Lactobacillus crispatus and application thereof

Abstract

The application discloses a lactobacillus crispatus and application thereof. The lactobacillus crispatus in the application is lactobacillus crispatus OH139 with a preservation number of GDMCC No.63856. The lactobacillus crispatus newly developed in the application has inhibiting effect on the growth of various common harmful bacteria in the genital tract, can effectively inhibit the growth of external pathogenic bacteria and conditional pathogenic bacteria, has strong adhesion to the vagina and the urinary tract, ensures the colonization of the strain on the vaginal epithelium, and has good drug resistance. The lactobacillus crispatus in the application can effectively improve the microecological environment of the female genital tract, and provides a new scheme and way for improving the female genital tract flora and infection prevention.

Description

Technical Field

[0001] This application relates to the field of Lactobacillus curvaturei technology, and in particular to a novel Lactobacillus curvaturei strain and its applications. Background Technology

[0002] Lactobacillus is closely related to human life and is one of the beneficial microorganisms widely used in food fermentation, industrial lactic acid fermentation and medical and health care.

[0003] Lactobacilli are the main colonizing bacteria in the female reproductive tract. Currently, the main endemic bacteria in the reproductive tract of healthy women include *Lactobacillus crispatus*, *Lactobacillus johnsonii*, and *Lactobacillus gasseri* (Fettweis et al., 2019; Jie et al., 2022; Serrano et al., 2019; Zhu et al., 2022). Various studies have clearly indicated that supplementing with *Lactobacillus crispatus* can reduce the proportion of various harmful bacteria in the reproductive tract, improve the reproductive tract microecology, and alleviate clinical manifestations caused by infections (Antonio et al., 2009; Cohen et al., 2020; Ngugi et al., 2011). In particular, a randomized, double-blind trial in the United States also confirmed that supplementing with *Lactobacillus crispatus* can, to some extent, reduce the recurrence rate of bacterial vaginosis (Cohen et al., 2020).

[0004] Currently, although there are lactobacillus products on the market for improving the female reproductive tract, they generally suffer from three problems: 1. Lactobacillus strains are generally sourced from abroad, and related research data is opaque, making it impossible for consumers to verify their authenticity. 2. Different lactobacillus strains exhibit heterogeneity, and there is no clear research data to support whether foreign strains are suitable for domestic women. 3. The strains used are diverse, lacking accurate understanding of the types of lactobacillus in the female reproductive tract, and exaggerating the research conclusions of publicly published literature.

[0005] In general, there is still a lack of more targeted and applicable lactobacillus products. Developing new lactobacillus products to improve the microecological environment of the female reproductive tract remains an important research topic in this field. Summary of the Invention

[0006] The purpose of this application is to provide a novel Lactobacillus curvature and its applications.

[0007] The following technical solution is adopted in this application:

[0008] One aspect of this application discloses a strain of *Lactobacillus curlis*, specifically *Lactobacillus curlis* OH139, with accession number GDMCC No. 63856 (the strain name used during patent accession was RS-MRS-139). It should be noted that this application incidentally isolated a novel strain of *Lactobacillus curlis* from a healthy female reproductive tract secretion sample. This strain exhibits inhibitory effects on the growth of various common harmful bacteria in the reproductive tract and demonstrates good drug resistance. The experimental number is OH139, and the accession number is GDMCC No. 63856. This *Lactobacillus curlis* provides a new approach and method for improving the microecological environment of the female reproductive tract and for the treatment and prevention of related diseases.

[0009] Another aspect of this application discloses a microbial agent containing the Lactobacillus curvature of this application.

[0010] Preferably, each dose of the bacterial agent contains at least 2 × 10⁻⁶ live *Lactobacillus cristata* of this application. 8 CFU.

[0011] Another aspect of this application discloses the use of the present application's *Lactobacillus curvaturei* or the present application's bacterial agent in the preparation of products that improve the microecological environment of the female reproductive tract.

[0012] Another aspect of this application discloses a product for improving the microecological environment of the female reproductive tract, wherein the active ingredient of the product includes the Lactobacillus curvature of this application or the bacterial agent of this application.

[0013] Preferably, the product of this application is a powder or tablet.

[0014] Preferably, the product of this application also contains pharmaceutically acceptable excipients.

[0015] Preferably, the product of this application includes 25-35 parts by weight of bacterial powder, 20-30 parts by weight of fructooligosaccharides, 20-30 parts by weight of galactooligosaccharides, 50-80 parts by weight of additives, and 60-100 parts by weight of packaging materials.

[0016] Preferably, the additive is at least one of monosodium glutamate, lysozyme, and skim milk.

[0017] Preferably, the packaging material is at least one of silicon dioxide and magnesium stearate.

[0018] Preferably, in the product of this application, each product contains at least 2 × 10⁻⁶ live Lactobacillus curvaturei. 8 CFU.

[0019] The beneficial effects of this application are as follows:

[0020] The newly developed *Lactobacillus curvatureus* exhibits inhibitory effects on the growth of various common harmful bacteria in the reproductive tract, effectively suppressing the growth of exogenous pathogens and opportunistic pathogens. It also demonstrates strong adhesion to the vagina and urinary tract, ensuring colonization of the strain on the vaginal epithelium. Furthermore, it exhibits good drug resistance. This *Lactobacillus curvatureus* effectively improves the microecological environment of the female reproductive tract, providing a new approach and strategy for improving the female reproductive tract flora and preventing infections. Attached Figure Description

[0021] Figure 1 This is the result of anaerobic culture of the isolated strains streaked on MRS solid plates in the embodiments of this application;

[0022] Figure 2 These are the microscopic observation results of the isolated strains in the embodiments of this application;

[0023] Figure 3 These are the antibacterial test results of Lactobacillus curvature OH139 in the embodiments of this application;

[0024] Figure 4 These are the acid resistance test results of Lactobacillus curvature OH139 in the embodiments of this application;

[0025] Figure 5 This refers to the hemolysis test results of Lactobacillus curvature OH139 in the embodiments of this application;

[0026] Figure 6 This is a flowchart illustrating the construction of a mouse bacterial vaginosis model in this application embodiment.

[0027] The Lactobacillus curvature OH139 of this application was deposited on October 24, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; accession number: GDMCC No. 63856; the strain name used for patent deposit was: RS-MRS-139 (i.e., OH139). Detailed Implementation

[0028] Lactobacillus curvature is a major strain inherent in the female reproductive tract. This application study discovered a new strain of Lactobacillus curvature, namely Lactobacillus curvature OH139, which can inhibit a variety of common harmful bacteria in the reproductive tract and has good drug resistance.

[0029] The *Lactobacillus curvature* OH139 of this application has the following properties:

[0030] (1) The Lactobacillus curvature OH139 of this application has good inhibitory effects on Gardnerella vaginalis ATCC14018, Fannyhessea vaginae DSM 15829, Prevotella bivia DSM 20514, Streptococcus agalactiae ATCC 27956, Streptococcus anginosus ATCC 33397 and Candida albicans ATCC 10231.

[0031] (2) The Lactobacillus curvature OH139 of this application is drug resistant and can grow normally in culture media containing commonly used drugs for reproductive tract infections such as metronidazole, tinidazole, clindamycin, ofloxacin or ciprofloxacin.

[0032] (3) The Lactobacillus curvature OH139 of this application has a high lactic acid production capacity.

[0033] (4) The Lactobacillus curvature OH139 of this application has a high efficiency in producing hydrogen peroxide.

[0034] The *Lactobacillus curvature* OH139 of this application can be formulated into powder or tablet products for improving the microecological environment of the female reproductive tract.

[0035] The preparation methods for powder or tablet products include:

[0036] (1) Bacterial strain resuscitation: The bacterial strain is resuscitated on MRS medium plates;

[0037] (2) The strain is inoculated into a liquid fermentation medium and cultured to obtain a fermentation broth;

[0038] (3) Centrifugation: The bacterial cells were separated using centrifugation technology and centrifuged at a low temperature of 4°C.

[0039] (4) After obtaining the bacterial precipitate, resuspend the bacterial cells;

[0040] (5) Using freeze-drying technology, freeze-dried bacterial powder was obtained;

[0041] (6) Combined with excipients, the product is obtained, and then packaged to obtain powder.

[0042] For tablet products, the lyophilized bacterial powder is combined with the excipients required for tablets to form a probiotic tablet raw material mixture. The mixture is then injected into a mold mechanism and extruded to form the tablet product.

[0043] The powder product of this application can be used for daily washing and care, and the tablet product can be placed directly into the vagina to balance the reproductive tract flora, prevent and improve problems such as increased secretions, fishy smell, and vulvar itching caused by reproductive tract infections, without side effects.

[0044] The present application will now be described in detail with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the application.

[0045] Example

[0046] This case presents a novel Lactobacillus crispatus OH139 strain that improves the reproductive health of Chinese women. This Lactobacillus crispatus OH139 strain was isolated from vaginal secretions of healthy women and has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No. 63856 (RS-MRS-139).

[0047] The culture medium involved in this example is as follows:

[0048] MRS solid medium (g / L): peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, diamine citrate 2.0 g / L, Tween-80 1.0 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.29 g / L, calcium carbonate 20.0 g / L, agar 15.0 g / L.

[0049] MRS liquid culture medium (g / L): peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, diamine citrate 2.0 g / L, Tween-80 1.0 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.29 g / L, calcium carbonate 20.0 g / L.

[0050] Experiment 1: Isolation and identification of Lactobacillus curvatureii strain OH139

[0051] 1. Isolation of Lactobacillus curvatureii strain OH139

[0052] Lactobacillus curvature was isolated from vaginal secretions of healthy women. Vaginal secretions were collected from the posterior fornix of the vagina using a vaginal swab. The vaginal swab was placed in ESWAB transport and preservation solution (Copan) and vortexed to mix. The mixture was then serially diluted with sterile PBS. Appropriate serial dilutions were then plated onto MRS solid medium; in this case, 10...2 The diluted solution was spread on a plate and anaerobically incubated at 37°C for 48 hours. Typical colonies were picked and streaked for isolation. Single colonies were picked and transferred to MRS liquid medium for enrichment. The bacterial culture was preserved and frozen at -80°C.

[0053] 2. Identification of Lactobacillus curvatureii OH139 strain

[0054] (1) Morphological identification

[0055] The isolated strains were streaked onto MRS solid plates and cultured anaerobically. The results are as follows: Figure 1 As shown, the colonies are approximately 2-3 mm in diameter, grayish-white, round, opaque, with a raised center, and regular edges, exhibiting stable size and shape. Gram staining of the smear yielded the following results: Figure 2 As shown, this bacterium is Gram-positive, non-flagellated, short rod-shaped, and can form long chains; it does not spore.

[0056] (2) Identification of 16S rDNA gene sequence

[0057] The 16S rDNA of Lactobacillus was amplified using PCR, and the 16S rRNA of Lactobacillus was sequenced. The obtained bacterial sequence was analyzed by BLAST in the NCBI database. The results showed that the strain was Lactobacillus crispatus, and the strain was named Lactobacillus crispatus OH139.

[0058] The 16S rDNA sequence of Lactobacillus curvature OH139 strain is shown in Seq ID No.1.

[0059]

[0060] Experiment 2: Biochemical reaction test of Lactobacillus curvature OH139 strain

[0061] 1. Catalase experiment

[0062] Pick a single colony from an MRS plate, place it on a clean glass slide, and add a few drops of 0.3% hydrogen peroxide solution. The appearance of a large number of bubbles immediately indicates a positive (+) result, while the absence of bubbles indicates a negative (-) result.

[0063] 2. Gelatin liquefaction experiment

[0064] Pick a single colony from an MRS plate using an inoculation needle, puncture it into gelatin solid medium, and puncture to a depth of about 2 / 3 of the culture medium. Incubate anaerobically at 20°C for 7 days. Before observation, place it at 4°C for 30 minutes, then remove it to observe the results. If the gelatin does not coagulate partially or completely, it is a positive (+) result; if the gelatin still coagulates completely, it is a negative (-) result.

[0065] 3. Hydrogen sulfide experiment

[0066] Pick a single colony from an MRS plate using an inoculation needle, puncture and inoculate it into lead acetate medium, and incubate anaerobically at 37°C for 24-48 hours. Observe whether a black precipitate appears. If the medium turns black, it is a positive (+) result; if the medium does not change color, it is a negative (-) result.

[0067] 4. Nitrate Reduction Experiment

[0068] Pick a single colony from an MRS plate using an inoculation needle and inoculate it into nitrate medium. Incubate anaerobically at 37°C for 1–4 days. Add 0.1 ml of a mixture of equal volumes of reagents A and B to a test tube and immediately observe the color change of the liquid in the test tube. A red color indicates a positive (+) result, while no color reaction after adding the reagents indicates a negative (-) result.

[0069] The biochemical test results of Lactobacillus curvature OH139 are shown in Table 1.

[0070] Table 1. Biochemical test results of Lactobacillus curvature OH139

[0071]

[0072] The results in Table 1 show that Lactobacillus curvature OH139 does not contain catalase, cannot decompose gelatin, does not reduce nitrate, and does not produce hydrogen sulfide.

[0073] Experiment 3: Detection of antibacterial activity of Lactobacillus curvature OH139

[0074] 1. Detection of lactic acid production capacity of Lactobacillus curvature OH139

[0075] Inoculation concentration: 2×104 10 μL of Lactobacillus curvature OH139 bacterial culture (CFU / mL) was added to MRS liquid medium and anaerobic cultured at 37℃ for 36 h. After centrifugation, the culture supernatant was collected, and the lactic acid (LD) content in the bacterial fermentation broth was detected using a lactic acid (LD) test kit (Nanjing Jiancheng, catalog number A019-2-1).

[0076] The results showed that the total lactic acid content in the fermentation broth of Lactobacillus curvature OH139 was 23.61 mg / mL.

[0077] 2. Detection of hydrogen peroxide production capacity of Lactobacillus curvature OH139

[0078] Lactobacillus curli OH139 was spread onto MRS agar plates containing 0.25 mg / mL 3,3',5,5'-tetramethylbenzidine and 0.01 mg / mL horseradish peroxidase, and anaerobically cultured at 37°C for 48 hours. The plates were then removed, and the bacteria were exposed to air. Colony color changes were observed, and the ability of the strain to produce hydrogen peroxide was assessed based on the time required for color change. The scoring criteria for hydrogen peroxide production by the tested strains were: high (change time <10 min), medium (change time 10–20 min), low (change time >20 min), and none (no blue production). Lactobacillus acidophilus ATCC 33820 was used as a positive control.

[0079] The results of the hydrogen peroxide production capacity determination of *Lactobacillus curvatureii* OH139 and *Lactobacillus acidophilus* ATCC 33820 in this example are shown in Table 2.

[0080] Table 2 Results of hydrogen peroxide capacity test

[0081]

[0082] The experimental results show that Lactobacillus curvature OH139 has a strong ability to produce hydrogen peroxide.

[0083] 3. Antibacterial activity test of Lactobacillus curvature OH139

[0084] The antibacterial activity of *Lactobacillus curvature* OH139 against *Staphylococcus aureus* (ATCC 6538), *Gardnerella vaginalis* (ATCC 14018), *Fannyhessea vaginae* (DSM 15829), *Prevotella bivia* (DSM 20514), *Escherichia coli* (ATCC 25922), *Streptococcus agalactiae* (ATCC 27956), *Streptococcus anginosus* (ATCC 33397), and *Candida albicans* (ATCC 10231) was detected by disk diffusion method.

[0085] The specific steps are as follows:

[0086] In a 96-well plate, equal volumes of Staphylococcus aureus, Gardnerella vaginalis, Atobacillus vaginalis, Prevotella bisporus, Escherichia coli, Streptococcus agalactiae, Streptococcus pharyngitis, and Candida albicans were added to each well, for a total of 10 wells. 5 Each bacterial count was mixed with different proportions (10%, 20%, 30%) of Lactobacillus culture supernatant, with a total volume of 200 μL. Culture medium was used instead of Lactobacillus supernatant as a positive control.

[0087] The 96-well plates were incubated at 37°C for 12 hours. After incubation, the bacterial cultures from each group were serially diluted 10-fold using the CFU counting method and spotted onto solid culture media. Once the colonies had grown to a suitable size for counting, the colonies were counted and analyzed.

[0088] The results are as follows Figure 3 As shown, Figure 3 Among them, Staphylococcus aureus (a), Gardnerella vaginalis (b), Atobora vaginalis (c), Prevotella ilex (d), Escherichia coli (e), Streptococcus agalactiae (f), Streptococcus pharyngis (g), and Candida albicans (h) were included, with p < 0.0001.

[0089] from Figure 3 The results showed that Lactobacillus OH139 could inhibit the growth of a variety of common harmful bacteria in the reproductive tract, including Staphylococcus aureus (a), Gardnerella vaginalis (b), Atobacillus vaginalis (c), Prevotella ileum (d), Escherichia coli (e), Streptococcus agalactiae (f), Streptococcus pharyngitis (g), and Candida albicans (h).

[0090] Experiment 4: Acid resistance test of Lactobacillus curvature OH139

[0091] Prepare a lactic acid gradient solution (NaCl 0.2%): pH 2-6, filter to sterilize. In this example, tests were conducted at pH 2, 3, 4, 5, and 6. Lactobacillus curvature OH139 was cultured to the logarithmic growth phase, and the OD of the bacterial culture was measured. 600 The bacterial count was recorded, and CFU counts were performed simultaneously, with the bacterial count at this point serving as a control. 1000 μL of bacterial culture was added to 9000 μL of lactate gradient solution and incubated at 37°C for 1.5 h, followed by CFU counting.

[0092] The results are as follows Figure 4 As shown, Lactobacillus curvature OH139 has a strong acid resistance and can exist stably in the acidic environment of the vagina to exert its function.

[0093] Experiment 5: Adhesion ability of Lactobacillus curvature OH139

[0094] Adhesion ability is an important component of the probiotic's beneficial properties. Strong adhesion ability helps probiotics colonize the human body, compete for space with pathogens, and prevent pathogen invasion. Vaginal probiotic strains should have good adhesion to vaginal and urinary tract epithelium, thereby preventing the adhesion and growth of urogenital pathogens.

[0095] 1. Adhesion of Lactobacillus curvatureii OH139 strain to HeLa cells

[0096] HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences. HeLa cells were cultured in cell culture flasks until the cell coverage reached approximately 80%. Then, 1 mL of 0.25% trypsin-EDTA solution was added for 2 min of digestion. The cells were gently detached from the culture flask by pipetting, and 3 mL of cell culture medium (DMEM high-glucose medium + 10% FBS + 1% P / S) was added and the cells were mixed by pipetting. 2 mL of the cell suspension (~4×10⁻⁶ cells / mL) was then collected. 4 ( / mL) was inoculated into 6-well plates and cultured at 37°C in a 5% CO2 incubator until a monolayer was formed.

[0097] Take the culture medium of Lactobacillus curvature OH139, centrifuge to remove the supernatant, and wash the bacterial precipitate three times with PBS. Resuspend the bacterial cells in an equal volume of DMEM high-glucose medium (Gibco), spread the bacterial suspension on MRS solid plates, and count CFU. Use the adhesion of Lactobacillus delbrueckii DM8909 as a control group.

[0098] Before the adhesion experiment, the culture medium was aspirated from the cell culture plate, and the cells were washed three times with 2 mL of PBS in each well. Then, 2 mL of bacterial suspension was added to each well, and the cell culture plate was gently shaken to ensure that the lactic acid bacteria were fully and evenly dispersed. The cell culture plate was then placed in a 37°C, 5% CO2 incubator for 2.5 h for adhesion.

[0099] After adhesion, the culture medium containing bacteria was aspirated, and 2 mL of PBS was added to each well to wash the cells three times. Then, 0.5 mL of 0.25% trypsin-EDTA solution was added to digest for 2 min. The digestion solution was aspirated, and 1 mL of PBS solution was added to gently pipette the cells to detach them from the bottom of the culture plate. 1 mL of 0.05% Triton X-100 was added to lyse the cells and prepare a bacterial suspension. The suspension was then spread on MRS solid plates for CFU counting.

[0100] The formula for calculating adhesion rate is as follows:

[0101] Adhesion rate = Strain survival rate (%) = Nl ÷ N0 × 100%;

[0102] In the formula, N1 is the number of viable bacteria (CFU / mL) after the strain adhesion treatment; N0 is the number of viable bacteria (CFU / mL) before the strain adhesion.

[0103] 2. Adhesion of Lactobacillus curvatureii OH139 strain to human bladder transitional cell carcinoma T24 cells.

[0104] Human bladder transitional cell carcinoma T24 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. T24 cells were cultured in cell culture flasks. When the cell coverage reached approximately 80%, 1 mL of 0.25% trypsin-EDTA solution was added for digestion for 2 min. The cells were gently detached from the culture flask by pipetting, and 3 mL of cell culture medium (McCoy's 5a + 10% FBS + 1% P / S) was added and the cells were mixed by pipetting. 2 mL of the cell suspension (~4×10⁻⁶) was then collected. 4 The cells were seeded in 6-well plates at 37°C and cultured at 37°C in a 5% CO2 incubator until a monolayer was formed.

[0105] Take the culture medium of Lactobacillus curvature OH139, centrifuge to remove the supernatant, and wash the bacterial precipitate three times with PBS. Resuspend the bacterial cells in an equal volume of McCoy's 5a medium, spread the bacterial suspension on MRS solid plates, and count CFU. Use the adhesion of Lactobacillus de Germanis DM8909 as a control group.

[0106] Before the adhesion experiment, the culture medium was aspirated from the cell culture plate, and the cells were washed three times with 2 mL of PBS in each well. Then, 2 mL of bacterial suspension was added to each well, and the cell culture plate was gently shaken to ensure that the lactic acid bacteria were fully and evenly dispersed. The cell culture plate was then placed in a 37°C, 5% CO2 incubator for 2.5 h for adhesion.

[0107] After adhesion, the culture medium containing bacteria was aspirated, and 2 mL of PBS was added to each well to wash the cells three times. Then, 0.5 mL of 0.25% trypsin-EDTA solution was added to digest for 2 min. The digestion solution was aspirated, and 1 mL of PBS solution was added to gently pipette the cells to detach them from the bottom of the culture plate. 1 mL of 0.05% Triton X-100 was added to lyse the cells and prepare a bacterial suspension. The suspension was then spread on MRS solid plates for CFU counting.

[0108] The formula for calculating adhesion rate is as follows:

[0109] Adhesion rate = Strain survival rate (%) = Nl ÷ N0 × 100%;

[0110] In the formula, N1 is the number of viable bacteria (CFU / mL) after the strain adhesion treatment; N0 is the number of viable bacteria (CFU / mL) before the strain adhesion.

[0111] The results of the cell adhesion test are shown in Table 3.

[0112] Table 3 Cell adhesion results

[0113]

[0114] As shown in Table 3, Lactobacillus curvatureii OH139 exhibits high adhesion to human cervical epithelial cells (HeLa cells) and human bladder transitional cell carcinoma cells (T24 cells), demonstrating better colonization ability.

[0115] Experiment 6: Antibiotic susceptibility test for Lactobacillus curvature OH139

[0116] The susceptibility of Lactobacillus curvature OH139 to polymyxin B, vancomycin, lincomycin, ciprofloxacin, norfloxacin, azithromycin, erythromycin, minocycline, tetracycline, streptomycin, kanamycin, gentamicin, cefoperazone, oxacillin, amikacin, penicillin, ampicillin, piperacillin, cefazolin, cefalexin, cefuroxime, ceftazidime, ceftriaxone, rifampin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, clindamycin, imipenem, levofloxacin, flubendioxonil, metronidazole, and clotrimazole was determined using the agar disk diffusion method.

[0117] The specific steps are as follows:

[0118] 1. Preparation of drug sensitivity test discs: Prepare round filter paper discs with a diameter of 5 mm and a thickness of no more than 4 mm. In this example, the thickness is about 3 mm. Sterilize them by high temperature and high pressure. Add an appropriate amount of the corresponding antibiotic solution to the filter paper disc. Place the filter paper disc flat in a sterile Petri dish and let it dry naturally at room temperature for later use.

[0119] 2. Inoculation of Lactobacillus curvature OH139: Take a bacterial suspension of Lactobacillus curvature OH139 cultured overnight (5×10⁻⁶).6 (CFU / mL) The test bacterial suspension was evenly spread on MRS solid medium using a glass rod.

[0120] 3. Placement of Antimicrobial Sensitivity Testing Discs: Use sterile forceps to place antimicrobial sensitivity testing discs onto the surface of the agar plate. The centers of each disc should be at least 25 mm apart, and the discs should be at least 15 mm away from the perimeter of the plate. After placement, gently press the discs with sterile forceps to ensure they adhere tightly to the plate surface. Cover the agar plate and incubate anaerobically at 37 ℃ for 24 h. Measure the diameter of the inhibition zone.

[0121] The antibacterial test results of different antibiotics against Lactobacillus curvature OH139 are shown in Table 4.

[0122] Table 4. Diameter of inhibition zones (mm) of different antibiotics against Lactobacillus curvature OH139

[0123]

[0124]

[0125] As shown in Table 4, Lactobacillus curvature OH139 is sensitive to a variety of antibiotics, but resistant to metronidazole and clotrimazole, commonly used drugs in the reproductive tract.

[0126] Experiment 7: Hemolysis test of Lactobacillus curvature OH139

[0127] The hemolysis test for bacteria reflects their pathogenicity. *Lactobacillus curvatureii* OH139 is inoculated onto 7% defibrinated sheep blood agar plates and cultured anaerobically. Once colonies form, their morphology is observed to determine hemolysis. A 1-2 mm greenish hemolytic ring around the colony indicates α-hemolysis, suggesting pathogenicity; a 2-4 mm clear hemolytic ring indicates β-hemolysis, indicating extremely high pathogenicity; no change in the culture medium around the colony indicates γ-hemolysis (non-hemolysis), suggesting non-pathogenicity. Figure 5 As shown, this Lactobacillus curvature OH139 strain exhibits α-hemolysis.

[0128] Experiment 8: Preparation of Lactobacillus curvature OH139 powder and tablets

[0129] 1. Preparation of Lactobacillus curvatureii OH139 bacterial powder

[0130] (1) Collection of bacterial cells: After fermentation of Lactobacillus curvature OH139, high-speed centrifugation was performed immediately. The centrifugation speed was 14,000 rpm and the operation time was completed within 13 hours. Live bacterial cells were collected by centrifugation. The entire process was carried out under strict aseptic conditions.

[0131] (2) Cell embedding: The cells are embedded by adding an embedding agent. The embedding agent formula is: 9% yeast extract, 8.4% skim milk powder, 7.4% soy protein isolate, 6.8% trehalose, and 1% sodium D-isoascorbate. The ratio of the embedding agent to the bacterial sludge is 1:1. The mixture is stirred evenly and the whole process is carried out under strict aseptic conditions.

[0132] (3) Freeze-drying: Pre-freeze by lowering the temperature to -40°C, then raise the temperature to -20°C and hold for 30 minutes, then raise the temperature to -10°C and hold for 30 minutes, and then raise the temperature to 26°C in increments of 5°C. Each heating process takes about 3 hours, and each temperature is held for 30 minutes. The freeze-drying time is 30-48 hours.

[0133] (4) Grinding: After grinding, the freeze-dried powder is a milky white to brown powder or granules with a special odor of lactic acid bacteria. Weigh the freeze-dried powder and store it.

[0134] 2. Preparation methods of Lactobacillus curvature OH139 powder and tablets

[0135] (1) Preparation of bacterial powder: 44wt% skim milk, 15wt% sodium glutamate and 41wt% trehalose were mixed and then mixed with an equal amount of freeze-dried Lactobacillus curvature OH139 powder to obtain the bacterial powder of this example.

[0136] (2) Tablet preparation: The probiotic tablet raw materials are mixed evenly and placed in the tableting mechanism, and the mold mechanism is fastened by two fastening mechanisms; the tableting mechanism injects the probiotic tablet raw materials into the mold mechanism and simultaneously extrudes the mold mechanism to form probiotic tablets. The raw materials include 46wt% lactose, 24wt% xylitol, 13wt% maltose, 15wt% isomaltooligosaccharide, 1wt% silicon dioxide, 0.9wt% magnesium stearate, and 0.1wt% lysozyme; after the above raw materials are mixed evenly, they are mixed with an equal amount of Lactobacillus OH139 lyophilized powder, and then used for tableting to obtain the tablets in this example.

[0137] Experiment 9: The therapeutic efficacy of Lactobacillus curvature OH139 against Gardnerella vaginalis model mice

[0138] Forty 6-8 week old SPF-grade female mice (weighing 14-18g) were randomly divided into four groups: healthy group, infected group, metronidazole group and Lactobacillus curvature OH139 group, with 10 mice in each group.

[0139] like Figure 6 As shown in the protocol, a mouse model of bacterial vaginosis caused by Gardnerella vaginalis infection was established. From Day 1 to Day 3, each mouse was estrogenized by daily injection of 0.5 mg of estradiol; from Day 4 to Day 8, 20 μL of Gardnerella vaginalis solution (approximately 1 × 10⁻⁶) was injected into the vagina of each mouse for four consecutive days. 8Healthy mice were injected with 20 μL of physiological saline (CFU / mL). On Day 8, a small amount of mucus was collected from the vagina of the mice using a sterile swab and stained under a microscope to observe whether Gardnerella vaginalis was present, thus ensuring the colonization of Gardnerella vaginalis in the mouse vagina and the establishment of a bacterial vaginosis model mouse.

[0140] From Day 8 to Day 14, mice were administered the drug vaginally once daily for 7 consecutive days. Healthy and infected mice received 20 μL of physiological saline vaginally daily; mice in the metronidazole group received 20 μL of metronidazole solution vaginally daily; and mice in the *Lactobacillus curvaturei* OH139 group received 20 μL of *Lactobacillus curvaturei* OH139 bacterial suspension vaginally daily (10 μL / mL). 9 (CFU / mL).

[0141] Mice vaginas were repeatedly flushed with 50 μL of physiological saline 5-6 times before drug administration (Day 8), Day 1 after drug administration (Day 9), and Day 7 after drug administration (Day 14). 30 μL of the flushing solution was collected for CFU counting of Lactobacillus and Gardnerella vaginalis. Lactobacillus was counted using MRS agar plates, and Gardnerella vaginalis was counted using Columbia blood agar plates supplemented with gentamicin sulfate (4 mg / L), nalidixinone acid (30 mg / L), and amphotericin B (2 mg / L). The test results are shown in Table 5.

[0142] Table 5. Number of Lactobacillus and Gardnerella vaginalis in the vagina of mice in different experimental groups (CFU / mL)

[0143]

[0144] The results in Table 5 show that Lactobacillus curvatureii strain OH139 has both preventive and therapeutic effects on bacterial vaginosis.

[0145] Experiment 10: Trial of Lactobacillus curvaturei OH139 powder and tablets for bacterial vaginosis in volunteers.

[0146] 1. Test materials

[0147] Active ingredient: Lactobacillus OH139 bacterial powder / tablet, prepared by the same method as in Experiment 8;

[0148] Excipient: Starch (Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.);

[0149] Lactobacillus curvature powder: 0.5g / packet, each packet containing no less than 2×10⁻⁶ live Lactobacillus curvature bacteria. 8 CFU;

[0150] Lactobacillus curvature tablets: 0.5 g / tablet, each tablet should contain no less than 2 × 10⁻⁶ live Lactobacillus curvature bacteria. 8 CFU;

[0151] After passing the viable bacteria count and tests for miscellaneous bacteria and mold, human trials will be conducted.

[0152] 2. Test subjects

[0153] Thirty-two female patients with bacterial vaginosis presented with symptoms such as abnormal discharge and itching. All volunteers had given their consent for this trial.

[0154] (1) Probiotic powder group: 11 people, who used the probiotic powder externally according to the instructions, once a day for 7 consecutive days;

[0155] (2) Probiotic tablet group: 11 people, who used probiotic tablets externally according to the instructions, once a day for 7 consecutive days;

[0156] (3) Control group: 10 people, applied externally without probiotic excipient tablets once a day for 7 consecutive days.

[0157] 3. Test Results

[0158] (1) User experience

[0159] The fact that 32 people experienced no obvious discomfort indicates that this Lactobacillus curvature OH139 is non-irritating.

[0160] Table 6. Distribution of participants' experiences in each group.

[0161]

[0162] (2) Effects on vaginal secretions

[0163] In the probiotic powder group, the vaginal discharge of all 10 patients tended to normalize, of which 7 had previously had abnormal discharge; in the probiotic tablet group, the vaginal discharge of all 11 patients tended to normalize, of which 8 had previously had abnormal discharge; in the control group, the vaginal discharge of all 10 patients did not improve and remained the same as before.

[0164] Table 7. Distribution of patients affected by the effects of probiotic powder / tablets on vaginal secretions

[0165]

[0166] (3) Impact on inflammation recovery

[0167] In the probiotic powder group, all 11 patients experienced relief of their inflammation, with 9 recovering to normal, 1 showing significant relief, and 1 showing slight relief. In the probiotic tablet group, all 11 patients experienced relief of their inflammation, with 9 recovering to normal and 2 showing significant relief. In the control group, the vaginal inflammation of 10 patients did not improve and remained unchanged.

[0168] Table 8. Distribution of patients affected by the effects of probiotic powder / tablets on the recovery of vaginal inflammation.

[0169]

[0170] In summary, the *Lactobacillus curvature* OH139 in this case showed resistance to *Gardnerella vaginalis* (ATCC 14018), *Fannyhessea vaginae* (DSM 15829), *Prevotella bivia* (DSM 20514), *Streptococcus agalactiae* (ATCC 27956), *Streptococcus anginosus* (ATCC 33397), and *Candida albicans* (ATCC 20514). Both 10231 and other drugs have good inhibitory effects; moreover, they are resistant to commonly used drugs for reproductive tract infections such as metronidazole, tinidazole, clindamycin, ofloxacin, or ciprofloxacin, and can adhere to the vagina and have strong colonization ability; they can be used to balance the reproductive tract flora, improve the female reproductive tract microecological environment, prevent and improve problems such as increased secretions, fishy smell, and vulvar itching caused by reproductive tract infections, and have no side effects.

[0171] The above description, in conjunction with specific embodiments, provides a detailed explanation of this application and should not be construed as limiting the implementation of this application to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the basic inventive concept of this application.

Claims

1. A strain of Lactobacillus crispatus, characterized by: The *Lactobacillus curvature* is *Lactobacillus curvature* OH139 with preservation number GDMCC No. 63856; the *Lactobacillus curvature* is resistant to metronidazole, tinidazole, levofloxacin and ciprofloxacin.

2. A microbial agent, characterized in that: The bacterial agent contains the *Lactobacillus curvatureus* as described in claim 1.

3. The microbial agent according to claim 2, characterized in that: Each dose of the microbial agent contains at least 2 × 10⁻⁶ live *Lactobacillus curvatureensis*. 8 CFU.

4. The use of the *Lactobacillus curvatureus* of claim 1 or the bacterial agent of claim 2 or 3 in the preparation of products for improving bacterial vaginosis.

5. A product for improving bacterial vaginosis, characterized in that: The active ingredient of the product includes the *Lactobacillus curvatureus* as described in claim 1 or the bacterial agent as described in claim 2 or 3.

6. The product according to claim 5, characterized in that: The product is in the form of powder or tablets.

7. The product according to claim 5, characterized in that: The product also contains pharmaceutically acceptable excipients.

8. The product according to claim 7, characterized in that: The product comprises 25-35 parts by weight of bacterial powder, 20-30 parts by weight of fructooligosaccharides, 20-30 parts by weight of galactooligosaccharides, 50-80 parts by weight of additives, and 60-100 parts by weight of packaging materials.

9. The product according to claim 8, characterized in that: The additive is at least one of monosodium glutamate and skim milk; the packaging material is at least one of silicon dioxide and magnesium stearate.

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