Lactobacillus crispatus X25B and application thereof

Abstract

The invention relates to lactobacillus crispatus X25B and application thereof. In particular, the present application relates to Lactobacillus crispatus, and compositions, pharmaceutical compositions, cultures, food products or dietary supplements and cleaning products comprising the same. The invention also relates to application of the lactobacillus crispatus and the composition containing the lactobacillus crispatus in preparation of medicines.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a Lactobacillus crispatus, and a composition, a pharmaceutical composition, a culture, a food product or a dietary supplement and a cleaning product containing the same. The present invention also relates to the use of the Lactobacillus crispatus and the composition containing the same in preparing medicines. Background Art

[0002] Bacterial vaginosis (BV) is a type of vaginal infection caused by an imbalance of vaginal flora. It is a common gynecological disease in women and has become a public health issue affecting women's daily lives worldwide. The main factors causing BV include decreased estrogen levels, sexual behavior, and the use of antibiotics. Its clinical manifestations include vulvar itching, burning sensation, odor, and abnormal vaginal discharge.

[0003] Under normal conditions, the vaginal flora is dominated by lactobacilli, among which Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus jensenii are the most common species. The hydrogen peroxide, bacteriocins and other substances produced by these dominant strains maintain the acidic and healthy environment of the vagina; if the balance of the vaginal flora is broken, the abundance of lactobacilli decreases and the pathogenic flora of the vagina increases significantly. Among them, Gardnerella vaginalis is the main pathogen of bacterial vaginitis. In addition, when the normal environment of the vagina is destroyed, Candida albicans, Staphylococcus aureus and Escherichia coli may multiply in large numbers and cause reproductive tract infections.

[0004] Conventional drug treatment for BV is mainly through oral or topical use of clindamycin and metronidazole or tinidazole. However, antibiotic therapy is ineffective and has a high recurrence rate; this may be related to the resistance of pathogenic bacteria and their biofilms to antibiotics and the failure to restore the acidic environment of the vagina. In recent years, probiotics have shown good antibacterial activity in the treatment of bacterial vaginitis and help restore the balance of vaginal microecology. There is evidence that hydrogen peroxide-producing lactobacilli have the effect of inhibiting bacterial infection and preventing the occurrence of bacterial vaginitis.

[0005] Currently, there are very limited microecological products on the market for the treatment or relief of bacterial vaginitis. For example, Dingjunsheng is a strain of Lactobacillus delbrueckii DM8909 screened from the vaginal secretions of healthy women by Professor Kang Bai of Dalian Medical University, but the study of the therapeutic effect of this strain on bacterial vaginitis has certain limitations. Therefore, it is particularly necessary to develop effective probiotic strains that are derived from the dominant strains in the vagina of healthy women of childbearing age in my country. Summary of the invention

[0006] The inventor of the present application screened a strain of Lactobacillus crispatus with significant strain-specific probiotic potential from more than 100 strains of healthy female sources through a large number of experiments. The inventor of the present application confirmed through a large number of experiments that the Lactobacillus crispatus has high acid production capacity and high hydrogen peroxide production capacity, can maintain the acidic and healthy environment of the female reproductive tract, and can maintain the normalization of the female reproductive tract microbiome. In addition, the Lactobacillus crispatus of the present application can also significantly inhibit the growth and biofilm formation of Gardnerella, the most important pathogenic bacteria of the vagina. Thus, the applicant has completed the present invention.

[0007] Lactobacillus crispatus

[0008] In a first aspect, the present application provides a Lactobacillus crispatus, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No.63707.

[0009] In certain embodiments, the Lactobacillus crispatus has the ability to produce greater than 15umol / l of hydrogen peroxide under effective culture conditions. In certain embodiments, the Lactobacillus crispatus has the ability to produce 15umol / l to 25umol / l, 25umol / l to 35umol / l, 35umol / l to 45umol / l or more of hydrogen peroxide under effective culture conditions.

[0010] In certain embodiments, the Lactobacillus crispatus is capable of lowering the pH of the female reproductive tract. In certain embodiments, the Lactobacillus crispatus lowers the pH of the female reproductive tract to a pH of pH 6.5 or less, a pH of pH 6 or less, a pH of pH 5.5 or less, a pH of pH 5 or less, a pH of pH 4.5 or less, a pH of pH 4 or less.

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

[0012] In certain embodiments, the Lactobacillus crispatus can reduce the level of proinflammatory factors (e.g., TNF-α, IL-1β, IL-6). In certain embodiments, the Lactobacillus crispatus reduces the level 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%, at least 70%.

[0013] In certain embodiments, the Lactobacillus crispatus can increase the level of anti-inflammatory factors (e.g., IL-10). In certain embodiments, the Lactobacillus crispatus reduces 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%, at least 70%.

[0014] In certain embodiments, the colonies of Lactobacillus crispatus are white, milky white and / or translucent.

[0015] In certain embodiments, the Lactobacillus crispatus is a Gram-positive bacterium.

[0016] It is easy to understand that in the growth and cultivation of microorganisms, especially in the growth and cultivation of bacteria, genetic material may undergo certain changes (for example, mutations of one or several bases), which may occur spontaneously or as a result of mutagenesis by chemical and / or physical agents (for example, mutagens) and / or recombinant DNA techniques known in the art. Therefore, herein, the Lactobacillus crispatus of the present invention includes mutants and / or offspring of the Lactobacillus crispatus with a deposit number of GDMCC No.63707. In certain embodiments, these mutants and / or offspring still retain the functions and / or physiological and biochemical characteristics (for example, one or more functions and / or physiological and biochemical characteristics mentioned above) of the Lactobacillus crispatus with a deposit number of GDMCC No.63707.

[0017] Composition

[0018] In a second aspect, the present application provides a composition comprising the Lactobacillus crispatus as described in the first aspect.

[0019] As used herein, the term "probiotic" is defined as any non-pathogenic microorganism that, when administered in sufficient amounts to a host in the form of live bacteria, can have a beneficial effect on the health of the host.

[0020] In certain embodiments, the composition further comprises a probiotic selected from the group consisting of bacteria, fungi (eg, yeast), or any combination thereof.

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

[0022] In certain embodiments, the bacteria of the genus Lactobacillus are selected from the group consisting of: 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, Lactobacillus reuteri, Lactobacillus spp. reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, or any combination thereof.

[0023] In certain embodiments, the Lactobacillus paracasei is Lactobacillus paracasei 207-27 having a microbial deposit number of GDMCC No.60960.

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

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

[0026] In certain embodiments, the bacterium of the genus Bacillus is selected from: Bacillus subtilis, Bacillus coagulans, or any combination thereof.

[0027] In certain embodiments, the Propionibacterium bacterium is selected from: Propionibacterium shermanii, Propionibacterium freudenreichii, Propionibacterium acidipropionici, or any combination thereof.

[0028] In certain embodiments, the bacteria of the genus Streptococcus are selected from: Streptococcus thermophilus, Streptococcus salivarius, or any combination thereof.

[0029] In certain embodiments, the Lactococcus bacterium is Lactococcus lactis.

[0030] In certain embodiments, the Enterococcus bacterium is selected from the group consisting of Enterococcus faecalis, Enterococcus faecium, or any combination thereof.

[0031] Pharmaceutical composition

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

[0033] In certain 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 certain embodiments, the pharmaceutically acceptable carrier and / or excipient comprises: a filler, a binder, a lubricant, a glidant, a thickener, a flavoring agent, an edible oil, a stabilizer, a suspending agent, a surfactant, or any combination thereof.

[0035] In certain 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), povidone (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, silicon dioxide and its derivatives, hydrolyzates, or any combination thereof.

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

[0037] In certain embodiments, the pharmaceutical composition further comprises glycerol, skim milk powder, soluble starch, polyethylene glycol, dextran, trehalose, sorbitol, xylo-oligosaccharides, glutathione, or any combination thereof.

[0038] In certain embodiments, the pharmaceutical composition includes an amino acid.

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

[0040] In certain embodiments, the pharmaceutical composition includes a vitamin.

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

[0042] In certain embodiments, the pharmaceutical composition includes a mineral.

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

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

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

[0046] The pharmaceutical composition of the present invention can be administered to mammals including humans by various routes. The mode of administration can be any mode commonly used in the art. For example, it can be administered orally, in vitro, intravenously, intramuscularly, subcutaneously, etc. In certain embodiments, the pharmaceutical composition is formulated for oral administration or in vitro administration.

[0047] In certain embodiments, the pharmaceutical composition comprises a preparation of Lactobacillus crispatus.

[0048] In certain embodiments, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), oil drops, film-coated tablets, orodispersible granules, liquids, suppositories, enema gels, and / or creams.

[0049] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutical excipient (eg, an excipient for preparing a powder, tablet, capsule, or oil drop).

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

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

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

[0053] In certain embodiments, when the pharmaceutical composition is a liquid, each milliliter of the composition contains 10 6 Up to 10 12CFU of Lactobacillus crispatus (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL).

[0054] In certain embodiments, the Lactobacillus crispatus in the pharmaceutical composition is present in the form of live bacteria or in the form of dead bacteria.

[0055] Culture

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

[0057] In certain embodiments, the culture further comprises components that provide nutrients (eg, solid or liquid culture medium, feeder cell layers).

[0058] In certain embodiments, the nutritional ingredients are selected from proteins (eg, enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0059] In certain embodiments, the culture further comprises a cell-free culture filtrate of Lactobacillus crispatus.

[0060] In certain embodiments, the culture further comprises a derivative of Lactobacillus crispatus; wherein the derivative is selected from a metabolite, an enzyme, a cell structure component (e.g., a cell wall or a component thereof), an exopolysaccharide, a bacteriocin, a compound containing an immunogenic component, or any combination thereof.

[0061] Food products or dietary supplements or health foods

[0062] In a fifth aspect, the present application provides a food product or a dietary supplement or a health food, comprising the Lactobacillus crispatus described in the first aspect, the composition described in the second aspect, or the culture described in the fourth aspect.

[0063] In the text, the term "food" is broad, including food and drinks for humans, and also covers food and drinks (i.e. feed) for animals. In certain embodiments, the food product is suitable for and designed for human consumption. In certain embodiments, the food product is selected from solid beverages, liquid beverages, tablet 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, lactobacillus beverages, cheese).

[0064] In the text, "dietary supplements" refer to edible products that can provide beneficial effects to consumers. They are also called nutritional supplements, nutrients, dietary supplements, etc. They are used as an auxiliary means of diet to supplement the amino acids, trace elements, vitamins, minerals, etc. required by the human body.

[0065] In certain embodiments, the food product, dietary supplement or nutraceutical further comprises a prebiotic.

[0066] In certain embodiments, the prebiotic is selected from fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharides, carrot nitrogen-containing polysaccharides, or any combination thereof.

[0067] In certain embodiments, the food product or dietary supplement or nutraceutical further comprises additional additives.

[0068] In certain embodiments, the additional additives are selected from proteins (eg, enzymes), carbohydrates, fats, vitamins, minerals, dietary fiber, amino acids, or any combination thereof.

[0069] In certain 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 certain embodiments, the amino acid is selected from tryptophan, phenylalanine, threonine, leucine, isoleucine, histidine, arginine, or any combination thereof.

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

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

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

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

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

[0076] In certain embodiments, the Lactobacillus crispatus is present in the food product, dietary supplement or health food in the form of live bacteria or in the form of killed bacteria.

[0077] Cleaning Products

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

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

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

[0081] use

[0082] In the seventh aspect, the present application provides the use of Lactobacillus crispatus as described in the first aspect, the composition as described in the second aspect, the pharmaceutical composition as described in the third aspect, the culture as described in the fourth aspect, the food product or dietary supplement or health food as described in the fifth aspect, or the cleaning product as described in the sixth aspect in the preparation of a medicament for preventing and / or treating diseases and / or symptoms related to the female urogenital tract in a subject.

[0083] In certain embodiments, the female urogenital tract-related disease is caused by an imbalance in the reproductive tract microbiota and / or an abnormality in the reproductive tract pH value.

[0084] In certain embodiments, the female urogenital tract-related disease is associated with a reduction in the level of a Lactobacillus bacterial strain in the reproductive tract ecosystem.

[0085] In certain embodiments, the female urogenital tract-related disease is associated with an increased proliferation of at least one microorganism belonging to the following species in the reproductive tract ecosystem: Gardnerella vaginalis, Candida albicans, Streptococcus aureus, Atopobium vaginae, or any combination thereof.

[0086] In certain embodiments, the female urogenital tract-related disease is associated with an increased proliferation of at least one microorganism belonging to 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 certain embodiments, the female urogenital tract-related disorder is associated with an elevated pH in the reproductive tract environment.

[0088] Therefore, in certain embodiments, the medicament is used to maintain the balance of the female reproductive tract microbiota, reduce the pH value of the female reproductive tract, increase the level of Lactobacillus bacterial strains in the female reproductive tract ecosystem and / or reduce the level of Gardnerella bacterial strains in the female reproductive tract.

[0089] In certain embodiments, the strain of Gardnerella is Gardnerella.

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

[0091] In certain embodiments, the symptom is selected from the group consisting of: changes in menstrual cycles, vaginal itching, vaginal redness, abnormal discharge, vaginal pain, or any combination thereof.

[0092] In certain embodiments, the medicament 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 certain embodiments, the medicament prevents and / or treats inflammatory diseases associated with the female urogenital tract by reducing sialidase activity and / or myeloperoxidase (MPO) activity.

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

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

[0096] In the eighth aspect, the present application provides the use of Lactobacillus crispatus as described in the first aspect, the composition as described in the second aspect, the pharmaceutical composition as described in the third aspect, the culture as described in the fourth aspect, the food product or dietary supplement or health food as described in the fifth 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 certain embodiments, the medicament inhibits the growth of a strain of the genus Gardnerella by increasing the level of hydrogen peroxide (H2O2).

[0098] In certain embodiments, the medicament inhibits the growth of the strain of the genus Gardnerella by lowering the pH level of the environment.

[0099] In certain embodiments, the medicament is used to inhibit biofilm formation of a strain of the genus Gardnerella, thereby inhibiting the growth of the strain of the genus Gardnerella.

[0100] In certain embodiments, the medicament is used to inhibit the growth and / or reduce the amount of Gardnerella in a female, or to inhibit the growth and / or reduce the amount of Gardnerella in the environment.

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

[0102] As used herein, the term "mucosa" refers to the membranes that line various cavities in the mammalian body and cover the surfaces of internal organs. It consists of one or more layers of epithelial cells covered by a layer of loose connective tissue. It is primarily of endodermal origin and is continuous with the skin at various body openings, such as the eyes, ears, inside of the nose and mouth, lips, vagina, urethral opening, and anus.

[0103] In certain embodiments, the environment includes a home, a workplace, a laboratory, an industrial environment, an aquatic environment, a medical instrument, and / or a gynecological examination tool.

[0104] method

[0105] In the ninth aspect, the present application provides a method for inhibiting the growth of Gardnerella in a female body and / or reducing its amount, the method comprising: administering to a female subject in need thereof an effective amount of Lactobacillus crispatus 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 food product or dietary supplement or health food as described in the fifth aspect, or the cleaning product as described in the sixth aspect.

[0106] In certain embodiments, the method comprises: implanting the Lactobacillus crispatus of the first aspect, the composition of the second aspect, or the pharmaceutical composition of the third aspect into the reproductive tract of a female subject.

[0107] In certain embodiments, the method comprises: applying (e.g., applying, spraying) the Lactobacillus crispatus as described in the first aspect, the composition as described in the second aspect, the pharmaceutical composition as described in the third aspect, or the cleaning product as described in the sixth aspect to the reproductive tract and / or perineum of a female subject.

[0108] In certain embodiments, the method comprises: allowing a female subject to orally (e.g., orally) administer Lactobacillus crispatus described in the first aspect, or the composition described in the second aspect, or the pharmaceutical composition described in the third aspect, or the food product, dietary supplement, or health food described in the fifth aspect.

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

[0110] In certain embodiments, the female body includes the reproductive tract and / or mucosa.

[0111] In certain embodiments, the Lactobacillus crispatus is present in an amount of 10 6 Up to 10 12 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 109 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the subject.

[0112] In the tenth aspect, the present application provides a method for inhibiting the growth of Gardnerella in an environment and / or reducing its amount, the method comprising: applying an effective amount of Lactobacillus crispatus 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 to the environment.

[0113] In certain embodiments, the environment includes a home, a workplace, a laboratory, an industrial environment, an aquatic environment, a medical instrument, and / or a gynecological examination tool.

[0114] In certain embodiments, the Lactobacillus crispatus is present in an amount of 10 6 Up to 10 12 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 10 9 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the environment.

[0115] In the eleventh aspect, the present 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 thereof an effective amount of Lactobacillus crispatus 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 food product or dietary supplement or health food as described in the fifth aspect, or the cleaning product as described in the sixth aspect.

[0116] In certain embodiments, the female urogenital tract-related disease is caused by an imbalance in the reproductive tract microbiota and / or an abnormal pH value in the reproductive tract.

[0117] In certain embodiments, the female urogenital tract-related disease is associated with a reduction in the level of a Lactobacillus bacterial strain in the reproductive tract ecosystem.

[0118] In certain embodiments, the female urogenital tract-related disease is associated with an increased proliferation of at least one microorganism belonging to the genus Gardnerella, Dorea, Streptococcus, Anaerococcus, Fingoldia, Prevotella, Other Steineria, Staphylococcus, Corynebacterium, or any combination thereof in the reproductive tract ecosystem.

[0119] In certain embodiments, the female urogenital tract-related disorder is associated with an elevated pH in the reproductive tract environment.

[0120] In certain embodiments, the strain of Gardnerella is Gardnerella.

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

[0122] In certain embodiments, the symptom is selected from the group consisting of: changes in menstrual cycles, vaginal itching, vaginal redness, abnormal discharge, vaginal pain, or any combination thereof.

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

[0124] In certain embodiments, the Lactobacillus crispatus is present in an amount of 10 6 Up to 10 12 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 10 9 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the subject.

[0125] Definition of terms

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

[0127] As used herein, the term "mucosa" refers to the membranes that line various cavities in the mammalian body and cover the surfaces of internal organs. It consists of one or more layers of epithelial cells covered by a layer of loose connective tissue. It is primarily of endodermal origin and is continuous with the skin at various body openings, such as the eyes, ears, inside of the nose and mouth, lips, vagina, urethral opening, and anus.

[0128] As used herein, the term "genital tract microbiota" or "genital tract ecosystem" refers to a group of microorganisms that reside in the female urogenital tract (preferably the vagina) and live in balance with each other and with the reproductive tract environment that accommodates them. Thus, the term "imbalance in the reproductive tract microbiota" 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 the present invention, the expression "pathogenic bacteria" refers to microorganisms that are able to exist in the vaginal ecosystem and have the potential to be pathogenic.

[0130] In this article, the term "therapeutically effective amount" or "effective amount" can refer to the amount of the strain in the composition or pharmaceutical composition, when applied as part of the desired dosage regimen (to humans or animals, preferably humans), according to the clinically acceptable criteria for the disorder or condition of the subject to be treated, such as with a reasonable benefit-risk ratio applicable to any medical treatment, to relieve symptoms, improve the condition or slow down the onset of the disease condition. The therapeutically effective amount can vary according to the age, weight, sex, dosage form, health status and severity of the disease of different subjects to be treated. In addition, the frequency can be determined by a doctor or pharmacist, and the dose can be administered one to several times a day at fixed time intervals.

[0131] As used herein, the term "CFU (Colony-Forming Units)" refers to the total number of microorganisms such as bacteria, fungi, yeast, etc. in a product, and is usually used for calculation of viable bacteria.

[0132] As used herein, the term "CFU / dose" means the amount of bacteria present in a composition / food product or dietary supplement or health food / pharmaceutical composition provided to a subject each day or each time. For example, in certain embodiments, the amount of Lactobacillus crispatus in the food product or dietary supplement or health food is 10 6 Up to 10 12 In this embodiment, if Lactobacillus crispatus is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject every day or each time may contain about 10 CFU / dose. 6 Up to 10 12Of course, alternatively, this amount of bacteria can be divided into multiple administrations, as long as the total amount of Lactobacillus crispatus received by the subject in any particular time (e.g., every 24-hour period) is from about 10 6 to about 10 12 CFU of bacteria, i.e. Lactobacillus crispatus in food products, dietary supplements or health foods meeting the above requirements, is 10 6 Up to 10 12 The amount of CFU / dose is present.

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

[0134] As used herein, the term "subject" is an animal. Animals may include, but are not limited to, primates, farm animals, sports 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; minks; goats; cattle; horses; and non-human primates, such as apes and monkeys.

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

[0136] As used herein, the term "sialidase" is also referred to as "neuraminidase", which is the main virulence factor of the pathogen Gardnerella of bacterial vaginosis (BV). Sialidase can be detected in cervicovaginal lavage fluid of human women. A large number of studies have shown that the level of sialidase activity in vaginal secretions of women with BV is higher than that of women without BV (e.g., healthy women). Therefore, in current clinical diagnosis, sialidase positivity in vaginal secretions has been used as an important diagnostic indicator for BV.

[0137] As used herein, the term "MPO" is an enzyme present in white blood cells. MPO is often used to evaluate the diagnosis and treatment of diseases such as inflammatory responses and infections. Generally, a high MPO level may mean that the host body has inflammation, infection, autoimmune diseases, etc. During the inflammatory process, white blood cells release a large amount of MPO to eliminate bacteria, viruses and other pathogens. Therefore, the MPO level in the cervical vaginal lavage fluid of patients with bacterial vaginosis is usually elevated.

[0138] Advantageous Effects of the Invention

[0139] The Lactobacillus crispatus of the present application has high acid production capacity and high hydrogen peroxide production capacity, can maintain the acidic and healthy environment of the female reproductive tract, and can maintain the balance of the female reproductive tract microbial community. In addition, the Lactobacillus crispatus of the present application can also significantly inhibit the growth and biofilm formation of the most important pathogenic bacteria of the reproductive tract-Gardnerella. And, it can significantly reduce the activity of sialidase and myeloperoxidase (MPO) in the vagina, significantly reduce the level of pro-inflammatory factors (for example, TNF-α, IL-1β and IL-6), and significantly improve the level of anti-inflammatory factors (for example, IL-10). Therefore, the Lactobacillus crispatus of the present application has a high application value in female reproductive health (especially female vaginal health).

[0140] Embodiments of the present invention will be described in detail below in conjunction with examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become implementable for those skilled in the art.

[0141] Notes on the Deposit of Biological Materials

[0142] Lactobacillus crispatus X25B has been preserved at the Guangdong Microbial Culture Collection Center (GDMCC) located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou. It has the preservation number GDMCC No. 63707 and the preservation time is August 2, 2023. DETAILED DESCRIPTION

[0143] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but 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 carried out basically according to 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 the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., ed., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH) (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), and ANIMALCELLCULTURE (RI Freshney, ed. (1987)).

[0145] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases 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 more than 100 strains of bacteria from vaginal swabs of 60 healthy women who underwent physical examinations at Dalian Maternal and Child Health Hospital, and screened out a strain of Lactobacillus crispatus, which was named Lactobacillus crispatus X25B. Among them, the inclusion criteria for sample donors are: age 22-35 years old; regular menstruation, and not in the menstrual period when sampling; no sexual life within 72 hours, no vaginal medication and vaginal washing; no diagnosis of gynecological inflammatory diseases and urinary inflammatory diseases within half a year; no positive signs found in gynecological examinations; no antibiotics taken within 1 month, no sex hormones taken within 3 months; no hysterectomy or traumatic treatment of the cervix and uterus; patients have not received immunosuppressive treatment recently; patients have no severe heart, liver, or kidney dysfunction and related complications. Vaginal swab samples are immediately stored in a -80℃ refrigerator after collection.

[0148] The strains were cultured in MRS liquid medium, modified MRS solid medium (adding 2% agar and 1% calcium carbonate to the prepared MRS liquid medium, autoclaving at 121°C for 15 min), BHI liquid medium, and BHI solid medium (adding 2% agar to the prepared liquid medium, autoclaving at 121°C for 15 min). Before use, when the temperature drops to about 50°C, add 10% sterile defibrinated sheep blood and lactobacillus selective agar.

[0149] Soak the vaginal swab in saline and mix thoroughly for 5 minutes. Take 100 μl of liquid from each sample and spread it on modified MRS, LBS, and BHI solid culture media, respectively. Culture anaerobically at 37°C for 48-72 hours, pick medium-sized, raised, milky white round colonies for Gram staining microscopy, and observe the bacterial morphology and staining under an optical microscope. Select monoclonal colonies of Gram-positive bacteria in the shape of long rods or clubs and continue to purify until they are determined to be pure bacteria, and pick monoclonal colonies for 16S rRNA sequencing.

[0150] Sequencing result shows that the bacterial strain separated by the present application is lactobacillus crispatus. Further, adopt PacBio Sequel2 sequencing platform to carry out whole genome determination respectively to lactobacillus crispatus X25B, and compare with the whole genome sequence of the lactobacillus crispatus bacterial strain recorded in the NCBI database. Wherein, the genome length of lactobacillus crispatus X25B is 2185354bp, and GC contains 36.87%. Chromosomal genome contains 2270 coding genes, 66 tRNA genes, 4 rRNA genes (comprising 5s rRNA, 16s rRNA and 23s rRNA), 166 other types of RNA genes. Comprehensive the experimental result of above-mentioned PCR identification and whole genome sequencing, the present application has obtained a brand-new lactobacillus crispatus X25B, and it has been preserved.

[0151] Example 2. Determination of probiotic function of strains in vitro

[0152] The strains used in this embodiment are all from the probiotics library of By-Health Women. Except for the Lactobacillus crispatus X25B mentioned in Example 1, the remaining strains tested are: positive control Lactobacillus delbrueckii DM8909 (its trade name is "Dingjunsheng") and 3 kinds of Lactobacillus crispatus M60A, L54H and L54F, which are all separated and identified by 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 culture medium at an inoculum rate of 2%, cultured anaerobically at 37°C, and the OD value after 24 hours of growth was measured. Acid production capacity determination: The pH value of the blank MRS liquid culture medium was measured. The isolated lactobacillus was inoculated into MRS liquid culture medium, cultured anaerobically at 37°C, and the pH value of the bacterial solution was measured for 24 hours. ΔpH = pH value of MRS liquid culture medium - pH value of bacterial solution at 24 hours, which is a quantitative indicator of the acid production capacity of lactobacillus.

[0155] 2.2. Determination of the ability of strains to produce hydrogen peroxide

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

[0157] Solution preparation: Prepare 100mM PIPES (piperazine N,N'-di-ethanesulfonic acid) and 20mM TMB (tetramethylbenzidine). Preparation of standard curve: First, dilute the 30% H2O2 standard solution to 1mol / L with 100mmol / L PIPES, and then dilute it to 0, 20, 40, 60, 80, 100μmol / L H2O2 working solution with 100mmol / L PIPES; take 100μL of working solution and mix with 100μL 20mmol / L TMB respectively, and finally add 2μL HRP (horseradish peroxidase 1mg / mL) to the mixed solution and mix well, incubate at room temperature for 10min, use a microplate reader to measure the OD value and make the corresponding standard curve.

[0158] Determination of H2O2 production by lactobacillus: thawed frozen lactobacillus strains to be tested and positive control "Dingjunsheng" Lactobacillus delbrueckii DM8909, inoculated into liquid culture medium, cultured anaerobically at 37°C for 24 hours, centrifuged, aspirated supernatant and mixed with the same volume of 20 mmol / L TMB, then added HRP (1 mg / mL) and mixed, incubated at room temperature for 10 minutes, measured OD value, and substituted into the above standard curve to obtain the concentration of H2O2 produced by lactobacillus.

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

[0160] Gardnerella vaginalis (also known as Gardnerella or vaginal Gardnerella) is the main pathogen of bacterial vaginitis. The formation of biofilm provides a safe growth environment for vaginal Gardnerella, allowing vaginal Gardnerella to survive in large quantities in the vaginal environment, causing damage to the vaginal acidic environment. In this example, 4 strains of Lactobacillus crispatus were tested by crystal violet staining to determine their inhibitory effect on vaginal Gardnerella biofilm.

[0161] Table 1. Results of in vitro probiotic function assay of 4 strains of Lactobacillus crispatus and positive control DM8909

[0162] Strain number OD value Acid production (ΔpH) <![CDATA[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 crispatus have large strain differences in growth, in vitro acid production, hydrogen peroxide production and Gardner biofilm inhibition rate, and Lactobacillus crispatus X25B of the present application has outstanding effects in acid production, hydrogen peroxide production and biofilm inhibition among the 4 Lactobacillus crispatus tested. The hydrogen peroxide produced by the strain can help maintain the acidic and healthy environment of the vagina, and the ability of Lactobacillus crispatus X25B of the present application to produce hydrogen peroxide is now higher than that of the positive control DM8909. Therefore, the comprehensive ability of Lactobacillus crispatus X25B of the present application in terms of hydrogen peroxide production and biofilm inhibition is stronger than that of the positive control DM8909.

[0164] Example 3. Effect of Lactobacillus crispatus X25B on the alleviation of vaginitis in mice

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

[0166] 3.1. Preparation of experimental strain resuspension

[0167] The lactobacilli stored in a -80°C refrigerator with 50% glycerol were activated for three generations using MRS medium, centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the precipitated bacteria were washed three times with PBS buffer and finally resuspended in PBS buffer to make the number of viable bacteria reach 1×10 8 CFU / mL, and the Lactobacillus crispatus bacterial solution of animal experiment intervention was obtained. Similarly, after Gardnerella vaginalis was activated for three generations in BHI liquid medium, it was centrifuged at 6000rpm for 10min, and the cells of Gardnerella vaginalis after three generations of activation were collected, washed three times, and resuspended in PBS buffer. Finally, a concentration of 1×10 9 CFU / mL vaginal Gardnerella liquid is used in the modeling period of animal experiments.

[0168] 3.2. Effect of oral administration of Lactobacillus crispatus X25B on the alleviation of 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°C, a humidity of 55±5%, and a light-dark cycle of 12 h alternating cycles.

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

[0172] Bacterial vaginosis model establishment: 3 days before infection (day -3) and on the day of infection (day 0), except for the control group, all other mice were subcutaneously injected with 100ul of estradiol benzoate (0.5mg dissolved in 100ul of sesame oil). The number of live bacteria inoculated in the vagina on day 0 was 10 9 20ul of vaginal Gardnerella suspension was added for 5 consecutive days until the end of vaginal Gardnerella inoculation on day 4. After inoculation, the mice were inverted for 1 min to prevent the bacterial solution from flowing out.

[0173] Oral experiment: Starting from the 5th day, the experimental mice were gavaged for 10 8 CFU / mL lactobacillus 100ul was gavaged once a day for 14 days. The control group mice were gavaged with 100ul PBS buffer once a day. After the last gavage, all experimental mice were killed by cervical dislocation.

[0174] 3.2.2. Mouse vaginal epithelial exfoliated cell assay

[0175] On the last day before intervention, 5ul of vaginal lavage fluid was collected from mice and transferred to a glass slide. The sample was gently smeared with a 10ul pipette tip until it was the size of a coin. After fixation with an alcohol lamp, Gram staining was performed. The number of epithelial cells shed in three microscope fields was randomly counted under a 40x microscope as the shed cell count. The morphology of the cells and the morphology and staining of the bacteria adhered to the cells were observed under a 400x and 1000x microscope. The results are shown in Table 2.

[0176] 3.2.3. Mouse vaginal histopathological analysis and epithelial thickness determination

[0177] The mouse vaginal tissue was fixed and preserved with 4% tissue fixative, embedded in paraffin, and sliced, and stained with hematoxylin and eosin (H&E). The HE staining pathological scoring standard is: according to the integrity of the tissue (clear structure, neatly arranged cells), a score of 0-5 is given, where 0 means that the tissue structure is intact and 5 means that the structure is completely destroyed. The sliced ​​specimens were observed under a microscope at 400 times magnification and the thickness of the epithelial layer of 3 random sample slices was measured.

[0178] Table 2. Mouse vaginal epithelial cell shedding and histopathological results

[0179]

[0180] Note: * Indicates significant difference from the blank group. # Indicates significant difference from the model group

[0181] From the results in Table 2, it can be seen that the vaginal epithelial cells of mice infected with BV shed a large number of times, the thickness of the vaginal epithelium was significantly reduced, and a more serious pathological state appeared; the intervention of positive drugs DM8909 and Lactobacillus crispatus X25B significantly improved the pathological state of mice, greatly reducing the amount of vaginal epithelial cell shedding, significantly increasing the thickness of the vaginal epithelium, and the intervention of Lactobacillus crispatus X25B significantly reduced the pathological score and significantly improved the health of the vagina of mice. However, the intervention of Lactobacillus crispatus M60A showed basically no effect, and the test results were significantly worse than those of Lactobacillus crispatus X25B.

[0182] 3.2.4. Determination of neuraminidase activity and cytokines in mouse vaginal lavage fluid

[0183] The mouse vaginal lavage fluid was centrifuged and 10ul of the supernatant was used for the experiment. The sialidase activity was determined by ELISA and the OD 450 nm Values ​​were compared with the standard curve.

[0184] The mouse vaginal tissue sample (50 mg) was placed in 300 ul of RIPA lysis buffer containing 1% protease inhibitor mixture, and crushed with a high-throughput tissue grinder (70 Hz, 30 s / time, 10 times) to obtain vaginal tissue homogenate. The sample was centrifuged at 14000 rpm for 15 min at 4°C, and the supernatant was taken for myeloperoxidase (MPO) activity determination and TNF-α, IL-1β, IL-6, and IL-10 concentration determination using the ELISA method.

[0185] Table 3. Determination of MPO, inflammatory cytokines and sialidase in vaginal lavage fluid of mice

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

[0187] Note: * Indicates significant difference from the blank group. # Indicates significant difference from the model group

[0188] As can be seen from the results in Table 3, the vagina of mice infected with BV showed a significant inflammatory response, which was specifically manifested by the increase of sialidase and MPO enzyme activities in the vagina, the increase of the levels of proinflammatory factors TNF-α, IL-1β, and IL-6, and the decrease of the level of anti-inflammatory factor IL-10; the intervention of the positive drug DM8909 significantly reduced the sialidase and MPO enzyme activities in the vagina, and improved the inflammatory response to a certain extent; while the intervention of Lactobacillus crispatus X25B not only significantly reduced the sialidase and MPO enzyme activities, but also significantly reduced the levels of proinflammatory factors TNF-α, IL-1β, and IL-6, and increased the level of anti-inflammatory factor IL-10, which greatly improved the inflammatory response, and the effect was better than that of the positive drug group. However, the intervention of Lactobacillus crispatus M60A showed basically no effect, and the test results were basically the same as those of the BV model group, which was significantly worse than that of Lactobacillus crispatus X25B. Therefore, oral administration of Lactobacillus crispatus X25B has a good effect in improving BV-induced vaginal inflammation in mice.

[0189] 3.3. Effect of topical application of Lactobacillus crispatus X25B on the alleviation of 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°C, a humidity of 55±5%, and a light-dark cycle of 12 h alternating cycles.

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

[0193] Bacterial vaginosis model establishment: 3 days before infection (day -3) and on the day of infection (day 0), except for the control group, all other mice were subcutaneously injected with 100ul of estradiol benzoate (0.5mg dissolved in 100ul of sesame oil). The number of live bacteria inoculated in the vagina on day 0 was 10 9 CFU / mL vaginal Gardnerella suspension 20ul, for 5 consecutive days, until the end of vaginal Gardnerella inoculation on day 4. After inoculation, the mice were inverted for 1min to prevent the bacterial solution from flowing out.

[0194] External use experiment: Starting from the 5th day, the experimental mice were inoculated with 10 8CFU / mL lactobacillus 20ul, invert the mouse, use a 200ul pipette tip to suck up 20ul of PBS resuspended bacterial solution and inject it into the mouse vagina, maintain the inverted posture for 1 minute to prevent the bacterial solution from flowing out. Inoculate once a day for a total of 14 days. The control group of mice was inoculated with 20ul PBS buffer in the vagina once a day. After the last inoculation, all experimental mice were killed by cervical dislocation.

[0195] 3.3.2. Mouse vaginal epithelial exfoliated cell assay

[0196] On the last day before intervention, 5ul of vaginal lavage fluid was collected from mice and transferred to a glass slide. The sample was gently smeared with a 10ul pipette tip until it was the size of a coin. After fixation with an alcohol lamp, Gram staining was performed. The number of epithelial cells shed in three microscope fields was randomly counted under a 40x microscope as the shed cell count. The morphology of the cells and the morphology and staining of the bacteria adhered to the cells were observed under a 400x and 1000x microscope. The results are shown in Table 2.

[0197] 3.3.3. Mouse vaginal histopathological analysis and epithelial thickness determination

[0198] The mouse vaginal tissue was fixed and preserved with 4% tissue fixative, embedded in paraffin, and sliced, and stained with hematoxylin and eosin (H&E). The HE staining pathological scoring standard is: according to the integrity of the tissue (clear structure, neatly arranged cells), a score of 0-5 is given, where 0 means that the tissue structure is intact and 5 means that the structure is completely destroyed. The sliced ​​specimens were observed under a microscope at 400 times magnification and the thickness of the epithelial layer of 3 random sample slices was measured.

[0199] Table 4. Mouse vaginal epithelial cell shedding and histopathological results

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

[0201] Note: * Indicates significant difference from the blank group. # Indicates significant difference from the model group

[0202] As can be seen from the results in Table 4, vaginal epithelial cells of BV-infected mice shed in large quantities, the thickness of the vaginal epithelium was significantly reduced, and a more serious pathological state appeared; the intervention of positive drug strains DM8909 and Lactobacillus crispatus X25B significantly improved the pathological state of mice, significantly reduced the amount of vaginal epithelial cell shedding, significantly reduced the pathological score, and significantly increased the thickness of the vaginal epithelium. Moreover, Lactobacillus crispatus X25B was superior to positive drug strains DM8909 in improving the thickness of epithelial cells, and significantly improved the health of the vagina of mice. However, the intervention of Lactobacillus crispatus M60A showed basically no effect, and the test results were significantly worse than those of Lactobacillus crispatus X25B.

[0203] 3.3.4. Determination of neuraminidase activity and cytokines in mouse vaginal lavage fluid

[0204] The mouse vaginal lavage fluid was centrifuged and 10ul of the supernatant was used for the experiment. The sialidase activity was determined by ELISA and the OD 450 nm Values ​​were compared with the standard curve.

[0205] The mouse vaginal tissue sample (50 mg) was placed in 300 ul of RIPA lysis buffer containing 1% protease inhibitor mixture, and crushed with a high-throughput tissue grinder (70 Hz, 30 s / time, 10 times) to obtain vaginal tissue homogenate. The sample was centrifuged at 14000 rpm for 15 min at 4°C, and the supernatant was taken for myeloperoxidase (MPO) activity determination and TNF-α, IL-1β, and IL-6 concentration determination using the ELISA method.

[0206] Table 5 Determination of MPO, inflammatory cytokines and sialidase in vaginal lavage fluid of mice

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

[0208] Note: * Indicates significant difference from the blank group. # Indicates significant difference from the model group

[0209] As can be seen from the results in Table 5, the vagina of mice infected with BV showed a significant inflammatory response, which was specifically manifested by the increase of sialidase activity and MPO enzyme activity in the vagina, and the increase of the levels of proinflammatory factors TNF-α, IL-1β, and IL-6; the intervention of the positive drug DM8909 significantly reduced the sialidase activity and the levels of proinflammatory factors TNF-α, IL-1β, and IL-6 in the vagina, and improved the inflammatory response to a certain extent; while the intervention of Lactobacillus crispatus X25B not only significantly reduced the sialidase activity and MPO enzyme activity, but also significantly reduced the levels of proinflammatory factors TNF-α, IL-1β, and IL-6, and significantly improved the inflammatory response. However, the intervention of Lactobacillus crispatus M60A showed basically no effect, and the test results were basically the same as those of the BV model group, which was significantly worse than Lactobacillus crispatus X25B. Therefore, topical Lactobacillus crispatus has a good effect in improving vaginal inflammation in mice caused by BV.

[0210] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A Lactobacillus crispatus, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No.63707.

2. The Lactobacillus crispatus of claim 1, having one or more characteristics selected from the following: (1) The Lactobacillus crispatus has the ability to produce greater than 15umol / l (e.g., 15umol / l to 25umol / l, 25umol / l to 35umol / l, 35umol / l to 45umol / l or greater) hydrogen peroxide under effective culture conditions; (2) the Lactobacillus crispatus is able to reduce the pH of the female reproductive tract (e.g., a pH reduction of 1 or more); (3) the Lactobacillus crispatus can inhibit the growth of strains of the genus Gardnerella and / or inhibit the biofilm formation of strains of the genus Gardnerella; (4) The Lactobacillus crispatus can reduce the levels of pro-inflammatory factors (e.g., TNF-α, IL-1β, IL-6); (5) The Lactobacillus crispatus can increase the level of anti-inflammatory factors (e.g., IL-10); (6) The colonies of the Lactobacillus crispatus are white, milky white and / or translucent; (7) The Lactobacillus crispatus is a Gram-positive bacterium.

3. A composition comprising the Lactobacillus crispatus according to claim 1 or 2; Preferably, the composition further comprises a probiotic selected from the group consisting of bacteria, fungi (e.g. yeast), or any combination thereof; Preferably, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria of the genus Lactobacillus are selected from: Lactobacillus paracasei, Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus rhamnosus, or any combination thereof; Preferably, the Lactobacillus paracasei is Lactobacillus paracasei 207-27 with a deposit number of GDMCC No.60960.

4. A pharmaceutical composition comprising the Lactobacillus crispatus according to claim 1 or 2 or the composition according to claim 3; Preferably, 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; Preferably, the pharmaceutically acceptable carrier and / or excipient comprises: Fillers, binders, lubricants, glidants, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof; Preferably, the pharmaceutical composition further comprises a cryoprotectant; Preferably, the pharmaceutical composition further comprises glycerol, skim milk powder, soluble starch, polyethylene glycol, dextran, trehalose, sorbitol, xylo-oligosaccharide, glutathione, or any combination thereof; Preferably, the pharmaceutical composition is formulated for oral administration or in vitro administration; Preferably, the pharmaceutical composition comprises a preparation of Lactobacillus crispatus; Preferably, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), oil drops, film-coated preparations, orally dissolving granules, liquids, suppositories, enema gels and / or creams; Preferably, the pharmaceutical composition further comprises formulation excipients (e.g., excipients for preparing powders, tablets, capsules, oil drops); Preferably, the pharmaceutical composition is used alone or in combination with other antifungal agents, antiviral agents, analgesics, anti-inflammatory drugs, healing agents and / or moisturizers; Preferably, the Lactobacillus crispatus in the pharmaceutical composition is present in an amount of 10 6 Up to 10 12 The amount of CFU / dose present (e.g., 10 7 Up to 10 9 CFU / ml); Preferably, when the pharmaceutical composition is solid, each gram of the pharmaceutical composition comprises 10 6 Up to 10 12 CFU of Lactobacillus crispatus (e.g., 10 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g); Preferably, when the pharmaceutical composition is a liquid, each milliliter of the liquid contains 10 6 Up to 10 12 CFU of Lactobacillus crispatus (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL); Preferably, the Lactobacillus crispatus in the pharmaceutical composition exists in the form of live bacteria or in the form of dead bacteria.

5. A culture comprising the Lactobacillus crispatus according to claim 1 or 2 or the composition according to claim 3; Preferably, the culture further comprises components that provide nutrients (e.g., solid or liquid culture medium, feeder cell layer); Preferably, the nutrient-providing ingredients are selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fibers, amino acids, or any combination thereof; Preferably, the culture further comprises a cell-free culture filtrate of Lactobacillus crispatus; Preferably, the culture further comprises a derivative of Lactobacillus crispatus; wherein, The derivative is selected from a metabolite, an enzyme, a cell structural component (eg, a cell wall or a component thereof), an exopolysaccharide, a bacteriocin, a compound containing an immunogenic component, or any combination thereof.

6. A food product or dietary supplement or health food comprising the Lactobacillus crispatus according to claim 1 or 2, the composition according to claim 3, or the culture according to claim 5; Preferably, 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); Preferably, the food product or dietary supplement or health food further comprises prebiotics; Preferably, the prebiotics are selected from fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharides, carrot nitrogen-containing polysaccharides, or any combination thereof; Preferably, the food product or dietary supplement or nutraceutical further comprises additional additives; Preferably, the additional additive is selected from proteins (e.g., enzymes), carbohydrates, fats, vitamins, minerals, dietary fibers, amino acids, or any combination thereof; Preferably, the Lactobacillus crispatus in the food product, dietary supplement or health food is present in an amount of 10 6 Up to 10 12 The amount of CFU / dose present (e.g., 10 7 Up to 10 9 CFU / ml); Preferably, when the food product, dietary supplement or health food is solid, it contains 10 6 Up to 10 12 CFU of Lactobacillus crispatus (e.g., 10 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g); Preferably, when the food product, dietary supplement or health food is a liquid, each milliliter of the liquid contains 10 6 Up to 10 12 CFU of Lactobacillus crispatus (e.g., 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL); Preferably, the Lactobacillus crispatus in the food product, dietary supplement or health food is present in the form of live bacteria or in the form of dead bacteria.

7. A cleaning product comprising the Lactobacillus crispatus of claim 1 or 2, the composition of claim 3, or the culture of claim 5; Preferably, the cleaning product is selected from solid soap, liquid soap, hand soap, shampoo, liquid conditioner, body cleanser, or any combination thereof; Preferably, the cleaning product is a vaginal douche, a vaginal gel and / or a vaginal cream.

8. Use of the Lactobacillus crispatus according to claim 1 or 2, or the composition according to claim 3, or the pharmaceutical composition according to claim 4, or the culture according to claim 5, or the food product or dietary supplement or health food according to claim 6, or the cleaning product according to claim 7 in the preparation of a medicament for preventing and / or treating diseases and / or symptoms related to the female urogenital tract in female subjects; Preferably, the female urogenital tract-related disease is caused by an imbalance of reproductive tract microbiota and / or an abnormal pH value in the reproductive tract; Preferably, the female urogenital tract-related disease is associated with one or more of the following conditions: (1) Reduction in the level of Lactobacillus bacterial strains in the reproductive tract ecosystem; (2) an increase in the proliferation of at least one microorganism belonging to the following species in the reproductive tract ecosystem: Gardnerella vaginalis, Candida albicans, Streptococcus aureus, Atopobium vaginae, or any combination thereof; (3) the female urogenital tract-related disease is associated with an increased proliferation of at least one microorganism belonging to the following genera in the genital 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; and / or (4) Increased pH in the reproductive tract environment; Preferably, the drug is used to maintain the balance of the female reproductive tract microbiota, reduce the pH value of the female reproductive tract, increase the level of Lactobacillus bacterial strains in the female reproductive tract ecosystem and / or reduce the level of Gardnerella bacterial strains in the female reproductive tract; Preferably, the strain of the genus Gardnerella is Gardnerella; Preferably, the disease is selected from: infection of the female urogenital tract, genital herpes, gonorrhea, inflammatory diseases related to the female urogenital tract (e.g., vaginitis, cervicitis), or any combination thereof; Preferably, the symptom is selected from: changes in menstrual cycle, vaginal itching, vaginal redness, abnormal discharge, vaginal pain, or any combination thereof.

9. The use according to claim 8, wherein The drug prevents and / or treats inflammatory diseases related to the female urogenital tract by reducing the level of pro-inflammatory factors and / or increasing the level of anti-inflammatory factors; Preferably, the drug prevents and / or treats inflammatory diseases related to the female urogenital tract by reducing sialidase activity and / or myeloperoxidase (MPO) activity; Preferably, the pro-inflammatory factor is selected from TNF-α, IL-1β, IL-6, or any combination thereof; Preferably, the anti-inflammatory factor is IL-10; Preferably, the subject is a mammal; Preferably, the subject is a human.

10. Use of the Lactobacillus crispatus according to claim 1 or 2, or the composition according to claim 3, or the pharmaceutical composition according to claim 4, or the culture according to claim 5, or the food product or dietary supplement or health food according to claim 6, or the cleaning product according to claim 7 in the preparation of a medicament for inhibiting the growth of Gardnerella and / or reducing its amount; Preferably, the drug inhibits the growth of strains of the genus Gardnerella by increasing the level of hydrogen peroxide (H2O2); Preferably, the drug inhibits the growth of strains of the genus Gardnerella by lowering the pH level of the environment; Preferably, the drug is used to inhibit biofilm formation of strains of the genus Gardnerella, thereby inhibiting the growth of strains of the genus Gardnerella; Preferably, the drug is used to inhibit the growth of Gardnerella in women and / or reduce its amount, or to inhibit the growth of Gardnerella in the environment and / or reduce its amount; Preferably, the female body includes mucous membranes and / or reproductive tract; Preferably, the environment includes a home, a workplace, a laboratory, an industrial environment, an aquatic environment, a medical instrument and / or a gynecological examination tool.

11. A method for inhibiting the growth of Gardnerella and / or reducing the amount thereof in a female, the method comprising: administering an effective amount of the Lactobacillus crispatus of claim 1 or 2, or the composition of claim 3, or the pharmaceutical composition of claim 4, or the culture of claim 5, or the food product or dietary supplement or health food of claim 6, or the cleaning product of claim 7 to a female subject in need thereof; Preferably, the method comprises: (1) implanting the Lactobacillus crispatus according to claim 1 or 2, the composition according to claim 3, or the pharmaceutical composition according to claim 4 into the reproductive tract of a female subject; (2) applying (e.g., applying) the Lactobacillus crispatus described in claim 1 or 2, the composition described in claim 3, the pharmaceutical composition described in claim 4, or the cleaning product described in claim 7 to the reproductive tract and / or perineum of a female subject; or (3) administering (e.g., orally) the Lactobacillus crispatus according to claim 1 or 2, the composition according to claim 3, the pharmaceutical composition according to claim 4, or the food product, dietary supplement, or health food according to claim 6 to a female subject; Preferably, the genus Gardnerella includes Gardnerella vaginalis; Preferably, the female body includes mucous membranes and / or reproductive tract; Preferably, the Lactobacillus crispatus is 10 6 Up to 10 12 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 10 9 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the subject.

12. A method for inhibiting the growth of Gardnerella in an environment and / or reducing its amount, the method comprising: Applying an effective amount of the Lactobacillus crispatus of claim 1 or 2, or the composition of claim 3, or the pharmaceutical composition of claim 4, or the culture of claim 5 to the environment; Preferably, the environment includes a home, a workplace, a laboratory, an industrial environment, an aquatic environment, a medical instrument and / or a gynecological examination tool; Preferably, the Lactobacillus crispatus is 10 6 Up to 10 12 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 10 9 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the environment.

13. A method for preventing and / or treating diseases and / or symptoms related to the female urogenital tract, the method comprising: administering an effective amount of the Lactobacillus crispatus of claim 1 or 2, or the composition of claim 3, or the pharmaceutical composition of claim 4, or the culture of claim 5, or the food product or dietary supplement or health food of claim 6, or the cleaning product of claim 7 to a female subject in need thereof; Preferably, the female urogenital tract-related disease is caused by an imbalance of reproductive tract microbiota and / or an abnormal pH value in the reproductive tract; Preferably, the female urogenital tract-related disease is associated with one or more of the following conditions: (1) Reduction in the level of Lactobacillus bacterial strains in the reproductive tract ecosystem; (2) an increase in the proliferation of at least one microorganism belonging to the genus Gardnerella, Dorea, Streptococcus, Anaerobicoccus, Fingoldia, Prevotella, Steinerella, Staphylococcus, Corynebacterium, or any combination thereof in the reproductive tract ecosystem; and / or (3) Increased pH in the reproductive tract environment; Preferably, the strain of the genus Gardnerella is Gardnerella; Preferably, the disease is selected from: infection of the female urogenital tract, genital herpes, gonorrhea, inflammatory diseases related to the female urogenital tract (e.g., vaginitis, cervicitis), or any combination thereof; Preferably, the symptom is selected from: menstrual cycle changes, vulvar itching, vaginal redness, vulvar burning sensation, abnormal discharge (e.g., abnormal leucorrhea), vaginal pain, or any combination thereof; Preferably, the subject is a mammal; Preferably, the subject is a human; Preferably, the Lactobacillus crispatus is 10 6 Up to 10 12 CFU / daily dose (e.g., 10 7 CFU / Daily Dose, 10 8 CFU / Daily Dose, 10 9 CFU / Daily Dose, 10 10 CFU / Daily Dose, 10 11 CFU / Daily Dose, 10 12 CFU / daily dose) is administered to the subject.

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