Lactobacillus crispatus x25b and uses thereof

Abstract

The present application relates to a Lactobacillus crispatus X25B and uses thereof. In particular, the present application relates to a Lactobacillus crispatus, as well as compositions, pharmaceutical compositions, cultures, food products or dietary supplements and cleaning products comprising the same. The present application also relates to the use of said Lactobacillus crispatus and compositions comprising the same for the manufacture of a medicament.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbiology technology, and particularly relates to a Lactobacillus crispatus, a composition, a pharmaceutical composition, a culture, a food product or a dietary supplement and a cleaning product comprising the same. The present application also relates to the use of the Lactobacillus crispatus and the composition comprising the same in the preparation of a medicament. BACKGROUND

[0002] Bacterial vaginosis (BV) is a type of vaginal infection caused by imbalance of vaginal flora, which is a common gynecological disease in women and has become a public health problem affecting women's daily life worldwide. The factors causing BV mainly include the decrease of estrogen level, sexual behavior, and the use of antibiotics, and its clinical manifestations include pruritus of vulva, burning sensation, odor and abnormal leucorrhea.

[0003] Under normal conditions, the vaginal flora has Lactobacillus as the dominant bacteria, among which Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii and Lactobacillus jensenii are the most common species. The substances such as hydrogen peroxide and bacteriocin produced by these dominant strains maintain the acidic and healthy environment of the vagina. If the balance of vaginal flora is broken, the abundance of Lactobacillus decreases, and the pathogenic flora of vagina increases significantly. Among them, Gardnerella vaginalis is the main pathogenic bacteria of bacterial vaginosis, in addition, when the normal environment of vagina is destroyed, Candida albicans, Staphylococcus aureus and Escherichia coli may multiply in large quantities to cause reproductive tract infection.

[0004] The conventional drug treatment of BV is mainly through oral or local use of clindamycin and metronidazole or tinidazole. However, the antibiotic therapy has poor effect and high recurrence rate, which may be related to the drug resistance of pathogenic bacteria and their biofilm to antibiotics, and the failure to restore the acidic environment of vagina. In recent years, probiotics have shown good antibacterial activity in the treatment of bacterial vaginosis, which helps to restore the balance of vaginal microecology. There is evidence that hydrogen peroxide-producing Lactobacillus has an inhibitory effect on bacterial infection and prevents the occurrence of bacterial vaginosis.

[0005] At present, there are still very limited microecological products on the market for treating or relieving bacterial vaginosis. For example, "Dingjunsheng" is a strain of Lactobacillus delbrueckii DM8909 selected from the vaginal secretions of healthy women by Professor Kangbai of Dalian Medical University, but the discussion on the treatment effect of the strain on bacterial vaginosis has certain limitations. Therefore, it is particularly necessary to develop effective probiotic strains and dominant strains derived from the vagina of healthy women of childbearing age in China. SUMMARY

[0006] The inventors of this application, through extensive experiments, screened a strain of *Lactobacillus curvaturei* with significant strain-specific probiotic potential from over 100 strains derived from healthy women. Extensive experiments have demonstrated that this *Lactobacillus curvaturei* possesses high acid-producing and hydrogen peroxide-producing capabilities, enabling it to maintain an acidic and healthy environment in the female reproductive tract and normalize the female reproductive tract microbiota. Furthermore, this *Lactobacillus curvaturei* significantly inhibits the growth and biofilm formation of *Gardnerella vaginalis*, the most prevalent pathogenic bacterium in the vagina. Thus, the applicant has completed this invention.

[0007] Lactobacillus crispatus

[0008] In a first aspect, this application provides a Lactobacillus crispatus, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC No. 63707.

[0009] In some embodiments, the *Lactobacillus curvatureii* has the ability to produce hydrogen peroxide greater than 15 μmol / L under effective culture conditions. In some embodiments, the *Lactobacillus curvatureii* has the ability to produce hydrogen peroxide at concentrations of 15 μmol / L to 25 μmol / L, 25 μmol / L to 35 μmol / L, 35 μmol / L to 45 μmol / L, or greater under effective culture conditions.

[0010] In some embodiments, the *Lactobacillus curvatureii* is capable of lowering the pH of the female reproductive tract. In some embodiments, the *Lactobacillus curvatureii* lowers the pH of the female reproductive tract to pH 6.5 or lower, pH 6 or lower, pH 5.5 or lower, pH 5 or lower, pH 4.5 or lower, or pH 4 or lower.

[0011] In some embodiments, the *Lactobacillus curvature* is capable of inhibiting the growth of *Gardnerella* strains and / or inhibiting biofilm formation by *Gardnerella* strains. In some embodiments, the *Gardnerella* strain is *Gardnerella vaginalis*. In some embodiments, the inhibition of biofilm formation can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%.

[0012] In some embodiments, the *Lactobacillus curvatureii* is able to reduce the levels of pro-inflammatory factors (e.g., TNF-α, IL-1β, IL-6). In some embodiments, the *Lactobacillus curvatureii* reduces the levels of TNF-α, IL-1β, and / or IL-6 by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, and at least 70%.

[0013] In some embodiments, the *Lactobacillus curvature* is able to increase the level of anti-inflammatory factors (e.g., IL-10). In some embodiments, the *Lactobacillus curvature* reduces or increases the level of IL-10 by 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, or at least 70%.

[0014] In some embodiments, the colonies of the *Lactobacillus curvatureensis* are white, milky white, and / or translucent.

[0015] In some embodiments, the *Lactobacillus curvatureii* is a Gram-positive bacterium.

[0016] It is readily understood that during the growth and culture of microorganisms, particularly bacteria, genetic material may undergo certain changes (e.g., mutations of one or more bases). These changes can occur spontaneously or as a result of mutagenesis induced by chemical and / or physical agents (e.g., mutagens) and / or recombinant DNA techniques known in the art. Therefore, in this document, *Lactobacillus curlis* of the present invention includes mutants and / or progeny of *Lactobacillus curlis* with accession number GDMCC No. 63707. In some embodiments, these mutants and / or progeny retain the functional and / or physiological and biochemical characteristics (e.g., one or more of the aforementioned functional and / or physiological and biochemical characteristics) of *Lactobacillus curlis* with accession number GDMCC No. 63707.

[0017] Composition

[0018] In a second aspect, this application provides a composition comprising *Lactobacillus curvatureensis* as described in the first aspect.

[0019] As used herein, the term “probiotic” is defined as any non-pathogenic microorganism that, when administered to a host in sufficient quantities in live form, can have a beneficial effect on the host’s health.

[0020] In some embodiments, the composition further comprises probiotics selected from bacteria, fungi (e.g., yeast), or any combination thereof.

[0021] In some embodiments, the bacteria are selected from Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.

[0022] In some embodiments, the bacteria of the genus *Lactobacillus* are selected from: *Lactobacillus paracasei*, *Lactobacillus acidophilus*, *Lactobacillus brevis*, *Lactobacillus jensenii*, *Lactobacillus iners*, *Lactobacillus casei*, *Lactobacillus crispatus*, *Lactobacillus curvatus*, *Lactobacillus delbrueckii*, *Lactobacillus fermentum*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *Lactobacillus plantarum*, and *Lactobacillus reuteri*. Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, or any combination thereof.

[0023] In some embodiments, the *Lactobacillus paracasei* is *Lactobacillus paracasei* 207-27 with the microbial preservation number GDMCC No. 60960.

[0024] In some embodiments, the yeast is selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces marxianus, or any combination thereof.

[0025] In some embodiments, the bacteria of the genus Bifidobacterium are selected from: Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis, or any combination thereof.

[0026] In some embodiments, the bacteria of the genus Bacillus are selected from Bacillus subtilis, Bacillus coagulans, or any combination thereof.

[0027] In some embodiments, the bacteria of the genus Propionibacterium are selected from: Propionibacterium shermanii, Propionibacterium freudenreichii, Propionibacterium acidipropionici, or any combination thereof.

[0028] In some embodiments, the Streptococcus bacteria are selected from Streptococcus thermophilus, Streptococcus salivarius, or any combination thereof.

[0029] In some embodiments, the bacteria of the genus *Lactococcus* is *Lactococcus lactis*.

[0030] In some embodiments, the Enterococcus species are selected from Enterococcus faecalis, Enterococcus faecium, or any combination thereof.

[0031] Pharmaceutical composition

[0032] In a third aspect, this application provides a pharmaceutical composition comprising Lactobacillus curvature as described in the first aspect or the composition described in the second aspect.

[0033] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient, a cryoprotectant, an amino acid, a vitamin, a mineral, a peptone, or any combination thereof.

[0034] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes: fillers, binders, lubricants, flow aids, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof.

[0035] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes: sugars (such as xylose, sucrose, fructose, lactose, trehalose), sugar alcohols (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gum, maltodextrin), polyethers (such as polypropylene glycol, polyethylene glycol, polybutylene glycol), povidones (such as povidone K30, K60, K90), oils (such as rapeseed oil, sunflower oil, soybean oil, sesame oil, olive oil), surfactants (such as Tween20, Tween40, Tween60, Tween80, fatty acids, polyoxyethylene fatty alcohol ethers), inorganic salts (such as phosphates, carbonates, citrates, chlorides, sulfates, borates, citrates), talc, silica and its derivatives, hydrolysis products, or any combination thereof.

[0036] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant.

[0037] In some embodiments, the pharmaceutical composition further comprises glycerin, skim milk powder, soluble starch, polyethylene glycol, dextran, trehalose, sorbitol, xylooligosaccharides, glutathione, or any combination thereof.

[0038] In some embodiments, the pharmaceutical composition comprises amino acids.

[0039] In some embodiments, the pharmaceutical composition includes tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

[0040] In some embodiments, the pharmaceutical composition includes vitamins.

[0041] In some embodiments, the pharmaceutical composition includes vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, and vitamin B1. 12 Vitamins C, D, E, and K, or any combination thereof.

[0042] In some embodiments, the pharmaceutical composition includes minerals.

[0043] In some embodiments, the pharmaceutical composition includes iron, manganese, zinc, copper, selenium, or any combination thereof.

[0044] In some embodiments, the pharmaceutical composition includes peptone.

[0045] In some embodiments, the pharmaceutical composition includes soy peptone, wheat peptone, whey peptone, or any combination thereof.

[0046] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, via various routes. The route of administration can be any method commonly used in the art. For example, it can be administered orally, in vitro, intravenously, intramuscularly, subcutaneously, etc. In some embodiments, the pharmaceutical compositions are formulated for oral or in vitro administration.

[0047] In some embodiments, the pharmaceutical composition comprises a formulation of Lactobacillus curvatureii.

[0048] In some embodiments, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), oil drops, capping films, orally soluble granules, liquids, suppositories, enemas, gels, and / or creams.

[0049] In some embodiments, the pharmaceutical composition further comprises formulation excipients (e.g., excipients for preparing powders, tablets, capsules, or oil drops).

[0050] In some embodiments, the pharmaceutical composition is used alone or in combination with other antifungal agents, antiviral agents, analgesics, anti-inflammatory agents, healing promoters, and / or moisturizers.

[0051] In some embodiments, the pharmaceutical composition contains Lactobacillus curvature at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 7 Up to 10 9 (CFU / ml).

[0052] In some embodiments, when the pharmaceutical composition is a solid, it contains 10 per gram. 6 Up to 10 12 CFU of Lactobacillus curvature (e.g., 10) 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g).

[0053] In some embodiments, when the pharmaceutical composition is a liquid, it contains 10 mg / ml. 6 Up to 10 12CFU of Lactobacillus curvature (e.g., 10) 7 CFU / mL, 10 8 CFU / mL, 10 9 (CFU / mL).

[0054] In some embodiments, the Lactobacillus curvature in the pharmaceutical composition is present in either live or dead form.

[0055] Culture

[0056] In a fourth aspect, this application provides a culture comprising Lactobacillus curvature as described in the first aspect or the composition described in the second aspect.

[0057] In some embodiments, the culture also contains nutrient-providing components (e.g., solid or liquid culture medium, feeding cell layer).

[0058] In some embodiments, the nutrient-providing components are selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0059] In some embodiments, the culture also comprises a cell-free culture filtrate of Lactobacillus curvature.

[0060] In some embodiments, the culture further comprises a derivative of *Lactobacillus curvatureensis*; wherein the derivative is selected from metabolites, enzymes, cellular structural components (e.g., cell walls or components thereof), extracellular polysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

[0061] Food product or dietary supplement or health food

[0062] In a fifth aspect, this application provides a food product or dietary supplement or health food containing Lactobacillus curvature as described in the first aspect, or the composition described in the second aspect, or the culture described in the fourth aspect.

[0063] In this text, the term "food" is used broadly to include human food and drink, as well as animal food and drink (i.e., feed). In some embodiments, the food product is suitable for and designed for human consumption. In some embodiments, the food product is selected from solid beverages, liquid beverages, compressed candies, puffed foods, protein bars, or nuts, or the food product is a dairy product (e.g., milk powder, milk tablets, yogurt, flavored fermented milk, lactic acid bacteria beverages, cheese).

[0064] In this text, "dietary supplements" refers to edible products that provide consumers with beneficial effects. Also known as nutritional supplements, nutrient solutions, or dietary supplements, they are used as an adjunct to a diet to supplement the body with essential amino acids, trace elements, vitamins, and minerals.

[0065] In some embodiments, the food product, dietary supplement, or health food also contains prebiotics.

[0066] In some embodiments, the prebiotic is selected from fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrophyllum, trachomatis polysaccharides, nitrogenous carotene, or any combination thereof.

[0067] In some implementations, the food product, dietary supplement, or health food may also include additional additives.

[0068] In some embodiments, the additional additive is selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0069] In some embodiments, the carbohydrate is selected from monosaccharides (glucose, fructose, xylose), disaccharides (maltose, lactose, sucrose, trehalose), sugar alcohols (such as glycerol, xylitol, sorbitol, mannitol, erythritol), polysaccharides (such as cellulose and its derivatives, starch and its derivatives, chitosan, gum, maltodextrin), or any combination thereof.

[0070] In some embodiments, the amino acid is selected from tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

[0071] In some embodiments, the vitamin is selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, and vitamin B1. 12 Vitamins C, D, E, and K, or any combination thereof.

[0072] In some embodiments, the mineral is selected from iron, manganese, zinc, copper, selenium, or any combination thereof.

[0073] In some embodiments, the food product, dietary supplement, or health food contains Lactobacillus flavus at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 7 Up to 10 9 (CFU / ml).

[0074] In some embodiments, when the food product, dietary supplement, or health food is a solid, it contains 10 per gram. 6 Up to 10 12 CFU of Lactobacillus curvature (e.g., 10) 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g).

[0075] In some embodiments, when the food product, dietary supplement, or health food is a liquid, it contains 10 [units of something] per milliliter. 6 Up to 10 12 CFU of Lactobacillus curvature (e.g., 10) 7 CFU / mL, 10 8 CFU / mL, 10 9 (CFU / mL).

[0076] In some embodiments, the Lactobacillus curvature in the food product, dietary supplement, or health food is present in either a live or dead form.

[0077] Cleaning product

[0078] In a sixth aspect, this application provides a cleaning product comprising Lactobacillus curvature as described in the first aspect, or a composition as described in the second aspect, or a culture as described in the fourth aspect.

[0079] In some embodiments, the cleaning product is selected from solid soap, liquid soap, hand sanitizer, shampoo, liquid conditioner, body cleanser, or any combination thereof.

[0080] In some embodiments, the cleaning product is a vaginal douche, vaginal gel, and / or vaginal cream.

[0081] Use

[0082] In a seventh aspect, this application provides the use of *Lactobacillus curvaturei* as described in the first aspect, or the composition as described in the second aspect, or the pharmaceutical composition as described in the third aspect, or the culture as described in the fourth aspect, or the cleaning product as described in the sixth aspect, in the preparation of a medicament for the prevention and / or treatment of female urogenital tract-related diseases and / or symptoms in a subject.

[0083] In some embodiments, the female urogenital tract-related diseases are caused by an imbalance of the reproductive tract microbiota and / or abnormal reproductive tract pH.

[0084] In some implementations, the female urogenital tract-related diseases are associated with reduced levels of Lactobacillus strains in the reproductive tract ecosystem.

[0085] In some embodiments, the female urogenital tract-related diseases are associated with an increased proliferation of at least one of the following microorganisms in the reproductive tract ecosystem: Gardnerella vaginalis, Candida albicans, Streptococcus aureus, Atopobium vaginae, or any combination thereof.

[0086] In some embodiments, the female urogenital tract-related diseases are associated with an increased proliferation of at least one of the following genera in the reproductive tract ecosystem: Prevotella spp., Megasphaera spp., Sneathia spp., Atopobium spp., Dialister spp., Anaerococcus spp., Eggerthella spp., Peptoniphilus spp., Aerococcus spp., Mobiluncus spp., or any combination thereof.

[0087] In some implementations, the female urogenital tract-related diseases are associated with elevated pH levels in the reproductive tract environment.

[0088] Therefore, in some embodiments, the drug is used to maintain the balance of the female reproductive tract microbiota, reduce the pH of the female reproductive tract, increase the level of Lactobacillus strains in the female reproductive tract ecosystem, and / or decrease the level of Gardnerella vaginalis strains in the female reproductive tract.

[0089] In some embodiments, the strain of the Gardnerella genus is Gardnerella vaginalis.

[0090] In some embodiments, the disease is selected from: female genitourinary tract infections, genital herpes, gonorrhea, inflammatory diseases associated with the female genitourinary tract (e.g., vaginitis, cervicitis), or any combination thereof. In some embodiments, female genitourinary tract infections may include or cause bacterial vaginosis, vaginal microbiota imbalance, and / or urinary tract infections.

[0091] In some implementations, the symptoms are selected from: changes in the menstrual cycle, vaginal itching, vaginal redness, abnormal discharge, vaginal pain, or any combination thereof.

[0092] In some embodiments, the drug prevents and / or treats inflammatory diseases associated with the female urogenital tract by reducing the level of pro-inflammatory factors and / or increasing the level of anti-inflammatory factors.

[0093] In some embodiments, the drug prevents and / or treats inflammatory diseases associated with the female urogenital tract by reducing sialidase activity and / or myeloperoxidase (MPO) activity.

[0094] In some embodiments, the pro-inflammatory factor is selected from TNF-α, IL-1β, IL-6, or any combination thereof. In some embodiments, the anti-inflammatory factor is IL-10.

[0095] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0096] In an eighth aspect, this application provides the use of *Lactobacillus curvaturei* as described in the first aspect, or the composition as described in the second aspect, or the pharmaceutical composition as described in the third aspect, or the culture as described in the fourth aspect, or the cleaning product as described in the sixth aspect, in the preparation of a medicament for inhibiting the growth of *Gardnerella* and / or reducing its amount.

[0097] In some embodiments, the drug inhibits the growth of Gardnerella vaginalis strains by increasing the level of hydrogen peroxide (H2O2).

[0098] In some embodiments, the drug inhibits the growth of Gardnerella vaginalis strains by lowering the pH level of the environment.

[0099] In some embodiments, the drug is used to inhibit biofilm formation in strains of the genus Gardnerella, thereby inhibiting the growth of strains of the genus Gardnerella.

[0100] In some embodiments, the drug is used to inhibit the growth of Gardnerella vaginalis in women and / or reduce its amount, or to inhibit the growth of Gardnerella vaginalis in the environment and / or reduce its amount.

[0101] In some embodiments, the female body includes mucous membranes and / or the reproductive tract.

[0102] In this article, the term "mucosa" refers to the membranes within various cavities of mammals and covering the surfaces of internal organs. It consists of one or more layers of epithelial cells covered by a loose connective tissue. It is primarily of endoderm origin and is continuous with the skin at various body openings, such as the inner sides of the eyes, ears, nose and mouth, lips, vagina, urethral opening, and anus.

[0103] In some implementations, the environment includes a home, workplace, laboratory, industrial environment, aquatic environment, medical instrument and / or gynecological examination equipment.

[0104] Method

[0105] In a ninth aspect, this application provides a method for inhibiting the growth of Gardnerella vaginalis and / or reducing its amount in a woman, the method comprising: administering to a female subject in need an effective amount of Lactobacillus curvatureus as described in the first aspect or a composition as described in the second aspect or a pharmaceutical composition as described in the third aspect or a culture as described in the fourth aspect or a cleaning product as described in the sixth aspect.

[0106] In some embodiments, the method includes implanting Lactobacillus curvatureus as described in the first aspect, or a composition as described in the second aspect, or a pharmaceutical composition as described in the third aspect into the reproductive tract of a female subject.

[0107] In some embodiments, the method includes applying (e.g., smearing, spraying) Lactobacillus curvature as described in the first aspect, or a composition as described in the second aspect, or a pharmaceutical composition as described in the third aspect, or a cleaning product as described in the sixth aspect to the female subject's reproductive tract and / or perineum.

[0108] In some embodiments, the method includes: administering, orally (e.g., orally) to a female subject Lactobacillus curvatureus as described in the first aspect, or a composition as described in the second aspect, or a pharmaceutical composition as described in the third aspect, or a dietary supplement or health food as described in the fifth aspect.

[0109] In some embodiments, the Gardnerella genus includes Gardnerella vaginalis.

[0110] In some embodiments, the female body includes the lining and / or mucous membranes of the reproductive tract.

[0111] In some embodiments, the *Lactobacillus curvature* is at 10 6 Up to 10 12 CFU / day dose (e.g., 10) 7 CFU / day dose, 10 8 CFU / day dose, 10 9 CFU / day dose, 1010 CFU / day dose, 10 11 CFU / day dose, 10 12 The dose (CFU / day) was administered to the subject.

[0112] In a tenth aspect, this application provides a method for inhibiting the growth of Gardnerella vaginalis in an environment and / or reducing its amount, the method comprising: applying to the environment an effective amount of Lactobacillus curvatureus as described in the first aspect, or a composition as described in the second aspect, or a pharmaceutical composition as described in the third aspect, or a culture as described in the fourth aspect.

[0113] In some implementations, the environment includes a home, workplace, laboratory, industrial environment, aquatic environment, medical instrument and / or gynecological examination equipment.

[0114] In some embodiments, the *Lactobacillus curvature* is at 10 6 Up to 10 12 CFU / day dose (e.g., 10) 7 CFU / day dose, 10 8 CFU / day dose, 10 9 CFU / day dose, 10 10 CFU / day dose, 10 11 CFU / day dose, 10 12 The amount of CFU / day dose is applied to the environment.

[0115] In the eleventh aspect, this application provides a method for preventing and / or treating diseases and / or symptoms related to the female urogenital tract, the method comprising: administering to a female subject in need an effective amount of *Lactobacillus curvularis* as described in the first aspect, or a composition as described in the second aspect, or a pharmaceutical composition as described in the third aspect, or a culture as described in the fourth aspect, or a cleaning product as described in the sixth aspect.

[0116] In some embodiments, the female urogenital tract-related diseases are caused by an imbalance of the reproductive tract microbiota and / or abnormal pH levels in the reproductive tract.

[0117] In some implementations, the female urogenital tract-related diseases are associated with reduced levels of Lactobacillus strains in the reproductive tract ecosystem.

[0118] In some embodiments, the female urogenital tract-related diseases are associated with an increased proliferation of at least one of the following genera in the reproductive tract ecosystem: Gardnerella spp., Dorebrospina spp., Streptococcus spp., Anaerobic cocci spp., Fingoldii spp., Prevotella spp., Sturgeonium spp., Staphylococcus spp., Corynebacterium spp., or any combination thereof.

[0119] In some implementations, the female urogenital tract-related diseases are associated with elevated pH levels in the reproductive tract environment.

[0120] In some embodiments, the strain of the Gardnerella genus is Gardnerella vaginalis.

[0121] In some embodiments, the disease is selected from: female genitourinary tract infections, genital herpes, gonorrhea, inflammatory diseases associated with the female genitourinary tract (e.g., vaginitis, cervicitis), or any combination thereof. In some embodiments, female genitourinary tract infections may include or cause bacterial vaginosis, vaginal microbiota imbalance, and / or urinary tract infections.

[0122] In some implementations, the symptoms are selected from: changes in the menstrual cycle, vaginal itching, vaginal redness, abnormal discharge, vaginal pain, or any combination thereof.

[0123] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0124] In some embodiments, the *Lactobacillus curvature* is at 10 6 Up to 10 12 CFU / day dose (e.g., 10) 7 CFU / day dose, 10 8 CFU / day dose, 10 9 CFU / day dose, 10 10 CFU / day dose, 10 11 CFU / day dose, 10 12 The dose (CFU / day) was administered to the subject.

[0125] Definitions of terms

[0126] In this article, the term "reproductive tract" includes the urinary tract and the vagina. The vagina comprises the elastic muscular portion of the female reproductive tract. In humans, the vagina extends from the vulva to the cervix.

[0127] In this article, the term "mucosa" refers to the membranes within various cavities of mammals and covering the surfaces of internal organs. It consists of one or more layers of epithelial cells covered by a loose connective tissue. It is primarily of endoderm origin and is continuous with the skin at various body openings, such as the inner sides of the eyes, ears, nose and mouth, lips, vagina, urethral opening, and anus.

[0128] In this paper, the term "reproductive tract microbiota" or "reproductive tract ecosystem" refers to a microbiome that exists within the female urogenital tract (preferably the vagina) and coexists in a balanced manner with each other and with the reproductive tract environment that houses them. Therefore, the term "reproductive tract microbiota imbalance" includes a decrease in the level of one or more dominant bacteria in the reproductive tract microbiota and / or an increase in the level of one or more pathogenic bacteria in the reproductive tract.

[0129] In the context of this invention, the term "pathogenic bacteria" refers to microorganisms that can exist in the vaginal ecosystem and have potential pathogenicity.

[0130] In this document, the term "therapeutic effective amount" or "effective amount" can refer to the amount of a strain in a composition or pharmaceutical composition, when administered as part of a desired dosing regimen (to a human or animal, preferably a human), based on clinically acceptable criteria for the condition or disease of the subject to be treated, such as a reasonable benefit-risk ratio applicable to any medical treatment, to relieve symptoms, improve the condition, or slow the onset of the disease. Therapeutic effective amounts can vary depending on the age, weight, sex, dosage form, health status, and severity of the disease of the subject to be treated. Furthermore, the frequency may be determined by a physician or pharmacist, administering the dose once or several times daily at fixed intervals.

[0131] As used in this article, the term "CFU (Colony-Forming Units)" refers to the total number of microbial communities such as bacteria, fungi, and yeast in a product, and is usually used to calculate the number of viable cells.

[0132] As used herein, the term "CFU / dosage" refers to the amount of bacteria present in a composition / food product or dietary supplement or health food / pharmaceutical composition administered to a subject daily or per dose. For example, in some embodiments, the *Lactobacillus flavus* in the food product or dietary supplement or health food is 10... 6 Up to 10 12 The amount of CFU / dose is present. In this embodiment, if *Lactobacillus curlis* is applied to a food product (e.g., in a solid beverage, yogurt), the food product (e.g., a solid beverage, yogurt) provided to the subject daily or per dose may contain approximately 10 CFU / dose. 6 Up to 10 12 CFU of *Lactobacillus curlis*. Alternatively, the amount of this bacteria can be divided into multiple administrations, provided that the total amount of *Lactobacillus curlis* received by the subject at any given time (e.g., every 24-hour period) is less than approximately 10... 6 To about 10 12 CFU of bacteria, namely Lactobacillus flavus in food products, dietary supplements, or health foods meeting the above criteria, at a concentration of 10... 6 Up to 10 12The amount of CFU / dose present.

[0133] As used herein, the term "pharmaceuticalally acceptable carrier" means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0134] As used herein, the term "subject" refers to an animal. Animals may include, but are not limited to, primates, farm animals, sporting animals, rodents, and pets. More specifically, animals may include mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; piglets; sows; poultry; turkeys; broilers; mink; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0135] As used herein, the term "inhibition" refers to controlling the growth of microorganisms or killing them. Initial attachment of microorganisms or further biofilm formation can be inhibited by inhibiting the growth of biofilm-forming microorganisms. In some embodiments, biofilm formation inhibition can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%.

[0136] As used herein, the term "sialidase," also known as "neuraminidase," is the major virulence factor of Gardnerella vaginalis, the causative agent of bacterial vaginosis (BV). Sialidase can be detected in cervical and vaginal douches from human women. Numerous studies have shown that women with BV have higher levels of sialidase activity in their vaginal secretions than women without BV (e.g., healthy women). Therefore, a positive sialidase test in vaginal secretions is currently used as an important diagnostic indicator for BV in clinical practice.

[0137] As used in this article, the term "MPO" refers to an enzyme found in white blood cells. MPO is commonly used to assess the diagnosis and treatment of diseases such as inflammation and infection. Generally, elevated MPO levels may indicate inflammation, infection, or autoimmune disease in the host. During inflammation, white blood cells release large amounts of MPO to eliminate bacteria, viruses, and other pathogens. Therefore, MPO levels in cervical and vaginal douches are typically elevated in patients with bacterial vaginosis.

[0138] Beneficial effects of the invention

[0139] The *Lactobacillus curvature* strain of this application possesses high acid-producing and hydrogen peroxide-producing capabilities, enabling it to maintain an acidic and healthy environment in the female reproductive tract and to maintain the balance of the female reproductive tract microbiota. Furthermore, this *Lactobacillus curvature* strain significantly inhibits the growth and biofilm formation of *Gardnerella vaginalis*, the most prevalent pathogenic bacterium in the reproductive tract. It also significantly reduces sialidase and myeloperoxidase (MPO) activities in the vagina, significantly reduces the levels of pro-inflammatory factors (e.g., TNF-α, IL-1β, and IL-6), and significantly increases the levels of anti-inflammatory factors (e.g., IL-10). Therefore, the *Lactobacillus curvature* strain of this application has high application value in women's reproductive health (especially women's vaginal health).

[0140] The embodiments of the present invention will now be described in detail with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of preferred embodiments.

[0141] Instructions on the Preservation of Biological Materials

[0142] Lactobacillus crispatus X25B has been deposited at the Guangdong Microbial Culture Collection Center (GDMCC), located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 63707 and deposit date of August 2, 2023. Detailed Implementation

[0143] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0144] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by F.M. Ausubel et al., 1987); and the *Methods in Enzymology* series (academic publishing company): *PCR2: A PRACTICAL APPROACH* (M.J. MacPherson, B.D. Hames, and G. G.R. Taylor (ed., 1995), and Animal Cell Culture (R.I. Freshney (ed., 1987)).

[0145] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0146] Example 1. Isolation and identification of strains

[0147] This application isolated over 100 strains of bacteria from vaginal swabs collected from 60 healthy women undergoing physical examinations at Dalian Women and Children's Health Hospital. One strain, *Lactobacillus curvatureii*, was selected and named *Lactobacillus curvatureii* X25B. The inclusion criteria for sample donors were: age 22-35 years; regular menstruation, and not menstruating at the time of sampling; no sexual activity, vaginal medication, or vaginal douching within 72 hours; no diagnosis of gynecological or urinary tract inflammatory diseases within the past six months; no positive signs found during gynecological examination; no use of antibiotics within the past month or sex hormones within the past three months; no history of hysterectomy or invasive treatment of the cervix or uterus; no recent immunosuppressive therapy; and no severe heart, liver, or kidney dysfunction or related complications. Vaginal swab samples were stored at -80°C immediately after collection.

[0148] The strains were cultured using MRS liquid medium, modified MRS solid medium (prepared MRS liquid medium with 2% agar and 1% calcium carbonate added, autoclaved at 121℃ for 15 min), BHI liquid medium, and BHI solid medium (prepared liquid medium with 2% agar added, autoclaved at 121℃ for 15 min). Before use, the temperature was lowered to approximately 50℃, and 10% sterile defibrinated sheep blood and lactobacillus selective agar were added.

[0149] Immerse vaginal swabs in physiological saline and mix thoroughly for 5 min. Take 100 μl of the liquid from each sample and spread it onto modified MRS, LBS, or BHI solid media, respectively. Incubate anaerobically at 37°C for 48-72 h. Select medium-sized, raised, milky-white, round colonies for Gram staining and microscopic examination. Observe bacterial morphology and staining properties under an optical microscope. Select single colonies of Gram-positive bacteria (rod-shaped or coccobacillus-shaped) for further purification until confirmed as pure bacteria. Select single colonies for 16S rRNA sequencing.

[0150] Sequencing results showed that the strain isolated in this application is *Lactobacillus curlis*. Furthermore, the whole genome of *Lactobacillus curlis* X25B was sequenced using the PacBio Sequel2 sequencing platform and compared with the whole genome sequences of *Lactobacillus curlis* strains recorded in the NCBI database. The genome length of *Lactobacillus curlis* X25B is 2185354 bp, with a GC content of 36.87%. The chromosomal genome contains 2270 coding genes, 66 tRNA genes, 4 rRNA genes (including 5S rRNA, 16S rRNA, and 23S rRNA), and 166 other types of RNA genes. Based on the above PCR identification and whole genome sequencing results, this application obtained a novel *Lactobacillus curlis* X25B strain, which has been deposited.

[0151] Example 2. In vitro probiotic function determination of strains

[0152] The strains used in this example were all from the female-derived probiotic library of By-Health. Apart from *Lactobacillus curlis* X25B mentioned in Example 1, the other tested strains were: the positive control *Lactobacillus delbrueckii* DM8909 (trade name "Dingjunsheng") and three *Lactobacillus curlis* strains M60A, L54H, and L54F. These *Lactobacillus curlis* strains were all isolated and identified using the method described in Example 1.

[0153] 2.1. Determination of strain growth and acid production capacity

[0154] The isolated Lactobacillus was inoculated into MRS liquid medium at a 2% inoculum and cultured anaerobically at 37°C. The OD value was measured after 24 hours of growth. Acid production capacity determination: The pH value of the blank MRS liquid medium was measured. The isolated Lactobacillus was inoculated into MRS liquid medium and cultured anaerobically at 37°C. The pH value of the bacterial culture was measured after 24 hours. ΔpH = pH value of MRS liquid medium - pH value of bacterial culture after 24 hours, which represents the quantitative indicator of the acid production capacity of Lactobacillus.

[0155] 2.2. Determination of the strain's ability to produce hydrogen peroxide

[0156] Referring to the method published by Wang Jiang's team, "Analysis of the diversity of vaginal lactobacilli in healthy women and screening of strains with probiotic characteristics," the ability of lactobacilli to produce hydrogen peroxide was determined.

[0157] Solution preparation: Prepare 100 mM PIPES (piperazine N,N'-di-ethanesulfonic acid) and 20 mM TMB (tetramethylbenzidine). Standard curve preparation: First, dilute the 30% H2O2 standard solution to 1 mol / L with 100 mmol / L PIPES. Then, dilute it again with 100 mmol / L PIPES to prepare 0, 20, 40, 60, 80, and 100 μmol / L H2O2 working solutions, respectively. Mix 100 μL of each working solution with 100 μL of 20 mmol / L TMB. Finally, add 2 μL of HRP (horseradish peroxidase 1 mg / mL) to the mixture and mix well. Incubate at room temperature for 10 min. Measure the OD value using a microplate reader and plot the corresponding standard curve.

[0158] Determination of H2O2 production by Lactobacillus: The frozen Lactobacillus strain to be tested and the positive control "Dingjunsheng" Lactobacillus delbrueckii DM8909 were revived and inoculated into liquid culture medium. After anaerobic culture at 37℃ for 24 h, the samples were centrifuged, and the supernatant was collected and mixed with the same volume of 20 mmol / L TMB. Then, HRP (1 mg / mL) was added and mixed well. The samples were incubated at room temperature for 10 min, and the OD value was measured. The concentration of H2O2 produced by Lactobacillus was obtained by substituting the OD value into the standard curve.

[0159] 2.3. Determination of the ability of strains to inhibit Gardnerella vaginalis biofilm formation

[0160] Gardnerella vaginalis (also known as Gardnerella vaginalis or vaginal Gardnerella) is a major pathogen causing bacterial vaginosis. Biofilm formation provides a safe growth environment for Gardnerella vaginalis, allowing it to survive in large numbers within the vaginal environment and disrupting the acidic vaginal environment. In this study, the inhibitory effects of four strains of Lactobacillus curvature on Gardnerella vaginalis biofilm were determined using crystal violet staining.

[0161] Table 1. Results of in vitro probiotic function assays for four strains of Lactobacillus curvature and the positive control DM8909

[0162] Strain number OD value Acid production (ΔpH) H2O2 production (umol / l) Biofilm inhibition rate % DM8909 1.42 1.6 11.7 42.5 M60A 0.77 1.3 4.5 6.5 L54H 0.64 1.5 4.8 5.9 L54F 0.84 1.6 7.8 7.0 X25B 1.66 1.6 34.5 33

[0163] The above results show that different *Lactobacillus curvature* strains exhibit significant differences in growth, in vitro acid production, hydrogen peroxide production, and Gardner biofilm inhibition rate. Among the four *Lactobacillus curvature* strains tested, the *Lactobacillus curvature* X25B strain of this application demonstrates outstanding effects in acid production, hydrogen peroxide production, and biofilm inhibition. The hydrogen peroxide produced by the strain helps maintain a healthy, acidic vaginal environment, and the hydrogen peroxide production capacity of the *Lactobacillus curvature* X25B strain of this application is currently higher than that of the positive control DM8909. Therefore, the combined ability of the *Lactobacillus curvature* X25B strain of this application in both hydrogen peroxide production and biofilm inhibition is stronger than that of the positive control DM8909.

[0164] Example 3. Alleviating effect of Lactobacillus crispatus X25B on vaginitis in mice

[0165] This embodiment uses a mouse vaginitis model infected with Gardnerella vaginalis.

[0166] 3.1. Preparation of resuspension of experimental strains

[0167] Lactobacillus cells stored at -80°C with 50% glycerol were activated three times using MRS medium. After centrifugation at 6000 rpm for 10 min, the supernatant was discarded, and the precipitated cells were washed three times with PBS buffer. Finally, the cells were resuspended in PBS buffer to achieve a viable count of 1 × 10⁻⁶ cells. 8 CFU / mL was used to obtain the *Lactobacillus curvatureensis* bacterial suspension for animal intervention. Similarly, *Gardnerella vaginalis* was activated three times in BHI liquid medium, centrifuged at 6000 rpm for 10 min, and the activated *Gardnerella vaginalis* cells were collected. The cells were washed three times and resuspended in PBS buffer. Finally, a concentration of 1×10⁻⁶ was obtained. 9 CFU / mL Gardnerella vaginalis solution is used for animal modeling during the modeling period.

[0168] 3.2. The alleviating effect of oral administration of Lactobacillus curvaturei X25B on vaginitis in mice.

[0169] 3.2.1. Animal Experiment Design

[0170] Seven-week-old female ICR mice were housed in an SPF animal room with a temperature of 20-23 ℃, humidity of 55±5%, and a light-dark cycle of 12 h.

[0171] Before the experiment, the mice were placed in their breeding environment for one week to acclimatize. The experiment included a blank control group, a model group, a Lactobacillus delbrueckii DM8909 group, and a Lactobacillus curvaturei X25B group, with 10 mice in each group. All mice were fed standard commercial feed with free access to food and water.

[0172] Establishment of a bacterial vaginosis model: Three days before infection (day -3) and on the day of infection (day 0), all mice except the control group were subcutaneously injected with 100 μL of estradiol benzoate (0.5 mg dissolved in 100 μL of sesame oil). On day 0, the vaginal inoculation count was 10 live bacteria. 9 Administer 20 μL of CFU / mL Gardnerella vaginalis suspension to mice for 5 consecutive days, continuing until day 4. After inoculation, invert the mice for 1 minute to prevent the bacterial suspension from flowing out.

[0173] Oral administration experiment: Starting from day 5, the experimental mice were administered 10 mg of oral medication via gavage. 8 Mice were administered 100 μL of CFU / mL Lactobacillus via gavage once daily for 14 days. Control group mice were administered 100 μL of PBS buffer via gavage once daily. After the final gavage, all experimental mice were euthanized by cervical dislocation.

[0174] 3.2.2. Assay of exfoliated vaginal epithelial cells in mice

[0175] Before the final day of intervention, vaginal lavage fluid was collected from mice. 5 μL of the lavage fluid was transferred to a glass slide and gently spread to a coin-sized size using a 10 μL pipette tip. After fixation with the outer flame of an alcohol lamp, Gram staining was performed. The number of exfoliated epithelial cells in three random microscopic fields under 40x magnification was recorded as the exfoliated cell count. Cell morphology and the morphology and staining properties of intracellularly adherent bacteria were observed under 400x and 1000x magnification. The results are shown in Table 2.

[0176] 3.2.3. Histopathological analysis and epithelial thickness measurement of mouse vaginal tissue

[0177] Mouse vaginal tissue was fixed and preserved with 4% tissue fixative, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The HE staining pathological scoring criteria were as follows: a score of 0-5 was assigned based on tissue integrity (clear structure, orderly cell arrangement), where 0 indicates intact tissue structure and 5 indicates complete tissue destruction. The sections were observed under a microscope at 400x magnification, and the epithelial thickness of three randomly selected sections was measured.

[0178] Table 2. Results of vaginal epithelial cell exfoliation and histopathology in mice

[0179] Blank group BV model group DM8909 group M60A X25B group Average number of exfoliated cells 18 173 *** ]] 43 ### ]] 135 65 ### ]] HE pathological score 0 2.7 *** ]]> 1.8 2.4 1.0 # ]]> Vaginal epithelial thickness / um 341.5 97.9 *** ]] 242.7 ### ]]> 155.7 # ]]> 266.9 ### ]]>

[0180] Note: * This indicates a significant difference compared to the control group. # This indicates a significant difference from the model group.

[0181] As shown in Table 2, BV-infected mice experienced significant vaginal epithelial cell shedding and a marked decrease in vaginal epithelial thickness, resulting in a severe pathological condition. Intervention with the positive control drug DM8909 and *Lactobacillus curvatureii* X25B significantly improved the pathological condition in mice, greatly reducing vaginal epithelial cell shedding and significantly increasing vaginal epithelial thickness. Furthermore, *Lactobacillus curvatureii* X25B intervention significantly reduced the pathological score and significantly improved the vaginal health of the mice. In contrast, intervention with *Lactobacillus curvatureii* M60A showed virtually no effect, with all test results significantly worse than those of *Lactobacillus curvatureii* X25B.

[0182] 3.2.4. Determination of sialidase activity and cytokines in mouse vaginal irrigation fluid

[0183] The mouse vaginal irrigation fluid was centrifuged, and 10 μL of the supernatant was used for experiments. Sialidase activity was measured using ELISA, and OD... 450 nm The values ​​are compared with the standard curve.

[0184] Mouse vaginal tissue samples (50 mg) were placed in 300 μL of RIPA lysis buffer containing a 1% protease inhibitor mixture and homogenized using a high-throughput tissue homogenizer (70 Hz, 30 s / time, 10 times) to obtain a vaginal tissue homogenate. The homogenate was centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was used for ELISA to determine myeloperoxidase (MPO) activity and the concentrations of TNF-α, IL-1β, IL-6, and IL-10.

[0185] Table 3. Determination of MPO, inflammatory cytokines, and sialidase in mouse vaginal tissue and vaginal irrigation fluid.

[0186] Blank group BV model group DM8909 group M60A X25B group MPO 37.6 52.2 *** ]]> 40.8 ### ]]> 47.1 40.8 ## ]]> TNF-α 302.7 445.4 *** ]]> 402.7 428.7 336.7 ### <!-- 13 -->]]> IL-10 117.3 95.0 *** ]]> 98.8 94.2 104.4 ## ]]> IL-1β 75.4 101.4 *** ]]> 88.8 98.6 75.8 ## ]]> IL-6 50.3 61.3 * ]]> 53.7 56.3 43.5 ### ]]> Sialidase 44.5 57.1 *** ]]> 47.4 ### ]]> 54.6 51.5 # ]]>

[0187] Note: * This indicates a significant difference compared to the control group. # This indicates a significant difference from the model group.

[0188] As shown in Table 3, the vaginas of BV-infected mice exhibited a significant inflammatory response, specifically manifested as increased sialidase and MPO enzyme activities, elevated levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6, and decreased levels of the anti-inflammatory factor IL-10. Intervention with the positive control drug DM8909 significantly reduced sialidase and MPO enzyme activities in the vagina, improving the inflammatory response to some extent. Intervention with *Lactobacillus curvaturei* X25B not only significantly reduced sialidase and MPO enzyme activities but also significantly reduced levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6, while increasing the level of the anti-inflammatory factor IL-10, greatly improving the inflammatory response and showing better efficacy than the positive control group. Intervention with *Lactobacillus curvaturei* M60A showed virtually no effect; the test results were essentially the same as the BV model group, significantly worse than those with *Lactobacillus curvaturei* X25B. Therefore, oral administration of *Lactobacillus curvaturei* X25B has a good effect in improving BV-induced vaginal inflammation in mice.

[0189] 3.3. The alleviating effect of topical Lactobacillus curvaturei X25B on vaginitis in mice.

[0190] 3.3.1. Animal Experiment Design

[0191] Seven-week-old female ICR mice were housed in an SPF animal room with a temperature of 20-23 ℃, humidity of 55±5%, and a light-dark cycle of 12 h.

[0192] Before the experiment, the mice were placed in their breeding environment for one week to acclimatize. The experiment included a blank control group, a model group, a Lactobacillus delbrueckii DM8909 group, and a Lactobacillus curvaturei X25B group, with 10 mice in each group. All mice were fed standard commercial feed with free access to food and water.

[0193] Establishment of a bacterial vaginosis model: Three days before infection (day -3) and on the day of infection (day 0), all mice except the control group were subcutaneously injected with 100 μL of estradiol benzoate (0.5 mg dissolved in 100 μL of sesame oil). On day 0, the vaginal inoculation count was 10 live bacteria. 9 Administer 20 μL of CFU / mL Gardnerella vaginalis suspension to mice for 5 consecutive days, continuing until day 4. After inoculation, invert the mice for 1 minute to prevent the bacterial suspension from flowing out.

[0194] External application experiment: Starting from day 5, experimental mice were vaginally inoculated with 10 8 20 μL of CFU / mL Lactobacillus was administered to mice. The mice were inverted, and 20 μL of the bacterial suspension, resuspended in PBS, was injected into the vagina using a 200 μL pipette tip. The mice were kept in the inverted position for 1 minute to prevent leakage. This was repeated daily for 14 days. Control mice were inoculated vaginally with 20 μL of PBS buffer once daily. After the final inoculation, all experimental mice were euthanized by cervical dislocation.

[0195] 3.3.2. Assay of exfoliated vaginal epithelial cells in mice

[0196] Before the final day of intervention, vaginal lavage fluid was collected from mice. 5 μL of the lavage fluid was transferred to a glass slide and gently spread to a coin-sized size using a 10 μL pipette tip. After fixation with the outer flame of an alcohol lamp, Gram staining was performed. The number of exfoliated epithelial cells in three random microscopic fields under 40x magnification was recorded as the exfoliated cell count. Cell morphology and the morphology and staining properties of intracellularly adherent bacteria were observed under 400x and 1000x magnification. The results are shown in Table 2.

[0197] 3.3.3. Histopathological analysis and epithelial thickness measurement of mouse vaginal tissue

[0198] Mouse vaginal tissue was fixed and preserved with 4% tissue fixative, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The HE staining pathological scoring criteria were as follows: a score of 0-5 was assigned based on tissue integrity (clear structure, orderly cell arrangement), where 0 indicates intact tissue structure and 5 indicates complete tissue destruction. The sections were observed under a microscope at 400x magnification, and the epithelial thickness of three randomly selected sections was measured.

[0199] Table 4. Results of vaginal epithelial cell exfoliation and histopathology in mice

[0200] Blank group BV model group DM8909 group M60A X25B group Average number of exfoliated cells 18 135 **** ]] 43 ### ]] 98 # ]] 46 ### ]] HE pathological score 0 2.8 **** ]]> 1.2 ## ]]> 2.7 0.8 ### ]] Vaginal epithelial thickness / um 307.8 134.6 **** ]]> 226.6 ### ]]> 152.6 293.5 ### ]]>

[0201] Note: * This indicates a significant difference compared to the control group. # This indicates a significant difference from the model group.

[0202] As shown in Table 4, BV-infected mice experienced significant vaginal epithelial cell shedding and a marked decrease in vaginal epithelial thickness, resulting in a severe pathological condition. Intervention with positive drug strain DM8909 and *Lactobacillus curvatureii* X25B significantly improved the pathological condition in mice, resulting in a significant reduction in vaginal epithelial cell shedding, a significant decrease in pathological scores, and a significant increase in vaginal epithelial thickness. Furthermore, *Lactobacillus curvatureii* X25B was superior to positive drug strain DM8909 in improving epithelial cell thickness, significantly enhancing the health of the mouse vagina. Intervention with *Lactobacillus curvatureii* M60A showed virtually no effect, with all test results significantly worse than those of *Lactobacillus curvatureii* X25B.

[0203] 3.3.4. Determination of sialidase activity and cytokines in mouse vaginal irrigation fluid

[0204] The mouse vaginal irrigation fluid was centrifuged, and 10 μL of the supernatant was used for experiments. Sialidase activity was measured using ELISA, and OD... 450 nm The values ​​are compared with the standard curve.

[0205] Mouse vaginal tissue samples (50 mg) were placed in 300 μL of RIPA lysis buffer containing a 1% protease inhibitor mixture and homogenized using a high-throughput tissue homogenizer (70 Hz, 30 s / time, 10 times) to obtain a vaginal tissue homogenate. The homogenate was centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was used for ELISA to determine myeloperoxidase (MPO) activity and the concentrations of TNF-α, IL-1β, and IL-6.

[0206] Table 5. Determination of MPO, inflammatory cytokines, and sialidase in mouse vaginal tissue and vaginal irrigation fluid.

[0207] Blank group BV model group DM8909 group M60A X25B group MPO 26.9 34.4 * ]]> 30.6 35.1 27.9 # ]]> TNF-α 545.3 709.4 *** ]] 565.2 ## ]]> 674.6 519.1 ## ]]> IL-1β 74.0 120.2 *** ]]> 79.6 ### ]]> 101.2 # ]]> 97.7 # ]] IL-6 58.1 82.2 ** ]]> 60.7 ## ]]> 76.1 64.7 # ]]> Sialidase 44.5 57.1 *** ]] 47.4 ### ]]> 57.4 51.5 # ]]>

[0208] Note: * This indicates a significant difference compared to the control group. # This indicates a significant difference from the model group.

[0209] As shown in Table 5, the vaginas of mice infected with BV exhibited a significant inflammatory response, specifically manifested as increased sialidase and MPO enzyme activities, and elevated levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6. Intervention with the positive control drug DM8909 significantly reduced sialidase activity and the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the vagina, thus improving the inflammatory response to some extent. Intervention with *Lactobacillus curvaturei* X25B not only significantly reduced sialidase and MPO enzyme activities but also significantly reduced the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6, significantly improving the inflammatory response. Intervention with *Lactobacillus curvaturei* M60A showed virtually no effect; the test results were essentially equivalent to those of the BV model group, significantly worse than those of *Lactobacillus curvaturei* X25B. Therefore, topical application of *Lactobacillus curvaturei* has a good effect on improving BV-induced vaginal inflammation in mice.

[0210] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A type of Lactobacillus crispatus, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 63707.

2. A composition comprising the *Lactobacillus curvatureus* of claim 1.

3. The composition of claim 2, wherein the composition further comprises probiotics; the probiotics are bacteria; the bacteria are selected from Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, or any combination thereof.

4. The composition of claim 3, wherein the Lactobacillus bacteria are selected from: Lactobacillus paracasei, Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus rhamnosus, or any combination thereof.

5. A pharmaceutical composition comprising Lactobacillus curvature of claim 1 or the composition of any one of claims 2-4.

6. The pharmaceutical composition of claim 5, wherein it has one or more of the following characteristics: (1) The pharmaceutical composition is formulated for oral or in vitro administration; (2) The pharmaceutical composition is in the form of pills, powders, capsules, tablets, orally soluble granules, liquids, suppositories, enemas, gels and / or creams; (3) The pharmaceutical composition contains Lactobacillus curvature at a concentration of 10 6 Up to 10 12 The amount of CFU / dose present.

7. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition comprises a cryoprotectant, amino acids, vitamins, minerals, peptone, or any combination thereof.

9. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient comprises: Fillers, binders, lubricants, flow aids, thickeners, flavoring agents, stabilizers, suspending agents, surfactants, or any combination thereof.

10. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition further comprises glycerin, skim milk powder, soluble starch, polyethylene glycol, dextran, trehalose, sorbitol, xylooligosaccharide, glutathione, or any combination thereof.

11. The pharmaceutical composition of claim 5, wherein the Lactobacillus curvature is present in the form of a live bacterium.

12. A culture comprising Lactobacillus curvature of claim 1 or a composition of any one of claims 2-4.

13. The culture of claim 12, wherein the culture further comprises a nutrient-providing component.

14. The culture of claim 13, wherein the nutrient-providing component is selected from proteins, carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

15. The culture of claim 12, wherein the culture further comprises a cell-free culture filtrate of Lactobacillus curvature.

16. The culture of claim 12, wherein the culture further comprises a derivative of *Lactobacillus curvatureii*; wherein, The derivative is a metabolite.

17. The culture of claim 12, wherein the culture further comprises a derivative of *Lactobacillus curvatureii*; wherein, The derivative is an enzyme and / or an extracellular polysaccharide.

18. A food product or dietary supplement comprising Lactobacillus curvature of claim 1, or a composition of any one of claims 2-4, or a culture of any one of claims 12-17.

19. The food product or dietary supplement of claim 18, wherein the food product is selected from solid beverages, liquid beverages, compressed candies, puffed foods, protein bars, or nuts.

20. The food product or dietary supplement of claim 18, wherein the food product is a dairy product.

21. The food product or dietary supplement of claim 19, wherein the food product or dietary supplement further comprises prebiotics.

22. The food product or dietary supplement of claim 21, wherein the prebiotic is selected from fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, spirulina, trachomatis polysaccharides, carrot nitrogenous polysaccharides, or any combination thereof.

23. The food product or dietary supplement of claim 19, wherein the Lactobacillus flavus comprises 10 6 Up to 10 12 The amount of CFU / dose present.

24. The food product or dietary supplement of claim 19, wherein the Lactobacillus curvature is present in the form of a live bacterium.

25. A health food product comprising Lactobacillus curvature of claim 1, or the composition of any one of claims 2-4, or the culture of any one of claims 12-17.

26. The health food product according to claim 25, wherein the health food product further comprises prebiotics.

27. The health food product of claim 26, wherein the prebiotic is selected from fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, spirulina, trachomatis polysaccharides, nitrogenous carotene, or any combination thereof.

28. The health food product according to claim 25, wherein the Lactobacillus curvature in the health food product is at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present.

29. The health food product according to claim 25, wherein the Lactobacillus curvature in the health food product exists in the form of live bacteria.

30. A cleaning product comprising Lactobacillus curvature of claim 1, or a composition of any one of claims 2-4, or a culture of any one of claims 12-17.

31. The cleaning product of claim 30, wherein the cleaning product is selected from solid soap, hand sanitizer, shampoo, or any combination thereof.

32. The cleaning product of claim 30, wherein the cleaning product is a vaginal douche, vaginal gel, or vaginal cream.

33. Use of the *Lactobacillus curvature* of claim 1, or the composition of any one of claims 2-4, or the culture of any one of claims 12-17, or the cleaning product of any one of claims 30-32, in the preparation of a medicament for the prevention and / or treatment of vaginitis and / or cervicitis caused by *Gardnerella vaginalis* in female subjects.

34. A method for inhibiting the growth of Gardnerella vaginalis and / or reducing its abundance in an environment, the method comprising: Applying an effective amount of the *Lactobacillus curvatureus* of claim 1, or the composition of any one of claims 2-4, or the pharmaceutical composition of any one of claims 5-11, or the culture of any one of claims 12-17 to the environment.

35. The method of claim 34, wherein the environment is selected from medical instruments.

36. The method of claim 34, wherein the environment is selected from gynecological examination equipment.

37. The method of claim 34, wherein the *Lactobacillus curvature* is at 10 6 Up to 10 12 The amount of CFU / day dose is administered to the environment.