Application of lactobacillus crispatus HC1261 in preparation of microbial inoculum for improving female reproductive health and composition for improving vagina inflammation

Abstract

The invention relates to the technical field of probiotic application, in particular to application of lactobacillus crispatus HC1261 in preparation of a microbial agent for improving female reproductive health and a composition for improving vagina inflammation. The lactobacillus crispatus HC1261 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 16, 2023, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.27355; the microbial inoculum for improving female reproductive health can reduce the load of the ureaplasma urealyticum in the vagina. The lactobacillus crispatus HC1261 can be used for pertinently reducing the load of the ureaplasma urealyticum in the vagina, and can be used for reducing the related health risks of urethritis, cervicitis, pelvic inflammation, infertility and the like caused by the ureaplasma urealyticum.

Description

Technical Field

[0001] This invention relates to the field of probiotic application technology, specifically to the application of a bacterial agent prepared from Lactobacillus curvature HC1261 for improving female reproductive health and a composition for improving vaginal inflammation. Background Technology

[0002] The vagina is one of the natural cavities connecting the human body to the outside world, and like the human intestines, it possesses a complex micro-ecosystem. The vaginal microecology of healthy women is colonized by various microorganisms, including lactobacilli, streptococci, staphylococci, corynebacteria, enterobacilli, enterococci, Candida albicans, mycoplasma, and viruses, which together constitute the normal vaginal flora. Scientific research indicates that lactobacilli are the most important normal flora in the vagina. The detection rate of lactobacilli in vaginal secretions can reach over 80%, with a viable count of up to 8 × 10⁻⁶. 7 CFU / mL. In the vaginal microecology dominated by lactobacilli, various microorganisms interact to form a dynamically balanced micro-ecosystem, maintaining a healthy vagina. Lactobacilli can utilize glycogen stored in shed vaginal epithelial cells as energy to grow and reproduce in the vaginal environment. They can inhibit the invasion and overgrowth of pathogenic microorganisms and regulate the vaginal microecological balance by competing with pathogens for nutrients; competing for adhesion receptors on the surface of vaginal epithelial cells; stimulating the body to produce local immune responses; and producing hydrogen peroxide, antibiotics, and lactic acid.

[0003] Bacterial vaginosis is one of the most common vaginal infections in women of reproductive age. It is characterized by typical vaginal microecological dysbiosis, increased anaerobic bacterial load, and increased production of volatile amines, leading to health problems such as gonorrhea, mycoplasma infection, herpes simplex virus type 2 infection, premature birth, pelvic inflammatory disease, and increased susceptibility to HIV. Mycoplasma is a common prokaryotic microorganism closely related to reproductive tract infections, with a high detection rate in the general population. Mycoplasma infection can cause various diseases such as urethritis, cervicitis, pelvic inflammatory disease, and infertility. The detection rate of Ureaplasma urealyticum is significantly higher in patients with bacterial vaginosis. Currently, bacterial vaginosis is mainly treated with antibiotics such as clindamycin, metronidazole, or tinidazole. However, standard antibiotic therapy cannot restore the acidic vaginal environment, exacerbating vaginal microecological dysbiosis. Once treatment ends, the corresponding pathogens will return and re-grow on the vaginal mucosa, leading to recurrence of bacterial vaginosis. Furthermore, antibiotic therapy can cause serious side effects, including nausea, vomiting, or gastrointestinal discomfort, and it is not suitable for pregnant or breastfeeding women.

[0004] Unlike traditional antibiotic therapy, probiotic treatment for bacterial vaginosis not only disrupts pathogen biofilms, reducing pathogen colonization in the vaginal environment and inhibiting biofilm formation, thus decreasing recurrence, but also regulates the body's immune system, restores the vaginal acidic environment, and modulates the vaginal microecological system. This avoids the problem of antibiotic overuse and is a safe and feasible treatment option for bacterial vaginosis. Currently, most existing Lactobacillus curvaturei treatments for bacterial vaginosis focus on inhibiting the growth and colonization of pathogens such as Gardnerella vaginalis, neglecting the concurrent high load of Ureaplasma urealyticum. Summary of the Invention

[0005] To address the current lack of technical solutions for Lactobacillus curvature that can reduce the viral load of Ureaplasma urealyticum in the vagina, this invention provides an application of Lactobacillus curvature HC1261 in the preparation of a bacterial agent for improving female reproductive health and a composition for improving vaginal inflammation.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides an application of Lactobacillus curvature HC1261 in the preparation of a bacterial agent for improving female reproductive health. Lactobacillus curvature ( Lactobacillus crispatus HC1261 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27355. Microbial agents used to improve women's reproductive health can reduce the load of Ureaplasma urealyticum in the vagina.

[0007] Furthermore, probiotics used to improve women's reproductive health can inhibit pathogenic bacteria in the vagina.

[0008] Furthermore, probiotics used to improve women's reproductive health can eliminate biofilms of vaginal pathogens in the vagina.

[0009] Furthermore, probiotics used to improve women's reproductive health can reduce the load of pathogenic bacteria in the vagina.

[0010] Furthermore, vaginal pathogens include Gardnerella vaginalis (… Gardnerella vaginalis ), Bilopredominus ( Prevotella bivia ), Timoprevotella ( Prevotella timonensis ), Fanny Hesse (vaginis) Fannyhessea vaginae , formerly known as Atopobium vaginae ) and / or amniotic fluid prevotella ( Prevotella amnii ).

[0011] Furthermore, microbial agents used to improve female reproductive health can reduce sialidase activity.

[0012] Furthermore, the bacterial agents used to improve female reproductive health can increase the level of the anti-inflammatory cytokine IL-10 and decrease the levels of the pro-inflammatory cytokines IL-1β and IL-8.

[0013] Secondly, the present invention also provides a composition for improving vaginal inflammation, comprising the above-mentioned *Lactobacillus curvatureii* (… Lactobacillus crispatus ) Live bacteria, inactivated bacteria and / or fermentation products of HC1261.

[0014] Furthermore, the composition is a non-therapeutic composition, such as vaginal cleansing products, vaginal care products, and vaginal hygiene products, specifically vulvar washes, sanitary napkins, and wet wipes containing live or inactivated Lactobacillus curvatureii HC1261 and / or its fermentation products.

[0015] The beneficial effects of this invention are as follows: The application of *Lactobacillus curvatureii* HC1261 provided by this invention in the preparation of bacterial agents for improving female reproductive health can specifically reduce the load of *Ureaplasma urealyticum* in the vagina, effectively filling the technological gap in this area compared to existing strains and metronidazole. This can reduce the health risks associated with *Ureaplasma urealyticum*-induced urethritis, cervicitis, pelvic inflammatory disease, and infertility. Simultaneously, the bacterial agent prepared from *Lactobacillus curvatureii* HC1261 can comprehensively inhibit the growth of various vaginal pathogens, eliminate biofilms formed by pathogens, reduce pathogen load, and disrupt the colonization basis of pathogens in the vagina. It can also regulate the vaginal microecological balance, rebuilding the acidic healthy environment of the vagina through hydrogen peroxide and acid production, providing a basic guarantee for vaginal health. Furthermore, it can reduce sialidase activity, reduce damage to the protective mucus layer of the vaginal epithelium, and further inhibit pathogen invasion. In addition, it can effectively alleviate vaginal tissue inflammation and improve the inflammatory state by regulating the levels of inflammatory factors, increasing the content of anti-inflammatory cytokines, and reducing the concentration of pro-inflammatory cytokines.

[0016] The use of Lactobacillus curvature HC1261 to prepare bacterial agents for improving women's reproductive health or compositions for improving vaginal inflammation offers flexibility and can be applied to a variety of vaginal care, cleaning, and hygiene products. It is convenient to use and highly safe, avoiding the problems of traditional antibiotic therapy, such as the inability to rebuild the vaginal microecology, easy recurrence of the disease, side effects, and limited applicable population. It provides a safer and more effective new solution for the prevention and adjuvant treatment of bacterial vaginosis in women, and comprehensively supports the maintenance of women's reproductive health. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a photograph of the inhibition zone of Lactobacillus curvature HC1261 inhibiting Gardnerella vaginalis in Example 1.

[0019] Figure 2 This is a colony morphology diagram of Lactobacillus curvature HC1261 in Example 1.

[0020] Figure 3 This is a microscopic photograph of Lactobacillus curvature HC1261 in Example 1.

[0021] Figure 4 This is an API 50 CHL carbon source metabolism identification map of Lactobacillus curvature HC1261 in Example 1.

[0022] Figure 5 This is the RAPD fingerprint of Lactobacillus curvature HC1261 in Example 1.

[0023] Figure 6 This is the rep-PCR fingerprint of Lactobacillus curvature HC1261 in Example 1.

[0024] Figure 7 This is a diagram showing the results of Lactobacillus curvature HC1261 eliminating vaginal pathogenic bacteria biofilm in Example 6.

[0025] Figure 8 The OD of H2O2 concentration in Example 7 600 nm Absorbance standard curve.

[0026] Figure 9 This is a graph showing the pH changes of the fermentation broth of Lactobacillus curvature HC1261 in Example 8.

[0027] Figure 10 The image shows the load of Lactobacillus curvaturei HC1261, vaginal pathogens, and Ureaplasma urealyticum in the vagina of rats in Example 10.

[0028] Figure 11 This is a graph showing the activity of rat vaginal sialysinase in Example 10.

[0029] Figure 12 This is a graph showing the results of rat inflammatory cytokines in Example 10.

[0030] In the diagram, * indicates P <0.05, ** indicates P <0.01. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0032] Example 1: Strain Identification I. Source of the strain In January 2023, it was collected from Hulunbuir City, Inner Mongolia Autonomous Region, and named strain C-3.

[0033] II. Anti-Gardnerella vaginalis test Gardnerella vaginalis was inoculated onto Columbia blood agar plates for activation. After single colonies grew, they were picked and transferred to modified BHI broth (containing 5% bovine serum) and cultured aerobically at 37°C for 24 h. Then, the colonies were transferred at a ratio of 1% (v / v) to fresh modified BHI broth and cultured aerobically at 37°C for 24 h to obtain a fresh Gardnerella vaginalis culture. The fresh culture was then mixed with an equal volume of PBS buffer at a 1:1 ratio to obtain a Gardnerella vaginalis suspension.

[0034] Prepare Columbia blood agar plates in advance, then spread 50 μL of Gardnerella vaginalis suspension onto the Columbia blood agar plates, place an Oxford cup on top, add 100 μL of fermentation broth of strain C-3 into the wells, incubate at 37℃ for 48 h, and observe for the presence or absence of inhibition zones.

[0035] The results are as follows Figure 1 As shown, the inhibition zone produced by strain C-3 reached a diameter of 27.5 ± 0.2 mm.

[0036] III. Colony Morphology Identification Colony morphology observation showed that strain C-3 colonies on MRS solid medium were 1-2 mm in diameter, round, with irregular edges, opaque, grayish-white on the front, raised in the center, smooth and bright, and moist in texture. Colony photographs are shown below. Figure 2 As shown.

[0037] Gram staining results as follows Figure 3 As shown, strain C-3 is a Gram-positive bacterium, and under a microscope, the bacterial cells are observed to be short rod-shaped, long filament-shaped, or short rod-shaped chains.

[0038] IV. Identification of Physiological and Biochemical Characteristics Under aseptic conditions, take an appropriate amount of fresh bacterial culture of strain C-3, centrifuge at 5000 r / min for 5 min, wash twice with PBS buffer, resuspend in the same volume of PBS buffer, and dilute 50 times to obtain the inoculum.

[0039] 1. Temperature growth range experiment Inoculate the inoculum into 10 mL of MRS liquid medium at an inoculation rate of 10% (v / v), with 10 mL of uninoculated MRS liquid medium as a control. Incubate the medium in constant temperature shaking incubators at 15℃, 45℃ and 60℃ for 7 days, and incubate it in a constant temperature shaking incubator at 37℃ for 2 days. Observe whether the culture medium becomes turbid.

[0040] The results showed that strain C-3 remained clear after 7 days of culture in constant temperature shaking incubators at 15℃, 45℃, and 60℃, but became turbid after 2 days of culture at 37℃. This indicates that strain C-3 cannot grow normally at 15℃ or above 37℃.

[0041] 2. Carbon source metabolism experiment The metabolic effects of strain C-3 on 49 carbon sources were determined using API 50 CHL reagent strips. Under aseptic conditions, an appropriate amount of fresh bacterial suspension of strain C-3 was centrifuged at 5000 r / min for 5 min, washed twice with PBS buffer (pH 7.0), and resuspended with the same volume of buffer to obtain a bacterial suspension. The bacterial suspension was added to the culture medium of the API kit at a concentration of 10% (v / v). The reagent strip was then incubated at 37℃ for 24–48 h according to the kit instructions. Color changes were observed and the experimental results were recorded.

[0042] The results of the identification are as follows Figure 4 As shown, strain C-3 was preliminarily identified as Lactobacillus curvature, with ID=99.7% and T value=0.87.

[0043] 3. Glucose acid and gas production experiment: Accurately weigh 0.5 g sodium acetate, 0.5 g peptone, 0.3 g yeast extract, 0.1 mL Tween 80, 2.5 g glucose, 0.05 mL 2% bromocresol green (w / v), 0.5 mL salt solution A, 0.5 mL salt solution B, and 100 mL distilled water to prepare the glucose acid production and gas generation test medium. Adjust the pH of the medium to 6.8–7.0. Salt solution A is prepared by accurately weighing 10 g KH₂PO₄ and 1.0 g K₂HPO₄, dissolving them in distilled water, and then bringing the volume to 100 mL with distilled water. Salt solution B is prepared by accurately weighing 11.5 g MgSO₄·7H₂O, 2.4 g MnSO₄·2H₂O, and 0.68 g FeSO₄·7H₂O, dissolving them in distilled water, and then bringing the volume to 100 mL with distilled water.

[0044] Dispense the prepared glucose acid production and gas production test medium into large test tubes containing inverted small test tubes, 3 mL per tube, and autoclave at 121℃ for 15 min. Under aseptic conditions, inoculate fresh bacterial suspension of strain C-3 into the glucose acid production and gas production test medium at an inoculation rate of 10% (v / v), using uninoculated glucose acid production and gas production test medium as a control. Then, seal the top with 2 mL of sterile liquid paraffin and incubate at 37℃ for 6 consecutive days, observing any changes in the color of the medium daily.

[0045] The results showed that after 6 days of incubation at 37℃, the culture medium changed from green to yellow, and there was no gas in the small inverted tube, indicating that strain C-3 can ferment glucose to produce acid without producing gas.

[0046] 4. Salinity tolerance test: Under aseptic conditions, the activated strain C-3 fresh bacterial suspension was inoculated at a rate of 10% into 5 mL of MRS liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 5 mL of uninoculated MRS liquid culture medium was used as a control. The medium was incubated at 37°C with shaking, and it was observed whether the medium became turbid.

[0047] The results showed that strain C-3 grew in culture media with salt concentrations of 1% to 3%, but did not grow in culture media with salt concentrations above 4%. The highest salt concentration that strain C-3 could tolerate was 3%.

[0048] 5. 16S rDNA gene sequence identification: 16S rDNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results are as follows:

[0049] The sequences and 16S rDNA genes were compared in the NCBI database, and a phylogenetic tree was constructed using MEGA 5.0 software. BLAST analysis showed that strain C-3 had the highest sequence similarity to existing Lactobacillus curvature, with 100% sequence similarity.

[0050] 6. RAPD fingerprint identification M13 (GAGGGTGGCGGTTCT, SEQ ID NO.2) was used as the primer. The RAPD reaction system consisted of 20 μL of RAPD, containing 0.2 μL of Taq DNA polymerase (5 U / μL) and 10× buffer (containing Mg). 2+ 2 μL of deoxyribonucleic acid (dNTPs, 2.5 mM), 0.8 μL of M13 primer (10 μM), 1 μL of DNA template, and 2 μL of double-distilled water (ddH2O) were added to prepare a 1.5% agarose gel plate. Using a DL2000 DNA Marker as a control, electrophoresis was performed at a constant voltage of 100 V for 80 min. The electrophoresis results were then analyzed using a gel imaging system. The RAPD fingerprint of strain C-3 is shown below. Figure 5 As shown.

[0051] 7. Rep-PCR fingerprint identification The primer sequence for rep-PCR is GTGGTGGTGGTGGTG (SEQ ID NO.3). The total reaction volume for rep-PCR is 20 μL, containing 0.2 μL of recombinant Taq DNA polymerase (rTaq) and 10×Ex Taq DNA buffer (containing Mg). 2+ 2 μL of deoxyribonucleic acid (dNTPs, 2.5 mM), 2 μL of primers (10 μM), 1 μL of DNA template, and 12.8 μL of double-distilled water (ddH2O) were added. A 1.5% agarose gel plate was prepared, and an DL2000 DNA Marker was used as a control. Electrophoresis was performed at a constant voltage of 100 V for 80 min. The electrophoresis results were then analyzed using a gel imaging system. The rep-PCR fingerprint of strain C-3 is shown below. Figure 6 As shown.

[0052] The strain C-3 was named *Lactobacillus curlis* HC1261 and deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27355. It was classified as *Lactobacillus curlis*. Lactobacillus crispatus .

[0053] Example 2: Determination of the antioxidant function of Lactobacillus curvature HC1261 A single colony of Lactobacillus curvature HC1261 was inoculated into 3 mL of MRS liquid medium and cultured at 37°C for 18-20 h. Using this culture medium as the inoculum, 2% (v / v) of the inoculum was inoculated into 50 mL of MRS liquid medium and cultured statically for 18 h to obtain the culture medium of the strain.

[0054] Centrifuge the culture medium at 4℃ for 10 min and collect the supernatant, which is the fermentation supernatant.

[0055] After collecting the bacterial cells by aspirating 1 mL of culture medium, wash the bacterial cells twice with 1 mL of PBS buffer, and then resuspend the bacterial cells in 2 mL of PBS buffer to obtain a bacterial suspension for later use.

[0056] The bacterial suspension was ultrasonically disrupted for 20 minutes to obtain an intracellular extract.

[0057] I. Determination of the ability to scavenge 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) Take 1 mL of Lactobacillus curlis HC1261 bacterial suspension and add 1 mL of freshly prepared 0.4 mM DPPH free radical solution. Mix well and incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm. 样品 Three parallel experiments were conducted, and the average value of the obtained data was taken.

[0058] An equal volume of PBS buffer and ethanol mixture was used as a blank for instrument zeroing; the absorbance was recorded as A. 空白 A mixture of an equal volume of PBS buffer and DPPH ethanol solution was used as a control, and the absorbance was recorded as A. 对照 DPPH free radical scavenging rate is calculated as scavenging rate % = [1 - (A...]]. 样品 -A 空白 ) / A 对照 Calculate by multiplying by 100%.

[0059] The results showed that the *Lactobacillus curvatureii* HC1261 provided by this invention had a DPPH free radical scavenging rate of up to 85.69% ± 4.80%.

[0060] II. Determination of hydroxyl radical scavenging (HRS) ability Take 200 μL of Lactobacillus curlii HC1261 bacterial suspension, mix it with 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, and 500 μL of deionized water, then add 100 μL of 3 mM hydrogen peroxide solution. After incubating at 37℃ for 15 min, measure the absorbance of the sample at a wavelength of 510 nm and record it as A. 样品 Replace the bacterial suspension or fermentation supernatant with an equal volume of deionized water, and mix with 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, and 500 μL of deionized water. Then add 100 μL of 3 mM hydrogen peroxide solution to each. After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm and record it as A. 控制 Replace the bacterial suspension or fermentation supernatant and hydrogen peroxide solution with an equal volume of deionized water, and mix with 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, and 500 μL of deionized water. After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm, and record it as A. 空白 HRS free radical scavenging rate is calculated as scavenging rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 Calculate by multiplying by 100%.

[0061] The results showed that the *Lactobacillus curvatureii* HC1261 provided by this invention had a scavenging rate of up to 79.43% ± 4.00% against HRS free radicals.

[0062] III. Determination of Anti-lipid Peroxidation Capacity Measure 0.1 mL of linoleic acid, 0.2 mL of Tween 20, 19.7 mL of deionized water, and 0.5 mL of PBS buffer (pH=7.4). Mix the above components thoroughly and stir until homogeneous to obtain a linoleic acid emulsion for later use.

[0063] Take 1 mL of the prepared linoleic acid emulsion, add 1 mL of FeSO4 solution (1%), and then add 0.5 mL of the sample to be tested (fermentation supernatant, bacterial suspension, or intracellular extract of Lactobacillus curvature HC1261). Gently shake to make the mixture homogeneous. Place the mixture in a 37℃ water bath and incubate for 1.5 h. After the reaction, add 0.2 mL of TCA solution (4%) and 2 mL of TBA solution (0.8%) to the mixture, mix well, and heat in a 100℃ water bath for 30 min. After color development, quickly cool the mixture and then centrifuge at 4000 r / min for 15 min to remove precipitated impurities. Collect the supernatant after centrifugation and measure the absorbance at 532 nm, denoted as A. Simultaneously, repeat the above operation with 0.5 mL of distilled water instead of the sample to be tested, and record the absorbance as A0. The lipid peroxidation inhibition rate is calculated as inhibition rate % = (A0 - A) / A0 × 100%.

[0064] The results are shown in Table 1. It can be seen that the fermentation supernatant, bacterial suspension and intracellular extract of Lactobacillus curvature HC1261 can resist lipid peroxidation. Among them, the anti-lipid peroxidation effect of fermentation supernatant and bacterial suspension is better than that of intracellular extract.

[0065] Table 1. Anti-lipid peroxidation level of Lactobacillus curvature HC1261

[0066] Based on the combined performance of Lactobacillus curvature HC1261 in DPPH clearance rate, HRS clearance rate and anti-lipid peroxidation, it can be concluded that Lactobacillus curvature HC1261 has excellent antioxidant levels, which is beneficial for clearing inflammatory responses in the body.

[0067] Example 3 Hydrophobic cell surface assay of Lactobacillus curvatureii HC1261 Purified Lactobacillus curvature HC1261 colonies were picked and inoculated into MRS liquid medium and cultured at 37°C with shaking for 24 h. Then, 1% (v / v) of the inoculum was transferred to fresh MRS liquid medium and cultured at 37°C with shaking for another 24 h. After centrifugation at 6000×g for 10 min, the bacterial cells were collected, washed twice with sterile physiological saline, and then resuspended in 1 mL of 0.1 M sterile KNO3 solution as the test solution.

[0068] Add 50 μL of the above-mentioned bacterial culture to 2450 μL of 0.1 M KNO3 solution and test the OD of the solution. 600 nmThe value is recorded as A0; 1.5 mL of the above-mentioned bacterial solution is mixed with 500 μL of xylene and allowed to stand at room temperature for 10 min. The resulting two-phase system is then vortexed for 2 min and allowed to stand for another 20 min to reform the aqueous and organic phases. The aqueous phase is carefully aspirated for OD testing. 600 nm The value is recorded as A1. Cell hydrophobicity is calculated as hydrophobicity% = (A0 - A1) / A1 × 100%. Three parallel experiments were conducted, and the average value of the obtained data was taken.

[0069] The results showed that the hydrophobicity of the cell surface of Lactobacillus curvature HC1261 was 85.23% ± 4.12%, indicating that Lactobacillus curvature HC1261 has a large adhesion potential.

[0070] Example 4: Antibiotic resistance test of Lactobacillus curvature HC1261 Gentamicin, streptomycin, ampicillin, clindamycin, erythromycin, and tetracycline were all prepared as stock solutions of 2048 μg / mL and stored at ~20℃ for later use. Before use, the stock solutions were serially diluted 2-fold with MRS liquid medium to prepare the working solutions, with concentrations ranging from 1 to 1024 μg / mL, for a total of 11 gradients.

[0071] After culturing Lactobacillus curvature HC1261 overnight at 37°C, an appropriate amount of fresh bacterial solution was centrifuged at 5000 r / min for 5 min, the bacterial cells were washed once with sterile physiological saline, and then resuspended with the same volume of physiological saline and diluted 50 times as the inoculum.

[0072] In a 96-well plate, antibiotic-free MRS liquid medium was added to the first column as a negative control. 190 μL of MRS liquid medium containing different concentrations of antibiotics was added sequentially to columns 2 through 12. Then, 10 μL of the above inoculum was inoculated into each well, creating three parallel wells. One well was left as a blank. 50 μL of paraffin oil was added to cover the plate and prevent moisture evaporation. The 96-well plate was incubated at 37°C with shaking for 48 h, and then the OD was measured. 600 The MIC values ​​of antibiotics against the strains were calculated using the results after 48 hours, and the results are shown in Table 2.

[0073] Table 2. MIC values ​​of antibiotics against Lactobacillus curvature HC1261 (unit: μg / mL)

[0074] As shown in Table 2, Lactobacillus curvature HC1261 is sensitive to common antibiotics such as erythromycin, streptomycin and tetracycline, and slightly sensitive to gentamicin and ampicillin, with good overall biosafety.

[0075] Example 5: Antibacterial effect test of Lactobacillus curvaturei HC1261 against vaginal pathogens Lactobacillus curvature HC1261 was inoculated into MRS liquid medium at an inoculum rate of 1% (v / v) and cultured at 37°C for 24 h. The fresh bacterial suspension was then centrifuged at 8000 r / min for 10 min, and the supernatant was discarded to obtain bacterial cells. The bacterial cells were washed twice with PBS buffer (pH 7.0) and then resuspended until the initial absorbance OD of the bacterial suspension was reached. 600 nm Reserved between 0.5 and 0.6.

[0076] The fresh bacterial broth of Lactobacillus curvature HC1261 after fermentation was homogenized under high pressure using an SPXFLOW homogenizer at a pressure of 1000 bar. After three rounds of high-pressure homogenization, the liquid was placed in an 80°C water bath for 30 minutes to completely kill the live cells in the fermentation broth, thus obtaining the fermentation lysate of Lactobacillus curvature HC1261.

[0077] Lactobacillus curvature CP515, a commercially available product, was used as a control strain. Its bacterial suspension and fermentation lysate were prepared in the same manner as those of Lactobacillus curvature HC1261.

[0078] Four pathogenic bacteria strains—*Prevotella biloba*, *Prevotella timonii*, *Fanny Hesse* vaginalis, and *Prevotella amniotic fluid*—were inoculated at a 1% inoculum into BHI broth (containing 5% bovine serum). After anaerobic incubation at 37°C for 48 h, the bacterial cells were collected by centrifugation at 8000 r / min for 10 min. The bacterial cells were washed twice with PBS buffer (pH 7.0), then resuspended, and the initial absorbance (OD) of the suspension was adjusted. 600 Reserve 0.5~0.6.

[0079] The inhibitory effects of *Lactobacillus curvatureii* HC1261 bacterial suspension and fermentation lysate on *Prevotella bisporus*, *Prevotella timomont*, *Fanny Hesse* of the vagina, and *Prevotella amniotic fluid* were determined using the Oxford cup double-layer plate method. BHI medium with 0.7% agar content was prepared and sterilized. After the medium temperature dropped below 47℃, 0.2% of the mixed pathogenic bacterial suspension was added and shaken well. 7 mL of medium was poured onto the bottom agar plate. After the medium solidified, Oxford cups were placed on top, and 150 μL of the bacterial suspension and fermentation supernatant were added to the wells of each Oxford cup. After incubation at 37℃ for 48 h, the presence of inhibition zones was observed. The diameters of the inhibition zones are shown in Table 3.

[0080] Table 3. Antibacterial effects of different *Lactobacillus curvatureii* strains against vaginal pathogens (unit: mm)

[0081] Table 3 shows that the suspension and fermentation lysate of *Lactobacillus curvatureii* HC1261 significantly inhibited *Prevotella bisporus*, *Prevotella timomont*, *Fanny Hesse* vaginalis, and *Prevotella amniotic fluid*, with inhibition zones exceeding 24.8 mm in diameter. In contrast, the control strain showed weaker inhibitory effects against the target pathogens; the inhibition zones produced by its suspension and fermentation lysate ranged from 4.8 to 8.0 mm in diameter, significantly lower than those of *Lactobacillus curvatureii* HC1261.

[0082] Example 6: Elimination of vaginal pathogenic bacteria biofilm by Lactobacillus curvatureii HC1261 Vaginal Gardnerella vaginalis suspension was prepared in the same manner as in Example 1. Lactobacillus curvularis HC1261 suspension, Prevotella bisporus suspension, Prevotella timontani suspension, vaginal Fanny Hesse suspension and amniotic fluid Prevotella suspension were prepared in the same manner as in Example 5.

[0083] Biofilms of *Gardnerella vaginalis*, *Prevotella biloba*, *Prevotella timomont*, *Fanny Hesse*, and *Prevotella amniotic fluid* were cultured using a well plate method. 300 μL of BHI medium supplemented with 2% sucrose was added to each well of a 24-well plate, followed by inoculation with activated vaginal pathogens at a 2% (v / v) inoculum. The plates were incubated under micro-anaerobic conditions at 37°C for 48 h, resulting in mature biofilms of *Gardnerella vaginalis* and other pathogens appearing on the inner walls and bottom of the wells. The culture medium and unadhered bacterial cells were then discarded, and the plates were washed twice with PBS buffer (pH 7.0). 300 µL of *Lactobacillus curvatureii* HC1261 was added to each well, and the plates were co-cultured for another 24 h. Three replicates were performed for each group, with an equal volume of MRS broth as a control. After culture, the supernatant containing planktonic cells was removed, while maintaining the integrity of the biofilm. The microplate was washed three times with sterile PBS buffer, fixed with methanol for 15 min, and then the microplate was emptied and air-dried for 45 min. The biofilm was stained with crystal violet (CV, 0.1% m / v) for 30 min, and then excess staining was removed. Finally, 300 µL of anhydrous ethanol was added to each well to release the bound crystal violet back into the well. The solution was transferred to a new microplate and its OD value was measured. 600 nm The absorbance at that point.

[0084] Lactobacillus curvature CP515, a commercially available product, was used as a control strain, and its bacterial suspension was prepared in the same manner as that of Lactobacillus curvature HC1261.

[0085] Biofilm elimination effects of two strains of *Lactobacillus curvature* on five vaginal pathogens, such as Figure 7As shown, compared with the control strain, *Lactobacillus curvaturei* HC1261 significantly reduced the elimination of *Gardnerella vaginalis* biofilm by 84.39%; significantly reduced the elimination of *Prevotella bisporus* biofilm by 89.99%; significantly reduced the elimination of *Prevotella timothymosa* biofilm by 80.19%; significantly reduced the elimination of *Fanny Hercetella* biofilm by 88.94%; and significantly reduced the elimination of *Prevotella moniliforme* biofilm in amniotic fluid by 87.32%.

[0086] As can be seen, compared with the control strain, *Lactobacillus curvatureii* HC1261 is significantly more effective in reducing biofilms of vaginal pathogens such as *Gardnerella vaginalis*, *Prevotella diversi*, *Prevotella timothy*, *Fanny Hesse*, and *Prevotella amniotic fluid*. Biofilm formation by vaginal pathogens facilitates their colonization and survival in the vaginal environment, making it difficult to restore the acidic environment. Therefore, inhibiting the formation of vaginal pathogen biofilms is an important pathway to alleviating bacterial vaginosis. This indicates that *Lactobacillus curvatureii* HC1261 can alleviate the symptoms of bacterial vaginosis by inhibiting the formation of vaginal pathogen biofilms.

[0087] Example 7: Determination of Hydrogen Peroxide Production Capacity of Lactobacillus curvature HC1261 A 30% H2O2 stock solution was diluted to 1 mol / L with 100 mmol / L piperazine-N,N'-di-ethanesulfonic acid (PIPES). Then, the 1 mol / L H2O2 solution was diluted with 100 mmol / L PIPES to prepare working solutions of 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, and 100 μmol / L, respectively. 100 μL of each of these working solutions was mixed with 100 μL of 20 mmol / L tetramethylbenzidine (TMB). Finally, 2 μL of horseradish peroxidase (1 mg / mL) was added to the mixture and mixed thoroughly. The mixture was incubated at 16°C for 10 min. Under the catalysis of horseradish peroxidase, H2O2 reacted with TMB to produce a colorimetric reaction. The darker the color of the reaction mixture, the higher the concentration of H2O2. OD was measured. 600 nm And draw the standard curve, such as Figure 8 As shown.

[0088] Activated Lactobacillus curvature HC1261 bacterial culture was inoculated into 10 mL of MRS liquid medium at a ratio of 10% (v / v). After static incubation at 37°C for 21 h, it was then incubated in a shaker at 37°C for 3 h at a rotation speed of 220 r / min. After shaking incubation, the culture was centrifuged at 12000 r / min for 2 min, and 100 μL of the supernatant was added to an EP tube containing 100 μL of 20 mmol / L TMB. After mixing, 2 μL of horseradish peroxidase (1 mg / mL) was added, and the mixture was incubated at 16°C for 10 min to catalyze the colorimetric reaction between TMB and H2O2. The above experimental steps were repeated using commercially available Lactobacillus curvature CP515 as a control strain.

[0089] The colors of the reaction systems of *Lactobacillus curvatureii* HC1261 and the control strain were observed after incubation for 10 min. The reaction system of *Lactobacillus curvatureii* HC1261 was a deep blackish-blue, while the reaction system of the control strain was a light blue. This indicates that the H2O2 production capacity of *Lactobacillus curvatureii* HC1261 is much higher than that of the control strain.

[0090] According to the regression equation y = 0.003x - 0.034 (R²), 2 =0.9896) Measure the OD of the two reaction systems 600 nm The concentration of H2O2 in the fermentation broth was calculated based on the standard curve. The calculated H2O2 yield of *Lactobacillus curvatureii* HC1261 was 1521 μmol / L, while the H2O2 yield of the control strain was 128 μmol / L. The measured values ​​were consistent with the color observation results. H2O2 is a strong oxidant, and its oxidation metabolites, such as H+, are produced by the fermentation of *Lactobacillus curvatureii*. + Free radicals can lower the vaginal pH environment. The absence of H2O2-producing strains in the vagina is closely related to the pathogenesis of bacterial vaginosis; the abundance of H2O2-producing strains in the vagina of healthy women is much higher than that in patients with bacterial vaginosis. The high H2O2 production characteristic of *Lactobacillus curvatureii* HC1261 helps to repair the vaginal acidic environment, which is beneficial to reducing the proliferation of pathogenic bacteria causing bacterial vaginosis and can play a probiotic role in the prevention or treatment of bacterial vaginosis.

[0091] Example 8: Determination of Acid-Producing Capacity of Lactobacillus curvature HC1261 Activated *Lactobacillus curvatureii* HC1261 was inoculated at a rate of 10% into 1 L of MRS liquid medium, and the pH was adjusted to 6.3. The culture was then incubated at 37°C for 36 h. Every 12 h, 10 mL of the bacterial culture was collected to measure the pH and assess the acid-producing capacity of *Lactobacillus curvatureii* HC1261. *Lactobacillus curvatureii* CP515 (a commercially available strain) was used as a control strain, and the above experimental steps were repeated.

[0092] After 36 hours of cultivation, the pH changes of the fermentation broth for both strains are as follows: Figure 9 As shown, the pH values ​​of the fermentation broth of *Lactobacillus curvaturei* HC1261 at 12 h, 24 h, and 36 h were 4.35, 3.54, and 3.06, respectively, while the pH values ​​of the fermentation broth of the control strain were 5.51, 4.44, and 4.23, respectively. The acid-producing capacity of *Lactobacillus curvaturei* HC1261 was significantly higher than that of the control strain. Studies have shown that the vaginal environment of healthy women has a pH of 3.5–4.5, and this acidic environment is unfavorable for the growth and colonization of pathogens causing bacterial vaginosis. The lipid-soluble carboxylic acids produced by lactobacilli can cross the acid-sensitive cell membranes of pathogens in a non-dissociated form, leading to the death of the pathogens. The superior acid-producing capacity of *Lactobacillus curvaturei* HC1261 helps maintain an acidic vaginal environment, inhibits the proliferation of pathogens causing bacterial vaginosis, and promotes the improvement of bacterial vaginosis symptoms.

[0093] Example 9: Adhesion of Lactobacillus curvature HC1261 to HeLa human vaginal epithelial cells HeLa human vaginal epithelial cells were resuscitated and cultured in liquid nitrogen to the required volume. When the cell density reached approximately 80%, they were digested with trypsin into a single-cell suspension and counted using a hemocytometer, with a cell count of 5 × 10⁶ cells / mL. 5 Cells / mL. Then, 500 μL of single-cell suspension was seeded into 24-well plates with cell spreaders at a seeding density of 2.5 × 10⁻⁶ cells / mL. 5 Cells per well. After overnight culture until fully adhered, discard the culture medium, rinse twice with fresh culture medium, and set aside.

[0094] Fresh Lactobacillus curvature HC1261 culture was washed twice with PBS buffer at pH 7.0, then resuspended in an equal volume of RPMI-1640 medium containing 10% fetal bovine serum. The absorbance was adjusted to allow the OD to reach the desired level. 600 nm = Between 0.8 and 1.0. Using commercially available Lactobacillus curvaturei CP515 as a control strain, the above experimental steps were repeated.

[0095] Add 500 μL of bacterial suspension to a prepared 24-well plate containing HeLa human vaginal epithelial cells and co-culture in a carbon dioxide incubator for 2 h; wash three times with PBS buffer at pH 7.0 to remove unadhered bacteria.

[0096] Cell slides were fixed with methanol for 15 min, stained with Giemsa stain for 5 min, washed thoroughly with PBS buffer (pH 7.0), and then transferred to a glass slide for observation under a microscope. The adhesion capacity of the strains was calculated using the formula: Adhesion capacity (CFU / cell) = Total number of bacteria adhering in each well / Total number of cells in each well.

[0097] The adhesion ability of *Lactobacillus curvaturei* HC1261 was 128.53 ± 3.50 CFU / cell, indicating that *Lactobacillus curvaturei* HC1261 has a certain degree of adhesion to HeLa human vaginal epithelial cells. The adhesion ability of the control strain was 7.81 ± 4.25 CFU / cell, indicating that the control strain had poor adhesion ability to HeLa human vaginal epithelial cells. Calculations showed that the adhesion ability of *Lactobacillus curvaturei* HC1261 to HeLa human vaginal epithelial cells was more than 16 times that of the control strain.

[0098] Whether probiotics or pathogens, adhesion and colonization are crucial for their survival in the body. Lactobacilli can achieve specific binding to body cells by expressing adhesion factors such as collagen-binding proteins, adhesion-promoting proteins, and specific glycoproteins. *Lactobacillus curvatureii* HC1261 has an adhesion capacity of 128.53 CFU / cell, which can strongly compete with vaginal pathogens for binding to adhesion receptors on vaginal epithelial cells, forming steric hindrance to prevent pathogen adhesion and colonization. Free bacterial vaginosis pathogens are easily expelled from the body with secretions, achieving an antibacterial effect.

[0099] Example 10: Experimental study on the alleviating effect of Lactobacillus curvaturei HC1261 on bacterial vaginosis in rats. Vaginal Gardnerella vaginalis suspension was prepared using the same method as in Example 1. Lactobacillus HC1261 suspension, Prevotella bisporus suspension, and Fannie Hercetella vaginalis suspension were prepared using the same method as in Example 5. The vaginal Gardnerella vaginalis suspension, Prevotella bisporus suspension, and Fannie Hercetella vaginalis suspension were mixed at a volume ratio of 1:1:1 to prepare a solution with a concentration of 1×10⁻⁶. 9 A mixed bacterial suspension of pathogenic bacteria at CFU / mL was used for animal modeling. The concentration of *Lactobacillus curvatureii* HC1261 bacterial suspension was adjusted to 1×10⁻⁶. 9 CFU / mL available for use.

[0100] Twenty-four SPF-grade female Wistar rats, weighing 150–200 g, were used in the experiment. Environmental conditions for animal husbandry and management included: room temperature 20–26°C, daily temperature difference ≤4°C, relative humidity 40–70%, and a light / dark cycle of 12 h / 12 ​​h. Animals were housed in standard rat cages, six rats per cage. All rats were fed a standard commercially available diet with free access to food and water.

[0101] After the rats completed their acclimatization period, they were randomly divided into four groups: a control group, a modeling group, an experimental group, and a control group. On days 0 and 3, rats in all groups except the control group were subcutaneously injected with 100 μL of estradiol valerate (5 μg / μL) to induce estrus. Starting from day 3, rats in all groups except the control group were vaginally inoculated with 20 μL of a mixed suspension of pathogenic bacteria for 5 consecutive days, ending on day 7. During inoculation, the tip of the pipette was inserted into the vagina to a depth of about 5 mm. After inoculation, the mice were inverted for 1-2 minutes to prevent the bacterial solution from flowing out. Starting from day 8, rats in the experimental group were vaginally inoculated once daily with 20 μL of Lactobacillus curvature HC1261 bacterial suspension, rats in the control group were vaginally inoculated once daily with 20 μL of metronidazole (100 mg / mL), and rats in the modeling group were inoculated once daily with 20 μL of sterile saline, for 12 consecutive days, ending on day 20. The control group received no treatment.

[0102] I. Detection of pathogenic bacteria and Lactobacillus curvature colonization in the rat vagina After the experiment, the rat vagina was rinsed with 500 μL of sterile saline and the vaginal irrigation fluid was collected. DNA from the rat vaginal flora samples was obtained using the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302). Subsequently, the changes in the load of *Lactobacillus curvaturei*, *Gardnerella vaginalis*, *D. pubescens*, *Fanny Hercetella vaginalis*, and *Ureaplasma urealyticum* in the rat vagina were quantified using real-time quantitative PCR (qPCR). The primers used are shown in Table 4.

[0103] Table 4 qPCR primer sequences

[0104] qPCR reactions were performed using a Thermo Fisher Scientific ABI 7500 real-time quantitative PCR instrument. The total reaction volume was 20 μL, containing 10 μL of 2×Fastfire qPCR premix, 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 0.4 μL of 50×ROX reference dye, 50 ng of gDNA, and double-distilled water (ddH2O) to make up the difference. One pre-denaturation cycle was performed at 95℃ for 1 min. The PCR reaction used a two-step method for a total of 40 cycles: denaturation at 95℃ for 5 s and annealing / extension at 60℃ for 15 s, during which fluorescence signals were collected. After the PCR reaction, melting curve analysis was performed to rule out non-specific amplification and primer dimers. The fluorescence threshold cycle value (Ct value) reached by the sample was recorded, and the result was analyzed according to Log. 10 The copy number / μL and Ct value standard curve were used to calculate the sample copy number. The sample copy number was used to replace the load of the target microorganism in the sample. Five copies of each sample were made.

[0105] Changes in the load of *Lactobacillus curvularia*, *Gardnerella vaginalis*, *D. prusporum*, *Fanny Hesse*, and *Ureaplasma urealyticum* in the vagina of rats are as follows: Figure 10 As shown.

[0106] For the load of Lactobacillus curlis HC1261, the control group was 11.69 log [value missing]. 10 Copy number / μL, modeling group is 0.18 Log 10 Copy number / μL, experimental group: 35.80 Log 10 Copy number / μL, control group was 0.30 Log 10 Copy number / μL. Compared with the control group, the load of *Lactobacillus curvaturei* HC1261 in the modeling group decreased by 98.46%, which was statistically significant, indicating that the beneficial *Lactobacillus curvaturei* load was significantly reduced in the rat model of bacterial vaginosis. Compared with the modeling group, the load of *Lactobacillus curvaturei* HC1261 in the experimental group and the control group increased by 197.9-fold and 0.68-fold, respectively, which were also statistically significant, indicating that *Lactobacillus curvaturei* HC1261 successfully colonized in the rat model of bacterial vaginosis, and the abundance of *Lactobacillus curvaturei* increased significantly. Antibiotic treatment failed to restore the abundance of *Lactobacillus curvaturei*.

[0107] For the vaginal Gardnerella vaginalis load, the control group had a load of 1.43 log. 10 Copy number / μL, modeling group was 23.71 Log 10 Copy number / μL, experimental group: 2.89 Log 10 Copy number / μL, control group was 13.72 Log 10 Copy number / μL. Compared with the control group, the Gardnerella vaginalis load in the modeling group increased by 15.56 times, which was statistically significant, indicating that the rat model of bacterial vaginosis was successfully established. Compared with the modeling group, the Gardnerella vaginalis load in the experimental group and the control group decreased by 87.80% and 42.12%, respectively, which were also statistically significant.

[0108] For the *Prevotella* load in the double-channel group, the control group had a load of 6.14 log. 10 Copy number / μL, modeling group was 33.54 Log 10 Copy number / μL, experimental group: 8.04 Log 10 Copy number / μL, control group was 22.78 Log 10 Copy number / μL. Compared with the control group, the Prevotella bisporus load in the modeling group increased by 4.46-fold, which was statistically significant, indicating that the rat model of bacterial vaginosis was successfully established. Compared with the modeling group, the Prevotella bisporus load in the experimental group and the control group decreased by 76.04% and 32.08%, respectively, which were also statistically significant.

[0109] For the vaginal Fanny Hesse bacterial load, the control group had a load of 7.41 log. 10 Copy number / μL, modeling group was 45.11 Log. 10 Copy number / μL, experimental group: 9.88 Log 10 Copy number / μL, control group was 33.22 Log 10 Copy number / μL. Compared with the control group, the vaginal Fannie H. N. necrotizing bacteria load in the modeling group increased by 5.08-fold, which was statistically significant, indicating that the rat model of bacterial vaginosis was successfully established. Compared with the modeling group, the vaginal Fannie H. N. necrotizing bacteria load in the experimental group and the control group decreased by 78.10% and 26.36%, respectively, which were also statistically significant.

[0110] For the Ureaplasma urealyticum viral load, the blank group was 3.63 Log. 10 Copy number / μL, modeling group 10.32 Log 10 Copy number / μL, experimental group: 4.05 Log 10 Copy number / μL, control group was 9.90 Log 10 Copy number / μL. Compared with the control group, the Ureaplasma urealyticum load in the modeling group increased by 1.84 times, which was statistically significant, indicating that Ureaplasma urealyticum exhibited a high load in the rat model of bacterial vaginosis. Compared with the modeling group, the Ureaplasma urealyticum load in the experimental group decreased by 60.73%, which was statistically significant, indicating that Lactobacillus HC1261 could reduce the Ureaplasma urealyticum load while treating bacterial vaginosis in rats. Compared with the modeling group, the Ureaplasma urealyticum load in the control group rats decreased by only 4.09%, which was not statistically significant, indicating that metronidazole was not effective in killing Ureaplasma urealyticum in treating bacterial vaginosis and could not reduce the risk of urethritis, cervicitis, and pelvic inflammatory disease caused by high Ureaplasma urealyticum load.

[0111] Therefore, a rat model of bacterial vaginosis was successfully established using a mixed bacterial suspension of pathogenic bacteria. The use of *Lactobacillus curvaturei* HC1261 not only reduced the abundance of *Gardnerella vaginalis*, *D. brevis*, and *Fanny H. Neisseria vaginalis* in the rat vagina, but also reduced the high load of *Ureaplasma urealyticum*, with better results than the antibiotic metronidazole. Furthermore, the abundance of *Lactobacillus curvaturei* in rats with bacterial vaginosis significantly increased after inoculation with *Lactobacillus curvaturei* HC1261, while in the control group, the abundance of *Lactobacillus curvaturei* failed to recover after metronidazole treatment, the load of *Ureaplasma urealyticum* did not decrease, and the normal vaginal flora remained absent.

[0112] II. Determination of Vaginal Sialidase Activity in Rats Women with bacterial vaginosis showed higher sialidase activity in their vaginal douches than healthy women. Sialidase can disrupt the protective mucus layer of the vaginal epithelium, exacerbating the adhesion of pathogens to the vaginal epithelium and promoting the formation of pathogenic biofilms. After the experiment, vaginal douches from mice were collected, centrifuged at 3600×g for 5 min at 4°C, and cell debris was removed. 10 μL of the centrifuge was then used to determine sialidase activity according to the enzyme-linked immunosorbent assay (ELISA) kit instructions.

[0113] Sialidase activity in vaginal irrigation fluid of rats in each group was as follows: Figure 11 As shown, compared with the control group, the sialidase activity in the modeling group reached 181.62 U / L, an increase of 115.97%, which was statistically significant. This indicates that the rat model of bacterial vaginosis was successfully established. The sialidase activities in the experimental group and the control group decreased by 45.44% and 31.57%, respectively, with statistically significant differences. This demonstrates that the use of *Lactobacillus curvaturei* HC1261 and metronidazole can improve the severity of bacterial vaginosis in rats, and that *Lactobacillus curvaturei* HC1261 is as effective as the antibiotic metronidazole in inhibiting sialidase activity.

[0114] III. Determination of Inflammatory Factors in Rat Vaginal Tissue Cells After the experiment, rat vaginal tissue was dissected, and the vaginal tissue sample (20 mg) was placed in 200 μL of RIPA lysis buffer containing a 2% (v / v) mixture of protease inhibitors and a 2% (v / v) mixture of phosphatase inhibitors. The tissue was homogenized using a high-throughput tissue homogenizer (65 Hz, 45 s / time, 8 times) to obtain a vaginal tissue homogenate. The vaginal tissue homogenate was centrifuged at 12000×g for 15 min at 4°C, and the vaginal tissue supernatant was obtained. The concentrations of IL-8, IL-10, and IL-1β were determined according to the kit instructions.

[0115] The levels of inflammatory cytokines IL-10, IL-1β, and IL-8 in each group of rats are as follows: Figure 12 As shown in the figure, for the anti-inflammatory cytokine IL-10, the level in the control group was 269.11 pg / mL. Compared with the control group, the IL-10 level in the modeling group decreased by 84.31%, down to 42.23 pg / mL, which was statistically significant. The IL-10 levels in the experimental group and the control group recovered to 236.00 pg / mL and 213.43 pg / mL, respectively, which were 4.58-fold and 4.05-fold higher than those in the modeling group, respectively, which were statistically significant.

[0116] Regarding the pro-inflammatory cytokine IL-1β, the level in the control group was 71.25 pg / mL. Compared with the control group, the IL-1β level in the modeling group increased 5.38-fold to 455.27 pg / mL, which was statistically significant. The IL-1β levels in the experimental group and the control group recovered to 206.10 pg / mL and 122.12 pg / mL, respectively, which were 54.73% and 73.18% lower than those in the modeling group, respectively, which were statistically significant.

[0117] Regarding the pro-inflammatory cytokine IL-8, the level in the control group was 83.09 pg / mL. Compared with the control group, the IL-8 level in the modeling group increased 3.66-fold to 387.15 pg / mL, which was statistically significant. The IL-8 levels in the experimental group and the control group recovered to 258.91 pg / mL and 148.24 pg / mL, respectively, which were 33.12% and 61.71% lower than those in the modeling group, respectively, which were statistically significant.

[0118] The levels of pro-inflammatory and anti-inflammatory cytokines in the modeling group rats showed significant negative changes compared to the control group, indicating the successful establishment of the bacterial vaginosis rat model. After intervention with *Lactobacillus curvaturei* HC1261 and metronidazole, the levels of inflammatory factors in the rats tended to normalize. In particular, *Lactobacillus curvaturei* HC1261 showed a greater improvement effect on anti-inflammatory cytokines than metronidazole. Therefore, *Lactobacillus curvaturei* HC1261 can regulate the tissue inflammation state caused by vaginal pathogens such as *Gardnerella vaginalis*, *Prevotella vaginalis*, and *Fanny Hercetella vaginalis*, restoring it to normal levels and promoting the improvement of bacterial vaginosis.

[0119] In summary, *Lactobacillus curvatureii* HC1261, as a strain derived from vaginal secretions of healthy women, can effectively adhere to and colonize the surface of HeLa vaginal epithelial cells, exhibiting significant hydrogen peroxide and acid-producing capabilities, which is beneficial for restoring the vaginal acidic environment. Furthermore, *Lactobacillus curvatureii* HC1261 can effectively inhibit the growth and biofilm formation of vaginal pathogens such as *Gardnerella vaginalis*, *Prevotella diffusa*, *Prevotella timothymosa*, *Fanny H. sec.*, and *Prevotella amniotic fluid*. In a rat bacterial vaginosis model constructed using *Gardnerella vaginalis*, *Prevotella diffusa*, and *Fanny H. sec.*, *Lactobacillus curvatureii* HC1261 not only effectively reduced the viral load of vaginal pathogens and *Ureaplasma urealyticum*, but also colonized the rat vaginal environment, bringing the rat vaginal flora closer to normal. Although metronidazole also inhibited pathogens, it failed to restore the *Lactobacillus curvatureii* viral load and reduce the *Ureaplasma urealyticum* viral load in rats. Furthermore, *Lactobacillus curvature* HC1261 can reduce sialidase activity, increase the level of anti-inflammatory cytokines, and decrease the level of pro-inflammatory cytokines. It can effectively improve bacterial vaginosis caused by *Gardnerella vaginalis*, *Diprelia bisporus*, and *Fanny Hesse*, and reduce urethritis, cervicitis, and pelvic inflammatory disease caused by *Ureaplasma urealyticum*. Therefore, *Lactobacillus curvature* HC1261 can be formulated into bactericides and antibacterial drugs and added to feminine hygiene products such as vulvar washes, sanitary napkins, and wet wipes, which will help prevent or treat bacterial vaginosis and maintain vaginal health in women.

[0120] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. An application of a microbial agent prepared from Lactobacillus curvature HC1261 for improving female reproductive health, characterized in that, Lactobacillus curvaturei ( Lactobacillus crispatus HC1261 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27355. Microbial agents used to improve women's reproductive health can reduce the load of Ureaplasma urealyticum in the vagina.

2. The application as described in claim 1, characterized in that, Antibiotics used to improve women's reproductive health can inhibit pathogenic bacteria in the vagina.

3. The application as described in claim 1, characterized in that, Microbial agents used to improve women's reproductive health can eliminate the biofilm of pathogenic bacteria in the vagina.

4. The application as described in claim 1, characterized in that, Microbial agents used to improve women's reproductive health can reduce the load of pathogenic bacteria in the vagina.

5. The application as described in any one of claims 2 to 4, characterized in that, Vaginal pathogens include Gardnerella vaginalis ( Gardnerella vaginalis ), Bilopredominus ( Prevotella bivia ), Timoprevotella ( Prevotella timonensis ), Fanny Hesse (vaginis) Fannyhessea vaginae ) and / or amniotic fluid prevotella ( Prevotella amnii ).

6. The application as described in claim 1, characterized in that, Microbial agents used to improve women's reproductive health can reduce sialidase activity.

7. The application as described in claim 1, characterized in that, Microbial agents used to improve women's reproductive health can increase the level of the anti-inflammatory cytokine IL-10 and decrease the level of the pro-inflammatory cytokine IL-1β and IL-8.

8. A composition for improving vaginal inflammation, characterized in that, Including Lactobacillus curvature ( Lactobacillus crispatus Live bacteria, inactivated cells and / or fermentation products of Lactobacillus HC1261 were deposited on May 16, 2023 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27355.

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