1L3-sub). Cell culture medium containing 5% (v / v) physiological saline, Lactobacillus reuteri CCFM1432 lysate (CCFM1432-sub) and Lactobacillus reuteri VJSWX1L3 lysate (VJSWX1L3-sub) was added to the corresponding groups respectively.

[0078] (2) The experiment of cell intervention with fermentation supernatant was divided into three groups: blank group, fermentation supernatant of Lactobacillus reuteri CCFM1432 (CCFM1432-sup), and fermentation supernatant of Lactobacillus reuteri VJSWX1L3 (VJSWX1L3-sup). Cell culture media containing 5% (v / v) MRS liquid medium, fermentation supernatant of Lactobacillus reuteri CCFM1432, and fermentation supernatant of Lactobacillus reuteri VJSWX1L3 were added respectively.

[0079] After adding the sample, the cells were incubated at 37°C with 5% CO2. After 24 hours of incubation, the cell culture supernatant was aspirated and the host defense peptide HBD2 was measured using an ELISA kit.

[0080] The results are as follows Figure 2 As shown, the lysates of *Lactobacillus reuteri* CCFM1432 and *Lactobacillus reuteri* VJSWX1L3 significantly increased the secretion of host defense peptide HBD2 by vaginal epithelial cells to 105.51 pg / mL and 87.13 pg / mL, respectively, compared with the control group (74.10 pg / mL), representing increases of 142.3% and 117.5%. The fermentation supernatants of *Lactobacillus reuteri* CCFM1432 and *Lactobacillus reuteri* VJSWX1L3 also significantly increased the secretion of HBD2 by 97.26 pg / mL and 85.54 pg / mL, respectively, compared with the control group (82.52 pg / mL), representing increases of 117.8% and 103.6%. Therefore, *Lactobacillus reuteri* CCFM1432 can significantly increase the content of host defense peptide HBD2.

[0081] Example 5: Effects of the postbiotic component of *Lactobacillus reuteri* CCFM1432 on the secretion of polymerized immunoglobulin receptor (pIgR) by vaginal epithelial cells

[0082] The specific steps are as follows:

[0083] Vaginal epithelial cells (VK2 / E6E7) were used at a rate of 2×10⁻⁶. 5 Seed cells per well in 6-well cell culture plates and culture overnight until cells adhere. Discard the old culture medium, wash three times with PBS buffer, and then add cells as follows:

[0084] (1) The experiment of cell intervention with bacterial lysate was divided into three groups: blank group, bacterial lysate group prepared by Lactobacillus reuteri CCFM1432 (CCFM1432-sub) and bacterial lysate group prepared by Lactobacillus reuteri VJSWX1L3 (VJSWX1L3-sub). Cell culture medium containing 5% (v / v) physiological saline, Lactobacillus reuteri CCFM1432 lysate (CCFM1432-sub) and Lactobacillus reuteri VJSWX1L3 lysate (VJSWX1L3-sub) was added to the corresponding groups respectively.

[0085] (2) The experiment of cell intervention with fermentation supernatant was divided into three groups: blank group, fermentation supernatant of Lactobacillus reuteri CCFM1432 (CCFM1432-sup), and fermentation supernatant of Lactobacillus reuteri VJSWX1L3 (VJSWX1L3-sup). Cell culture media containing 5% (v / v) MRS liquid medium, fermentation supernatant of Lactobacillus reuteri CCFM1432, and fermentation supernatant of Lactobacillus reuteri VJSWX1L3 were added respectively.

[0086] After adding the sample, the cells were incubated at 37°C with 5% CO2. After 24 hours of incubation, the cell culture supernatant was collected and used for ELISA to determine the content of polymerized immunoglobulin receptor (pIgR) secreted by vaginal epithelial cells.

[0087] Experimental results:

[0088] pIgR, an important membrane protein, 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 mucosal immune defense. pIgR is of great significance for understanding mucosal immune mechanisms, developing new immunotherapies, and improving public health.

[0089] like Figure 3 As shown, the cell lysates of *Lactobacillus reuteri* CCFM1432 and *Lactobacillus reuteri* VJSWX1L3 increased the pIgR levels secreted by vaginal epithelial cells to 109.02 pg / mL and 82.01 pg / mL, respectively, which were 127.9% and 96.2% of the control group (85.26 pg / mL). The fermentation supernatants of *Lactobacillus reuteri* CCFM1432 and *Lactobacillus reuteri* VJSWX1L3 increased the pIgR levels secreted by vaginal epithelial cells to 107.46 pg / mL and 76.95 pg / mL, respectively, which were 184.7% and 132.3% higher than the control group (58.17 pg / mL). Overall, *Lactobacillus reuteri* CCFM1432 significantly increased the pIgR levels.

[0090] Example 6: Application of topical Lactobacillus reuteri CCFM1432 postbiotic components in enhancing mouse defense against pathogenic bacterial infections

[0091] Laboratory animals and strains:

[0092] 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 Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK(Beijing)2012-0001). The animal experiment protocol and grouping are shown in Table 2. The specific steps are as follows:

[0093] Candida albicans SC5314 was used, which was purchased from the Culture Collection Center of Guangdong Institute of Microbiology, GDMCC. Figure 4 This is the flow chart of the animal experiment.

[0094] Table 2: Animal experiment protocol and grouping

[0095]

[0096] According to body weight, the mice were randomly divided into 5 groups, and all groups of mice were normally fed throughout the experiment. The preventive model was used in the experiment, that is, first intervened with probiotics and then modeled. In the experimental group of the topical application group, the mice were respectively intravaginally inoculated with 20 μL of cell lysates of Lactobacillus delbrueckii DM8909, mucilaginous Lactobacillus reuteri CCFM1432, and fermentation supernatant of mucilaginous Lactobacillus reuteri CCFM1432 prepared from a bacterial suspension with a concentration of 5×10 9 CFU / mL for 17 consecutive days. The specific operation steps were to use a pipette to aspirate 20 μL of the corresponding sample and slowly inject it into the vagina of the mice. The mice were held upside down for 1-2 minutes. The blank group and the model group were replaced with 20 μL of normal saline for vaginal intervention. On the 12th - 15th days, 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. On the 15th - 17th days, the model group and the experimental group used a pipette to aspirate 20 μL of a Candida albicans bacterial suspension with a concentration of 5×10 8 CFU / mL and slowly injected it into the vagina of the mice. The mice were held upside down for 1-2 minutes. The blank group was intravaginally inoculated with normal saline.

[0097] The experimental period was 17 days. On the 18th day, all experimental mice were sacrificed and the vaginal tissues were dissected for detecting the content of mouse β-defensin 3 (homologous to human β-defensin 2), polymeric immunoglobulin receptor, and immunoglobulin in the vaginal tissues, as well as subsequent histopathological analysis of the experimental tissues.

[0098] Assay Method: At the end of the experiment, blood was collected from the eyeballs and centrifuged to obtain serum for IgG content determination. Mice were euthanized and vaginal tissue was dissected. A portion of the tissue was homogenized with a mixture of pre-cooled RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) and protease inhibitors. The sample was centrifuged at 12000 rpm for 15 min at 4°C, and the vaginal tissue supernatant was collected for mBD3, pIgR, and sIgA content determination according to the kit instructions (Nanjing Senbega Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in 4% paraformaldehyde solution for histopathological examination.

[0099] Histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned into 5 mm thick sections, stained with hematoxylin and eosin (H&E). Vaginal tissue samples were observed under a pathological slide scanner (Panoramic MIDI, 3D Histech Ltd, Budapest, Hungary) at 20x magnification.

[0100] Experimental results:

[0101] (1) Host defense peptide (mBD3) in mouse vagina

[0102] Mucosal immunity is one of the most important immune defense mechanisms in the body, primarily occurring on the surface of mucous membranes, including the vaginal mucosa in women. Host defense peptides (HDPs) are a class of polypeptides less than 50 amino acids long, possessing a positive charge and hydrophobicity, enabling them to interact with microbial cell membranes. HBD2 is the most important host defense peptide in humans, sharing homology with mBD3 in mice. HDPs play a crucial role in mucosal immunity, enhancing immune responses by directly killing pathogens or regulating immune cell activity.

[0103] like Figure 5 As shown, the model group exhibited a significant reduction in defensin levels, with mBD3 secretion at 32.44 ng / L, compared to 50.65 ng / L in the control group. Topical application of *Lactobacillus delbrueckii* DM8909 cell lysate increased mBD3 secretion, reaching 48.70 ng / L (a 48.70% increase compared to the model group). Topical application of *Lactobacillus reuteri* CCFM1432 cell lysate and fermentation supernatant produced mBD3 levels of 55.29 ng / L and 52.03 ng / L, respectively (70.41% and 60.37% increases compared to the model group). Therefore, topical application of *Lactobacillus reuteri* CCFM1432 can significantly increase the content of the host defensive peptide mBD3 in mice.

[0104] (2) Secretion of polymerized immunoglobulin receptor (pIgR) and secretory immunoglobulin (sIgA) in mouse vaginal tissue

[0105] Polyimmunoglobulin receptor (pIgR) is a protein expressed on the surface of mucosal epithelial cells. It transports polyimmunoglobulin A (pIgA) from the blood to the mucosal surface through transcellular transport, forming secretory immunoglobulin A (sIgA). Detecting sIgA in the vagina is beneficial for enhancing local vaginal immunity, maintaining the immune protective layer on the mucosal surface, and preventing the invasion of pathogens.

[0106] like Figure 6 As shown in Figure A, compared with the control group (146.61 pg / mL), the model group significantly reduced the level of polymerized immunoglobulin receptor (pIgR), with a pIgR secretion of 116.61 pg / mL. Topical application of *Lactobacillus delbrueckii* DM8909 cell lysate increased pIgR secretion, reaching 179.25 pg / mL (a 53.70% increase compared to the model group). Topical application of *Lactobacillus reuteri* CCFM1432 cell lysate and fermentation supernatant produced pIgR levels of 241.85 pg / mL and 212.99 pg / mL, respectively (107.40% and 82.65% increases compared to the model group).

[0107] like Figure 6 As shown in Figure B, compared with the control group (0.83 μg / mL), the model group showed a significant decrease in secretory immunoglobulin A (sIgA), with a secretion level of 0.54 μg / mL. Topical application of *Lactobacillus delbrueckii* DM8909 cell lysate increased sIgA secretion, with a secretion level of 0.62 μg / mL (a 15.46% increase compared to the model group). Topical application of *Lactobacillus reuteri* CCFM1432 cell lysate and fermentation supernatant produced sIgA levels of 0.85 μg / mL and 0.73 μg / mL, respectively (57.79% and 35.22% increases compared to the model group). In conclusion, topical application of *Lactobacillus reuteri* CCFM1432 can enhance the protein expression of secretory immunoglobulin A by increasing the secretion of polymerized immunoglobulins, thereby improving the body's anti-infection ability.

[0108] (3) Histopathological analysis of mouse vaginal tissue

[0109] Vaginal histopathological studies can be used to evaluate the efficacy of different treatments for vaginal infections. For example... Figure 7As shown, for the mice in the blank control group, the vaginal mucosa structure remained intact and the surface keratinized layer was normal. In the model group, the keratinized layer of the vaginal mucosa disappeared, squamous hyperplasia of epithelial cells occurred, and a large number of inflammatory cells infiltrated the mucosa. After the intervention of the cell lysate of Lactobacillus delbrueckii DM8909, the surface of the vaginal tissue was intact, but there were a small number of infiltrating inflammatory cells; after the intervention of the cell lysate and fermentation supernatant of Lactobacillus mucosae CCFM1432, the surface cells of the vaginal tissue were relatively smooth, and the infiltration of inflammatory cells was significantly reduced compared with the model group. It shows that after the prevention with Lactobacillus mucosae, the damage of the vaginal tissue surface is greatly reduced, and it has a good effect of preventing the invasion of pathogenic bacteria.

[0110] Example 7: Application of Postbiotics Component of Oral Lactobacillus mucosae CCFM1432 in Enhancing the Defense of Mice against Pathogenic Bacteria Infection

[0111] Experimental animals and strains:

[0112] Twenty-five 7-week-old SPF-grade 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 Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK(Beijing)2012 - 0001), and the animal experiment plan and grouping are shown in Table 2. The specific steps are as follows:

[0113] Candida albicans SC5314 was used, which was purchased from the GDMCC of Guangdong Institute of Microbiology. The flow chart of the animal experiment is shown in Figure 4 .

[0114] Table 3: Animal Experiment Plan and Grouping

[0115]

[0116] According to body weight, the mice were randomly divided into 5 groups, and all groups of mice were normally raised throughout the experiment. The prevention model was used in the experiment, that is, the probiotics were first intervened and then the model was established. The experimental groups in the oral administration group were continuously gavaged with 200 μL of live bacteria of Lactobacillus delbrueckii DM8909, live bacteria of Lactobacillus mucosae CCFM1432, and inactivated bacterial suspension of Lactobacillus mucosae CCFM1432 with a concentration of 5×10 9 CFU / mL for 17 days. On the 12th - 15th days, the blank group was subcutaneously injected with 50 μL of normal saline, and the model group and the experimental groups were subcutaneously injected with 50 μL of estradiol to induce estrus. On the 16th - 17th days, the model group and the experimental groups used a pipette to aspirate 20 μL of Candida albicans suspension with a concentration of 5×10 8 CFU / mL and slowly injected it into the vagina of the mice, and the mice were inverted for 1 - 2 minutes. The blank group was vaginally inoculated with normal saline in the same way.

[0117] The experiment lasted 17 days. On day 18, all experimental mice were sacrificed and vaginal tissue was dissected to detect mouse β-defensin 3, polymerized immunoglobulin receptor, and immunoglobulin content in the vaginal tissue, as well as for subsequent histopathological analysis.

[0118] Assay Method: At the end of the experiment, blood was collected from the eyeballs and centrifuged to obtain serum for IgG content determination. Mice were euthanized and vaginal tissue was dissected. A portion of the tissue was homogenized with a mixture of pre-cooled RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) and protease inhibitors. The sample was centrifuged at 12000 rpm for 15 min at 4°C, and the vaginal tissue supernatant was collected for mBD3, pIgR, and sIgA content determination according to the kit instructions (Nanjing Senbega Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in 4% paraformaldehyde solution for histopathological examination.

[0119] Histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned into 5 mm thick sections, stained with hematoxylin and eosin (H&E). Vaginal tissue samples were observed under a pathological slide scanner (Panoramic MIDI, 3D Histech Ltd, Budapest, Hungary) at 20x magnification.

[0120] Experimental results:

[0121] (1) Host defense peptide (mBD3) in mouse vagina

[0122] like Figure 8 As shown, the model group exhibited a significant decrease in defensin levels, with mBD3 secretion decreasing from 49.67 ng / L to 33.33 ng / L. Oral administration of live *Lactobacillus delbrueckii* DM8909 resulted in mBD3 secretion of 49.88 ng / L (a 49.68% increase compared to the model group), while oral administration of live and inactivated *Lactobacillus reuteri* CCFM1432 suspensions produced mBD3 levels of 58.42 ng / L and 55.99 ng / L, respectively (75.31% and 68.02% increases compared to the model group). Therefore, *Lactobacillus reuteri* CCFM1432 can significantly increase the content of the host defensive peptide mBD3 in mice.

[0123] (2) The secretion of polymerized immunoglobulin receptor (pIgR) and secretory immunoglobulin (sIgA) in mouse vaginal tissue is as follows: Figure 9As shown in Figure A, compared with the control group (136.02 pg / mL), the model group significantly reduced the level of polymerized immunoglobulin receptor (pIgR), with a pIgR secretion of 120.92 pg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 increased pIgR secretion to 130.53 pg / mL (a 7.95% increase compared to the model group). Oral administration of live and inactivated Lactobacillus reuteri CCFM1432 produced pIgR levels of 146.41 pg / mL and 176.80 pg / mL, respectively (21.08% and 46.21% increases compared to the model group).

[0124] like Figure 9 As shown in Figure B, compared with the control group (0.70 μg / mL), the model group showed a significant decrease in secretory immunoglobulin A (sIgA), with a secretion level of 0.45 μg / mL. Oral administration of live *Lactobacillus delbrueckii* DM8909 increased sIgA secretion to 0.62 μg / mL (a 38.22% increase compared to the model group). Oral administration of live and inactivated *Lactobacillus reuteri* CCFM1432 suspensions produced sIgA levels of 1.22 μg / mL and 0.86 μg / mL, respectively (171.15% and 91.16% increases compared to the model group). In conclusion, oral administration of *Lactobacillus reuteri* CCFM1432 can enhance the protein expression of secretory immunoglobulin A by increasing the secretion of polymerized immunoglobulins, thereby improving the body's anti-infection ability.

[0125] (3) Secretion of immunoglobulin G (IgG) in mouse serum

[0126] like Figure 10 As shown, compared with the blank group (7.17 μg / mL), the model group significantly reduced immunoglobulin (IgG), with an IgG secretion level of 6.21 μg / mL. Oral administration of live Lactobacillus delbrueckii DM8909 increased IgG secretion, with the secretion level increasing to 6.53 μg / mL (5.08% higher than the model group). Oral administration of live and inactivated Lactobacillus reuteri CCFM1432 produced IgG levels of 7.42 μg / mL and 7.72 μg / mL, respectively (19.41% and 24.25% higher than the model group, respectively). In summary, oral administration of Lactobacillus reuteri CCFM1432 can significantly increase the serum IgG content and enhance systemic immunity to resist pathogenic bacterial infection. (4) Histopathological analysis of mouse vaginal tissue

[0127] Vaginal histopathological studies can be used to evaluate the efficacy of different treatments for vaginal infections. For example... Figure 11As shown, in the control group mice, the vaginal mucosa structure remained intact, with a clear squamous epithelial cell layer and basal layer, and a balanced distribution of immune cells within the tissue. In the model group mice, the vaginal mucosal epithelial cell layer disappeared, and a large number of inflammatory cells showed 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 tissue surface was slightly damaged. Intervention with live and inactivated *Lactobacillus reuteri* CCFM1432 suspension resulted in an intact tissue surface, reduced inflammatory cell infiltration, and restoration of the keratinized layer on the mucosal surface, leading to a smoother surface.

[0128] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the examples described above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus reuteri CCFM1432, which was deposited in Guangdong Provincial Microbiological Culture Collection 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 food, medicine or 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 the 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 the 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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