CN119530074A

CN119530074A - Lactobacillus gasseri M17C and composition thereof

Info

Publication number: CN119530074A
Application number: CN202411724999.4A
Authority: CN
Inventors: 吕洁; 迮晓雷; 李良
Original assignee: BY Health Co Ltd
Current assignee: BY Health Co Ltd
Priority date: 2024-11-28
Filing date: 2024-11-28
Publication date: 2025-02-28

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus gasseri M17C, and a composition, a pharmaceutical composition, a culture, a food product or a dietary supplement and a cleaning product containing the same. The invention also relates to the use of the lactobacillus gasseri and compositions comprising the same in the manufacture of a medicament.

Description

Lactobacillus gasseri M17C and composition thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus gasseri, and a composition, a pharmaceutical composition, a culture, a food product or a dietary supplement and a cleaning product containing the same. The invention also relates to the use of the lactobacillus gasseri and compositions comprising the same in the manufacture of a medicament.

Background

Bacterial vaginitis (Bacterial vaginosis, BV) is a type of vaginal infection disease caused by vaginal flora imbalance, is a common gynecopathy for women, and has become a public health problem affecting the daily life of women worldwide. Factors for BV production mainly include estrogen level reduction, sexual behavior, antibiotic use, etc., which are clinically manifested by pruritus vulvae, burning sensation, abnormal odor and leucorrhea.

Under normal conditions, the vaginal flora is dominated by lactobacillus, with lactobacillus crispatus, lactobacillus grignard, lactobacillus johnsonii and lactobacillus jensenii being the most common species. The hydrogen peroxide, bacteriocin and other substances produced by the dominant strains maintain the acidic and healthy environment of the vagina, and if the vaginal flora balance is broken, the abundance of lactobacillus is reduced, and the pathogenic flora of the vagina is obviously increased. Among them, gardnerella vaginalis is a major pathogenic bacterium for bacterial vaginitis, and in addition, candida albicans, staphylococcus aureus and escherichia coli may proliferate in large amounts to cause genital tract infection when the normal environment of the vagina is destroyed.

Conventional pharmaceutical treatment of BV is mainly by oral or topical administration of clindamycin and metronidazole or tinidazole. But antibiotic therapy is not effective and has a high recurrence rate, which may be related to the resistance of pathogenic bacteria and their biofilms to antibiotics, and failure to restore the acidic environment of the vagina by antibiotic administration. In recent years, probiotics have shown good antibacterial activity in the aspect of treating bacterial vaginitis, are helpful for restoring the microecological balance of vagina, and have evidence that hydrogen peroxide generated by lactobacillus has the effect of inhibiting bacterial infection and preventing the bacterial vaginitis from occurring.

The current microecological products for treating or relieving bacterial vaginitis are very limited, for example, dingjunsheng is a Lactobacillus delbrueckii DM8909 screened from vaginal secretion of healthy females taught by Dalian medical university Kang Bai, but the strain has a certain limitation on the discussion of the effect of treating bacterial vaginitis. Therefore, it is particularly necessary to develop advantageous strains of probiotics that are effective and derived from the vagina of healthy female of the child-bearing age in China.

Disclosure of Invention

The inventor of the application screens a lactobacillus gasseri strain with obvious strain-specific probiotic potential from strains from more than 100 healthy females through a large number of experiments. The inventor of the present application has confirmed through a large number of experiments that the lactobacillus gasseri and the composition comprising the same have high acid-producing ability and high hydrogen peroxide-producing ability, can maintain the acidic and healthy environment of the female genital tract, and can maintain the normalization of the female genital tract microbiota. In addition, the lactobacillus gasseri and the composition containing the same can also obviously inhibit the growth of the most main pathogenic bacteria of the vagina, namely gardnerella and the formation of a biological film. Thus, the applicant has completed the present application.

Lactobacillus gasseri

In a first aspect, the present application provides a lactobacillus gasseri (Lactobacillus gasseri) deposited with the cantonese collection of microbial strains under accession number GDMCC No.63872.

In certain embodiments, the lactobacillus gasseri has the ability to produce greater than 5umol/l of hydrogen peroxide under effective culture conditions. In certain embodiments, the lactobacillus gasseri has the ability to produce 5to 8, 8 to 12, 12 to 15 or more hydrogen peroxide under effective culture conditions.

In certain embodiments, the lactobacillus gasseri is capable of lowering the pH of the female genital tract. In certain embodiments, the lactobacillus gasseri reduces the pH of the female genital tract to a pH value of 6.5 or less, a pH value of 6 or less, a pH value of 5.5 or less, a pH value of 5 or less, a pH value of 4.5 or less, a pH value of 4 or less.

In certain embodiments, the lactobacillus gasseri is capable of inhibiting growth of and/or inhibiting biofilm formation of a strain of gardnerella. In certain embodiments, the strain of gardnerella is gardnerella (GARDNERELLA VAGINALIS). In certain embodiments, the inhibition of biofilm formation can be at least 5% biofilm inhibition, 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.

In certain embodiments, the lactobacillus gasseri is capable of reducing the level of a proinflammatory factor (e.g., TNF- α, IL-1β, IL-6). In certain embodiments, the lactobacillus gasseri 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%.

In certain embodiments, the lactobacillus gasseri is capable of increasing the level of an anti-inflammatory factor (e.g., IL-10). In certain embodiments, the lactobacillus gasseri 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%, at least 70%.

In certain embodiments, the colony of lactobacillus gasseri is white, milky white, and/or translucent.

In certain embodiments, the lactobacillus gasseri is a gram positive bacterium.

It will be readily appreciated that in the growth and cultivation of microorganisms, and in particular bacteria, genetic material may undergo certain changes (e.g., mutations of one or more bases) which may occur spontaneously or as a result of mutagenesis by chemical and/or physical agents (e.g., mutagens) and/or recombinant DNA techniques known in the art. Thus, herein, the lactobacillus gasseri of the present invention includes mutants and/or offspring of lactobacillus gasseri having deposit number GDMCC No. 63872. In certain embodiments, these mutants and/or offspring still retain the functional and/or physiochemical properties (e.g., one or more of the functional and/or physiochemical properties mentioned above) of lactobacillus gasseri having deposit number GDMCC No. 63872.

Composition and method for producing the same

In a second aspect, the present application provides a composition comprising lactobacillus gasseri as described in the first aspect.

As used herein, the term "probiotic" is defined as any non-pathogenic microorganism, and when administered to a host in a viable form in sufficient quantity, can have a beneficial effect on the health of the host.

In certain embodiments, the composition further comprises a probiotic selected from bacteria, fungi (e.g., yeast), or any combination thereof.

In certain embodiments, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof.

In certain embodiments, the bacterium of the genus Lactobacillus is selected from the group consisting of Lactobacillus paracasei (Lactobacillus paracasei), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus brevis (Lactobacillus brevis), lactobacillus jensenii (Lactobacillus jensenii), lactobacillus inertia (Lactobacillus iners), lactobacillus casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus curvatus (Lactobacillus curvatus), lactobacillus delbrueckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentum), lactobacillus gasseri (Lactobacillus gasseri), lactobacillus helveticus (Lactobacillus helveticus), lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus sake (Lactobacillus sakei), lactobacillus salivarius (Lactobacillus salivarius), or any combination thereof.

In certain embodiments, the lactobacillus paracasei is lactobacillus paracasei 207-27 having the microorganism deposit No. GDMCC No. 60960.

In certain embodiments, the yeast is selected from the group consisting of saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces boulardii (Saccharomyces boulardii), kluyveromyces marxianus (Kluyveromyces marxianus), or any combination thereof.

In certain embodiments, the bacterium of the genus bifidobacterium is selected from the group consisting of bifidobacterium animalis (Bifidobacterium animalis), bifidobacterium bifidum (Bifidobacterium bifidum), bifidobacterium breve (Bifidobacterium breve), bifidobacterium infantis (Bifidobacterium infantis), bifidobacterium longum (Bifidobacterium longum), bifidobacterium adolescentis (Bifidobacterium adolescentis), or any combination thereof.

In certain embodiments, the bacteria of the genus Bacillus are selected from the group consisting of Bacillus subtilis (Bacillus subtilis), bacillus coagulans (Bacillus coagulans), or any combination thereof.

In certain embodiments, the bacteria of the genus Propionibacterium are selected from the group consisting of Propionibacterium xie (Propionibacterium shermanii), propionibacterium freudenreichii (Propionibacterium freudenreichii), propionibacterium propionicum (Propionibacterium acidipropionici), or any combination thereof.

In certain embodiments, the Streptococcus bacteria are selected from Streptococcus thermophilus (Streptococcus thermophilus), streptococcus salivarius (Streptococcus salivarius), or any combination thereof.

In certain embodiments, the bacterium of the genus lactococcus is lactococcus lactis (Lactococcus lactis).

In certain embodiments, the bacterium of the enterococcus genus is selected from enterococcus faecalis (Enterococcus faecalis), enterococcus faecium (Enterococcus faecium), or any combination thereof.

In certain embodiments, the composition comprises Lactobacillus crispatus having deposit No. GDMCC No.63705, lactobacillus crispatus having deposit No. GDMCC No.63707, and Lactobacillus gasseri having deposit No. GDMCC No. 63872. In certain embodiments, the composition consists of Lactobacillus crispatus having accession number GDMCC No.63705, lactobacillus crispatus having accession number GDMCC No.63707, and Lactobacillus gasseri having accession number GDMCC No. 63872.

In certain embodiments, the ratio of Lactobacillus crispatus having deposit number GDMCC No.63705, lactobacillus crispatus having deposit number GDMCC No.63707, and Lactobacillus gasseri having deposit number GDMCC No.63872 included in the composition is 1-10:1-10:1-10 (e.g., 1:1:1,10-1:1:1,1:10-1:1,1:1:10-1,10-1:10-1, 10-1:1:10-1, 1:10-1:10-1).

In certain embodiments, the composition has the ability to produce greater than 10umol/l of hydrogen peroxide under effective culture conditions. In certain embodiments, the lactobacillus gasseri has the ability to produce 5 to 8, 8 to 12, 12 to 15 to 20 or more hydrogen peroxide under effective culture conditions.

In certain embodiments, the composition is capable of lowering the pH of the female genital tract. In certain embodiments, the lactobacillus gasseri reduces the pH of the female genital tract to a pH value of 6.5 or less, a pH value of 6 or less, a pH value of 5.5 or less, a pH value of 5 or less, a pH value of 4.5 or less, a pH value of 4 or less.

In certain embodiments, the composition is capable of inhibiting the growth of and/or inhibiting the biofilm formation of a strain of gardnerella. In certain embodiments, the strain of gardnerella is gardnerella (GARDNERELLA VAGINALIS). In certain embodiments, the inhibition of biofilm formation can be at least 5% biofilm inhibition, 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.

In certain embodiments, the compositions are capable of reducing the level of a proinflammatory factor (e.g., TNF- α, IL-1β, IL-6). In certain embodiments, the composition 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%.

In certain embodiments, the compositions are capable of increasing the level of an anti-inflammatory factor (e.g., IL-10). In certain embodiments, the composition 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%, at least 70%.

Pharmaceutical composition

In a third aspect, the present application provides a pharmaceutical composition comprising lactobacillus gasseri as described in the first aspect or a composition as described in the second aspect.

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

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

In certain embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sugar (e.g., xylose, sucrose, fructose, lactose, trehalose), a sugar alcohol (e.g., glycerol, xylitol, sorbitol, mannitol, erythritol), a polysaccharide (e.g., cellulose and derivatives thereof, starch and derivatives thereof, chitosan, gums, maltodextrin), a polyether (e.g., polypropylene glycol, polyethylene glycol, polytetramethylene glycol), a povidone (e.g., povidone K30, K60, K90), an oil (e.g., rapeseed oil, sunflower seed oil, soybean oil, sesame oil, olive oil), a surfactant (e.g., tween20, tween40, tween60, tween80, fatty acids, polyoxyethylene fatty alcohol ethers), an inorganic salt (e.g., phosphates, carbonates, citrates, chlorides, sulfates, borates, citrates), talc, silica and derivatives thereof, hydrolysates, or any combination thereof.

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

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.

In certain embodiments, the pharmaceutical composition comprises an amino acid.

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

In certain embodiments, the pharmaceutical composition comprises a vitamin.

In certain embodiments, the pharmaceutical composition comprises vitamin a, vitamin B ₁, vitamin B ₂, vitamin B ₃, vitamin B ₆, vitamin B ₉, vitamin B ₁₂, vitamin C, vitamin D, vitamin E, vitamin K, or any combination thereof.

In certain embodiments, the pharmaceutical composition comprises a mineral.

In certain embodiments, the pharmaceutical composition comprises iron, manganese, zinc, copper, selenium, or any combination thereof.

In certain embodiments, the pharmaceutical composition comprises peptone.

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

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

In certain embodiments, the pharmaceutical composition comprises a formulation of lactobacillus gasseri.

In certain embodiments, the pharmaceutical composition is in the form of a pill, powder, capsule, tablet (e.g., effervescent tablet), oil drop, caplet, mouth-soluble granule, liquid, suppository, enema gel, and/or cream.

In certain embodiments, the pharmaceutical composition further comprises a formulation adjuvant (e.g., an adjuvant for preparing a powder, tablet, capsule, oil drop).

In certain embodiments, the pharmaceutical composition is used alone or in combination with other antifungal agents, antiviral agents, analgesic agents, anti-inflammatory agents, healing promoting agents, and/or moisturizers.

In certain embodiments, the lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect is present in an amount of 10 ⁶ to 10 ¹² CFU/dose (e.g., 10 ⁷ to 10 ⁹ CFU/ml).

In certain embodiments, when the pharmaceutical composition is a solid, it comprises 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri as described in the first aspect or a composition as described in the second aspect (e.g., 10 ⁷CFU/g,10⁸CFU/g,10⁹ CFU/g) per gram.

In certain embodiments, when the pharmaceutical composition is a liquid, it comprises 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri as described in the first aspect or a composition as described in the second aspect (e.g., 10 ⁷CFU/mL,10⁸CFU/mL,10⁹ CFU/mL) per milliliter.

In certain embodiments, the lactobacillus crispatus and/or lactobacillus gasseri in the pharmaceutical composition are present in a live form or in a dead form.

Culture of

In a fourth aspect, the present application provides a culture comprising lactobacillus gasseri as described in the first aspect or a composition as described in the second aspect.

In certain embodiments, the culture further comprises a nutrient providing component (e.g., a solid or liquid medium, a feeder cell layer).

In certain embodiments, the nutrient-providing ingredient is selected from the group consisting of a protein (e.g., an enzyme), a carbohydrate, a fat, a vitamin, a mineral, a dietary fiber, an amino acid, or any combination thereof.

In certain embodiments, the culture further comprises a cell-free culture filtrate of lactobacillus crispatus and/or lactobacillus gasseri.

In certain embodiments, the culture further comprises a derivative of Lactobacillus crispatus and/or Lactobacillus gasseri, wherein the derivative is selected from the group consisting of a metabolite, an enzyme, a cell structural component (e.g., a cell wall or component thereof), an extracellular polysaccharide, a bacteriocin, a compound comprising an immunogenic component, or any combination thereof.

Food product or dietary supplement and health food

In a fifth aspect, the present application provides a food product or dietary supplement or health food comprising lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect or the culture as described in the fourth aspect.

In the context of the present specification, the term "food" is meant to be broad, including human foods and drinks, as well as animal foods and drinks (i.e. feed). In certain embodiments, the food product is suitable and designed for human feeding. In certain embodiments, the food product is selected from a solid beverage, a liquid beverage, a tabletted candy, an expanded food product, a protein bar or nut, or the food product is a dairy product (e.g., milk powder, milk flakes, yogurt, flavored fermented milk, a lactic acid bacteria beverage, cheese).

In the context of this document, a "dietary supplement" refers to an edible product that is capable of providing a benefit to the consumer. It is also called nutritional supplement, nutritional agent, dietary supplement, etc., and is an auxiliary means for diet to supplement amino acids, trace elements, vitamins, minerals, etc. required by human body.

In certain embodiments, the food product or dietary supplement or health food further comprises a prebiotic.

In certain embodiments, the prebiotic is selected from the group consisting of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, spirulina, arthrospira, coriolus versicolor polysaccharides, nitrogen-containing polysaccharides of the genus of the species, or any combination thereof.

In certain embodiments, the food product or dietary supplement or health food further comprises additional additives.

In certain embodiments, the additional additive is selected from a protein (e.g., an enzyme), a carbohydrate, a fat, a vitamin, a mineral, a dietary fiber, an amino acid, or any combination thereof.

In certain embodiments, the carbohydrate is selected from the group consisting of sugar (monosaccharides, glucose, fructose, disaccharides, maltose, sucrose, polysaccharides), cyclodextrin, sugar alcohols (xylitol, sorbitol, erythritol).

In certain embodiments, the lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect in the food product or dietary supplement or health food is present in an amount of 10 ⁶ to 10 ¹² CFU/dose (e.g., 10 ⁷ to 10 ⁹ CFU/ml).

In certain embodiments, when the food product or dietary supplement or health food is a solid, it comprises 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect (e.g., 10 ⁷CFU/g,10⁸CFU/g,10⁹ CFU/g) per gram.

In certain embodiments, when the food product or dietary supplement or health food is a liquid, it comprises 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect (e.g., 10 ⁷CFU/mL,10⁸CFU/mL,10⁹ CFU/mL) per milliliter.

In certain embodiments, the lactobacillus crispatus and/or lactobacillus gasseri in the food product or dietary supplement or health food are present in a live or dead bacterial form.

Cleaning products

In a sixth aspect, the present application provides a cleaning product comprising lactobacillus gasseri as described in the first aspect or a composition as described in the second aspect or a culture as described in the fourth aspect.

In certain embodiments, the cleansing product is selected from the group consisting of solid soaps, liquid soaps, hand washes, shampoos, liquid conditioners, body cleansers, or any combination thereof.

In certain embodiments, the cleaning product is a vaginal lavage, vaginal gel, and/or vaginal cream.

Use of the same

In a seventh aspect, the present application provides the use of a lactobacillus gasseri as described in the first aspect or the composition of the second aspect or the pharmaceutical composition of the third aspect or the culture of the fourth aspect or the food product or dietary supplement or health food product as described in the fifth aspect or the cleaning product as described in the sixth aspect for the manufacture of a medicament for the prevention and/or treatment of female genitourinary related diseases and/or symptoms in a subject.

In certain embodiments, the female genitourinary tract-related disease is caused by an imbalance in the genital tract microbiota and/or an abnormality in the genital tract pH.

In certain embodiments, the female genitourinary tract-related disease is associated with a reduction in the level of a lactobacillus bacterial strain in the genital tract ecosystem.

In certain embodiments, the female genitourinary tract-related disease is associated with an increase in proliferation of at least one microorganism belonging to the group consisting of gardnerella vaginalis (GARDNERELLA VAGINALIS), candida albicans (Candida albicans), staphylococcus aureus (Streptococcus aureus), atobacter vaginalis (Atopobium vaginae), or any combination thereof.

In certain embodiments, the female genitourinary tract-related disease is associated with an increase in proliferation of at least one microorganism belonging to the genus Prevolella (Prevolella spp.), pediococcus (Megasphaera spp.), sinapis (Sneathia spp.), aphanotheca (Atopodium spp.), listeria (DIALISTER spp.), anaerococcus (Anaerococcus spp.), egger (EGGERTHELLA spp.), peptone bacterium (Peptoniphilus spp.), balloon (Aerococcus spp.), campylobacter (Mobiluncus spp.), or any combination thereof.

In certain embodiments, the female genitourinary tract-related disease is associated with an increase in pH in the genital tract environment.

Thus, in certain embodiments, the medicament is for maintaining balance of microbiota in the female genital tract, lowering pH in the female genital tract, increasing the level of lactobacillus bacterial strain in the female genital tract ecosystem, and/or lowering the level of gardnerella bacterial strain in the female genital tract.

In certain embodiments, the strain of gardnerella is gardnerella.

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

In certain embodiments, the symptom is selected from the group consisting of altered menstrual cycle, vaginal itching, vaginal redness, abnormal secretions, vaginal pain, or any combination thereof.

In certain embodiments, the medicament prevents and/or treats inflammatory disorders associated with the female genitourinary tract by reducing the level of a pro-inflammatory factor and/or increasing the level of an anti-inflammatory factor.

In certain embodiments, the medicament prevents and/or treats inflammatory disorders associated with the female genitourinary tract by reducing sialidase activity and/or Myeloperoxidase (MPO) activity.

In certain embodiments, the proinflammatory factor is selected from the group consisting of TNF- α, IL-1β, IL-6, or any combination thereof. In certain embodiments, the anti-inflammatory factor is IL-10.

In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

In an eighth aspect, the present application provides the use of a lactobacillus gasseri as described in the first aspect or the composition of the second aspect or the pharmaceutical composition of the third aspect or the culture of the fourth aspect or the food product or dietary supplement or health food product as described in the fifth aspect or the cleaning product as described in the sixth aspect for the manufacture of a medicament for inhibiting the growth and/or reducing the amount of gardnerella.

In certain embodiments, the agent inhibits growth of a strain of gardnerella by increasing the level of hydrogen peroxide (H ₂O₂).

In certain embodiments, the agent inhibits growth of a strain of gardnerella by lowering the level of the pH of the environment in which it is located.

In certain embodiments, the agent is used to inhibit biofilm formation by a strain of gardnerella, thereby inhibiting growth of the strain of gardnerella.

In certain embodiments, the medicament is for inhibiting gardnerella growth and/or reducing the amount thereof in a female, or for inhibiting gardnerella growth and/or reducing the amount thereof in an environment.

In certain embodiments, the female body comprises within the mucosa and/or genital tract.

As used herein, the term "mucosa" refers to membranes within various cavities and covering the surfaces of internal organs in a mammal. It consists of one or more layers of epithelial cells covered with a layer of loose connective tissue. It is mainly of endodermal origin and is continuous with the skin at various body openings, such as the inner side of the eyes, ears, nose and mouth, lips, vagina, urethral orifice and anus.

In certain embodiments, the environment comprises a home, workplace, laboratory, industrial environment, aquatic environment, medical instrument, and/or gynecological examination appliance.

Method of

In a ninth aspect, the present application provides a method of inhibiting the growth and/or reducing the amount of gardnerella in a female, the method comprising administering to a female subject in need thereof an effective amount of a lactobacillus gasseri 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 food product as described in the fifth aspect or a dietary supplement or a health food or a cleaning product as described in the sixth aspect.

In certain embodiments, the method comprises implanting a lactobacillus gasseri 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 genital tract of a female subject.

In certain embodiments, the method comprises topically applying (e.g., painting, spraying) the lactobacillus gasseri as described in the first aspect or the composition of the second aspect or the pharmaceutical composition as described in the third aspect or the cleansing product as described in the sixth aspect to the genital tract and/or perineum of a female subject.

In certain embodiments, the method comprises orally (e.g., orally) administering to a female subject the lactobacillus gasseri of the first aspect or the composition of the second aspect or the pharmaceutical composition of the third aspect, such as the food product of the fifth aspect or the dietary supplement or the health food.

In certain embodiments, the gardnerella comprises gardnerella (GARDNERELLA VAGINALIS).

In certain embodiments, the female body comprises an internal genital tract and/or a mucosal membrane.

In certain embodiments, the lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect is administered to the subject in an amount of 10 ⁶ 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).

In a tenth aspect, the present application provides a method of inhibiting the growth and/or reducing the amount of gardnerella in an environment, the method comprising administering to the environment an effective amount of lactobacillus gasseri according to the first aspect or a composition according to the second aspect or a pharmaceutical composition according to the third aspect or a culture according to the fourth aspect.

In certain embodiments, the lactobacillus gasseri as described in the first aspect or the composition as described in the second aspect is administered to the environment in an amount of 10 ⁶ 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).

In an eleventh aspect, the present application provides a method of preventing and/or treating a female genitourinary tract-related disease and/or symptom, the method comprising administering to a female subject in need thereof an effective amount of lactobacillus gasseri according to the first aspect or the composition according to the second aspect or the pharmaceutical composition according to the third aspect or the culture according to the fourth aspect or the food product or dietary supplement or health food product according to the fifth aspect or the cleansing product according to the sixth aspect.

In certain embodiments, the female genitourinary tract-related disease is caused by an imbalance in the microbiota of the genital tract and/or wherein the pH of the genital tract is abnormal.

In certain embodiments, the female genitourinary tract-related disease is associated with an increase in proliferation of at least one microorganism belonging to the genus Gardnerella, tourette, streptococcus, anaerococcus, fingered, prevotella, pianobacteria, staphylococcus, corynebacterium, or any combination thereof in the genital tract ecosystem.

In certain embodiments, the strain of gardnerella is gardnerella.

Definition of terms

In this context, the term "genital tract" includes urinary tract and vagina. The vagina includes an elastic muscle portion of the female genital tract. In humans, the vagina extends from the vulva to the cervix.

In this context, the term "genital microbiota" or "genital ecosystem" refers to a microbiome that is in the female genitourinary tract (preferably the vagina) and survives in equilibrium with each other and with the genital environment in which they are housed. Thus, the term "imbalance of the genital microbiota" includes a decrease in the level of one or more dominant bacteria in the genital microbiota and/or an increase in the level of one or more pathogenic bacteria in the genital tract.

In the context of the present invention, the expression "pathogenic bacteria" refers to microorganisms that can be present in the vaginal ecosystem and are potentially pathogenic.

As used herein, the term "therapeutically effective amount" or "effective amount" may refer to an amount of a composition or strain in a pharmaceutical composition that, when administered as part of a desired dosage regimen (to a human or animal, preferably a human), alleviates symptoms, ameliorates a condition, or slows the onset of a disease condition, according to clinically acceptable criteria for the disorder or condition of the subject to be treated, e.g., at a reasonable benefit risk ratio applicable to any medical treatment. The therapeutically effective amount may vary depending on the age, weight, sex, dosage form, health condition and severity of the disease of the subject to be treated. In addition, the frequency may be determined by a doctor or pharmacist to administer the dose in divided doses at regular intervals of time one to several times per day.

As used herein, the term "CFU (color-Forming Units)" refers to the total population of microorganisms such as bacteria, fungi, yeast, etc., in a product, typically used as a viable count calculation.

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 daily or every time. For example, in certain embodiments, the lactobacillus gasseri is present in the food product or dietary supplement or health food in an amount of 10 ⁶ to 10 ¹² CFU/dose. In such embodiments, if lactobacillus gasseri is administered in a food product (e.g., in a solid beverage, yogurt), the food product provided to the subject daily or each time (e.g., solid beverage, yogurt) may contain from about 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri. Of course, alternatively, the amount of such bacteria may be divided into multiple administrations, provided that the total amount of lactobacillus gasseri received by the subject at any particular time (e.g., every 24 hours) is from about 10 ⁶ to about 10 ¹² CFU of bacteria, i.e., the amount of lactobacillus gasseri present in the food product or dietary supplement or health food product described above is from 10 ⁶ to 10 ¹² CFU/dose.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, as 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 including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, e.g., pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, e.g., tween-80, ionic strength enhancers include, but are not limited to sodium chloride.

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.

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 may be inhibited by inhibiting the growth of biofilm-forming microorganisms. In certain embodiments, the inhibition of biofilm formation can be at least 5% biofilm inhibition, 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.

As used herein, the term "sialidase" also referred to as "neuraminidase" is the major virulence factor of the genus gardnerella, the causative agent of Bacterial Vaginosis (BV). Sialidases are detectable in cervical-vaginal lavage fluid of human females. Numerous studies have shown that sialidase activity levels in vaginal secretions of women with BV are higher than those without BV (e.g., healthy women). Therefore, in the current clinical diagnosis, sialidase positivity in vaginal secretion has been used as an important diagnostic index for BV.

As used herein, the term "MPO" is an enzyme present in leukocytes. MPO is commonly used in the diagnosis and treatment of diseases such as inflammatory response, infection, and the like. In general, high MPO may mean that the host organism is suffering from inflammation, infection, autoimmune diseases, and the like. During inflammation, leukocytes release large amounts of MPO to destroy bacteria, viruses and other pathogens. Thus, bacterial vaginosis patients often experience elevated levels of MPO in cervical-vaginal lavage fluids.

Advantageous effects of the invention

The lactobacillus gasseri and the composition containing the same have high acid-producing capability and high hydrogen peroxide-producing capability, can maintain the acidity and healthy environment of female genital tract, and can maintain the balance of female genital tract microbiota. In addition, the lactobacillus gasseri and the composition containing the same can also significantly inhibit the growth of the most main pathogenic bacteria of the genital tract, namely gardnerella and the formation of biological films. Also, sialidase and Myeloperoxidase (MPO) activities in the vagina can be significantly reduced, proinflammatory factor (e.g., TNF-alpha, IL-1β, and IL-6) levels can be significantly reduced, and anti-inflammatory factor (e.g., IL-10) levels can be significantly increased. Therefore, the lactobacillus gasseri and the composition containing the same have higher application value in female reproductive health (especially female vaginal health).

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment.

Description of preservation of biological Material

Lactobacillus crispatus M58D (Lactobacillus crispatus M D) was deposited with the Guangdong province microorganism strain collection (GDMCC, guangdong Microbial Culture Collection Center) located in building 5 of Dai No. 59, 100, martyr, guangzhou, having accession number GDMCC No.63705 and a time of 2023, 8, 2.

Lactobacillus crispatus X25B (Lactobacillus crispatus X B) has been deposited with the Guangdong province microorganism strain collection (GDMCC, guangdong Microbial Culture Collection Center) located in building 5 of Dai No. 59, line 100, martyr, guangzhou, having accession number GDMCC No.63707 and a time of 2023, 8, 2.

Lactobacillus gasseri M17C (Lactobacillus gasseri M C) has been deposited with the guangdong province microorganism strain collection (GDMCC, guangdong Microbial Culture Collection Center) located in institute No. 59 building 5 of university No. 100 in the city martyr of guangzhou with deposit No. GDMCC No.63872 for 2023, month 10 and 12 days.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, the conventional techniques of immunology, biochemistry, molecular biology, microbiology, cytobiology, genomics and recombinant DNA used in the present invention can be seen in Samson (Sambrook), french (Fritsch) and Meniere's (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), 2 nd time edition (1989), current generation molecular biology laboratory Manual (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (F.M. Ausubel et al edition (1987)), enzyme method (METHODS IN ENZYMOLOGY) series (academic Press): PCR 2:practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. Fresen (M.J. Mackerson), B.D. Hames) and G.R. Taylor edition (1995)), and animal cell culture (French (R.494) (French.4).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 isolation and identification of strains

1. Isolation and identification of Lactobacillus gasseri

The application separates more than 100 strains of bacteria from vaginal swabs of 60 healthy women in the women and child health care hospital in Dalian city, screens a strain of lactobacillus grignard from the bacteria, and names the strain of lactobacillus grignard as lactobacillus grignard M17C. The inclusion criteria of the sample donor are 22-35 years old, regular menstruation, no menstrual period during sampling, no sexual life, no vaginal administration and vaginal irrigation within 72 hours, no diagnosis of gynecological inflammation diseases and urinary system inflammation diseases within half a year, no positive signs in gynecological examination, no antibiotics within 1 month, no sex hormone within 3 months, no hysterectomy or traumatic treatment of cervical uterus, no recent immunosuppressive treatment of the patient, and no serious cardiac, hepatic and renal insufficiency and related complications of the patient. The vaginal swab samples were stored in a-80 ℃ refrigerator immediately after collection.

The strain was cultured using MRS liquid medium, modified MRS solid medium (2% agar and 1% calcium carbonate were added to the prepared MRS liquid medium, and autoclaved at 121℃for 15 minutes), BHI liquid medium, and BHI solid medium (2% agar was added to the prepared liquid medium, and autoclaved at 121℃for 15 minutes). Before use, 10% sterile defibrinated sheep blood and lactobacillus selective agar were added to the mixture after the temperature had fallen to about 50 ℃.

The vaginal swab is soaked in physiological saline and thoroughly mixed for 5 minutes. 100 μl of liquid is absorbed by each sample and is respectively coated on a modified MRS, LBS, BHI solid culture medium, anaerobic culture is carried out for 48-72 hours at 37 ℃, medium-sized, convex and milky circular colonies are selected for gram-color detection, and bacterial morphology and dyeing property are observed under an optical microscope. The monoclonal colonies of gram-positive bacteria in the form of long rods or globules are selected for further purification until pure bacteria are determined, and the monoclonal colonies are picked for 16S rRNA sequencing.

Sequencing results show that the strain isolated by the application is lactobacillus gasseri. Further, whole genome assays were performed on lactobacillus gasseri M17C, respectively, using the PacBio sequence 2 sequencing platform, and aligned with the whole genome sequences of lactobacillus gasseri strains described in NCBI database. Wherein, the genome length of the lactobacillus gasseri M17C is 2002365bp, and the GC content is 34.6 percent. The chromosomal genome contains 1962 coding genes, 50 tRNA genes, 5 rRNA genes (including 5s rRNA, 16s rRNA and 23s rRNA) and 56 other types of RNA genes.

The application obtains a brand new lactobacillus gasseri M17C by combining the experimental results of PCR identification and whole genome sequencing, and stores the brand new lactobacillus gasseri M17C.

2. Isolation and identification of Lactobacillus crispatus

The application separates more than 100 strains of bacteria from vaginal swabs of 60 healthy women in the women and child health care hospital in Dalian city, and two strains of lactobacillus crispatus are selected from the vaginal swabs and named as lactobacillus crispatus M58D and X25B. Wherein, the inclusion standard of the sample donor is the same as the above. The vaginal swab samples were stored in a-80 ℃ refrigerator immediately after collection.

Sequencing results show that both strains isolated by the application are Lactobacillus crispatus. Further, whole genome assays were performed on lactobacillus crispatus M58D and X25B, respectively, using PacBio sequence 2 sequencing platform, and aligned with the whole genome sequences of lactobacillus crispatus strains described in NCBI database. Wherein, the genome length of the Lactobacillus crispatus M58D is 2188407bp, and the GC content is 36.86%. The chromosomal genome contains 2264 coding genes, 65 tRNA genes, 4 rRNA genes (including 5s rRNA, 16s rRNA, and 23s rRNA), 167 other types of RNA genes. Wherein, the genome length of the Lactobacillus crispatus X25B is 2185354bp, and the GC content is 36.87%. The chromosomal genome contains 2270 coding genes, 66 tRNA genes, 4 rRNA genes (including 5srRNA, 16s rRNA, and 23s rRNA), 166 other types of RNA genes.

By combining the experimental results of PCR identification and whole genome sequencing, two brand-new Lactobacillus crispatus M58D and X25B are obtained and preserved.

Example 2 in vitro probiotic function assay of strains

The strains used in this example were all from a pool of probiotics from Shang Chen times the female source. The strains tested were positive control Lactobacillus delbrueckii DM8909 (trade name "Dijunsheng"), lactobacillus gasseri M17C, L F and M17B (both isolated and identified by the method described in example 1.1), respectively.

2.1 Determination of acid producing ability of Strain

The pH of the blank MRS broth was measured. The separated lactobacillus is inoculated in MRS liquid culture medium, anaerobic culture is carried out at a constant temperature of 37 ℃ for 24 hours, and the pH value of the bacterial liquid is measured. Δph=ph of MRS broth-24 hours pH of the broth, indicating a quantitative indicator of the acidogenic capacity of lactobacillus.

2.2 Determination of Hydrogen peroxide production ability of Strain

Hydrogen peroxide produced by the strain is beneficial to maintaining the acidic environment of the vagina. Referring to the method published by Wang Jiang team of "healthy women vaginal lactobacillus diversity analysis and screening of strains with probiotic characteristics", the hydrogen peroxide production capacity of lactobacillus is determined.

Preparation of solution 100mM PIPES (piperazine N, N' -di-ethane sulfonic acid) and 20mM TMB (tetramethylbenzidine) were prepared. The standard curve is prepared by firstly diluting H ₂O₂ standard solution with the concentration of 30% to 1mol/L by using 100mmol/LPIPES, respectively diluting the H ₂O₂ standard solution to 0, 20, 40, 60, 80 and 100 mu mol/LH ₂O₂ working solution by using 100mmol/L PIPES, respectively mixing 100 mu L working solution with 100 mu L of 20mmol/L TMB, finally adding 2 mu L HRP (horseradish peroxidase 1 mg/mL) into the mixed solution, uniformly mixing, incubating for 10 minutes at room temperature, measuring OD value by using an enzyme-labeled instrument, and making a corresponding standard curve.

The method comprises the steps of determining the H ₂O₂ production of lactobacillus, namely resuscitating a frozen lactobacillus strain to be detected and positive control 'Dingjunsheng' lactobacillus delbrueckii DM8909, inoculating the lactobacillus strain to be detected and positive control 'Dingjunsheng' lactobacillus strain in a liquid culture medium, performing anaerobic culture at 37 ℃ for 24 hours, centrifuging, absorbing supernatant, mixing with 20mmol/L TMB with the same volume, adding HRP (1 mg/mL), uniformly mixing, incubating at room temperature for 10 minutes, measuring an OD value, and substituting the OD value into the standard curve to obtain the concentration of the H ₂O₂ production of lactobacillus.

2.3 Determination of the ability of the Strain to inhibit the formation of the Gardnerella vaginalis biofilm

Gardnerella (GARDNERELLA VAGINALIS), also known as gardnerella or gardnerella vaginalis, is the primary pathogen for bacterial vaginitis, and the formation of a biofilm provides a safe growing environment for gardnerella vaginalis, so that gardnerella vaginalis can survive in a large amount in the vaginal environment, and the acidic environment of the vagina is destroyed. In this example, 3 strains of Lactobacillus gasseri were subjected to crystal violet staining to determine their inhibition on the Gardnerella vaginalis biofilm.

Comprehensively considering acid production capacity, H ₂O₂ production capacity and capacity of inhibiting formation of gardnerella vaginalis biomembrane of the strain, and primarily screening lactobacillus gasseri M17C with good probiotics. In particular, the H ₂O₂ -producing ability and the gardnerella vaginalis biofilm-forming inhibiting ability of the lactobacillus gasseri M17C are significantly better than those of the other two strains belonging to the same lactobacillus gasseri. The results were as follows:

TABLE 1.3 in vitro probiotic function determination results of Lactobacillus gasseri

Strain numbering	OD value	Acid production (delta pH)	Product H ₂O₂ (umol/l)	Biofilm inhibition%
					DM8909	1.42	1.6	11.7	42.5
M17C	1.4	1.9	12.6	20.0
					L61F	1.1	1.3	5.4	5.6
M17B	1.5	1.6	5.3	6.0

Example 3 remission of Lactobacillus gasseri M17C and three Mixed probiotics on mouse vaginitis

This example uses the mouse vaginitis model infected with gardnerella vaginalis.

3.1 Preparation of the Experimental Strain heavy suspension

After lactobacillus (Lactobacillus crispatus X25B, lactobacillus crispatus M58D and Lactobacillus gasseri M17C) preserved in a refrigerator with 50% glycerol at-80 ℃ is activated for three times by using MRS culture medium, centrifuging for 10 minutes at 6000rpm, discarding supernatant, washing the precipitated thallus with PBS buffer for 3 times, finally suspending the thallus in the PBS buffer to make the number of viable bacteria reach 1X 10 ⁸ CFU/mL, and obtaining the animal experiment intervention lactobacillus bacterial liquid. Similarly, after the gardnerella vaginalis is activated in the BHI liquid medium for three generations, the gardnerella vaginalis is centrifuged at 6000rpm for 10 minutes, and after the three generations of the gardnerella vaginalis is activated, the gardnerella vaginalis is collected, washed for 3 times, and resuspended in PBS buffer. Finally, the gardnerella vaginalis bacterial liquid with the concentration of 1X 10 ⁹ CFU/mL is obtained and is used in the molding period of animal experiments.

3.2 Alleviation of vaginitis in mice by oral administration of Lactobacillus gasseri M17C and three probiotic combinations

3.2.1 Animal Experimental design

Female ICR mice of 7 weeks old are bred in SPF animal houses with the temperature of 20-23 ℃ and the humidity of 55+/-5% and the light-dark period of 12 hours in an alternating cycle.

Mice were placed in a feeding environment for one week prior to the start of the experiment. The experiments set up a blank group, a BV model group, a Lactobacillus delbrueckii DM8909 group, a Lactobacillus gasseri M17C group, a Lactobacillus crispatus X25B, a Lactobacillus crispatus M58D, a mixed strain group of Lactobacillus delbrueckii M17C=1:1:1, 10 mice per group. All mice were fed with standard formula commercial feed, fed ad libitum and drinking.

Bacterial vaginitis models were established 3 days before infection (day-3) and on the day of infection (day 0), and the remaining mice except the blank group were subcutaneously injected with 100ul of estradiol benzoate (0.5 mg in 100ul of sesame oil). The viable bacteria count of the vaginal inoculation is 10 ⁹ CFU/mL, 20ul of gardnerella vaginalis suspension is continued for 5 days until the end of the vaginal gardnerella vaginalis inoculation on the 4 th day. After inoculation, the mice were inverted for 1 minute to prevent bacterial fluid from flowing out.

Oral experiments, starting from day 5, the experimental mice were gavaged for 10 ⁸ CFU/m L lactobacillus solution 100ul, once daily for 14 days. Mice in the placebo group were perfused with 100ul of PBS buffer once a day. After the last gastric lavage, all experimental mice were sacrificed using cervical dislocation.

3.2.2 Mouse vaginal epithelial shed cell assay

The mice were collected for vaginal lavage and 5ul of vaginal lavage was transferred to a slide prior to the last day of intervention, and gently smeared with a 10ul tip into a round shape of approximately 2 cm diameter. After fixation with an alcohol burner flame, gram staining was performed. The number of epithelial cells shed in 3 microscopic fields was randomly counted under a 40-fold microscope as shed cell count. And observing the morphology of the cells and the morphology and staining of the intracellular adherent bacteria under a 400-fold mirror and a 1000-fold mirror. The results are shown in Table 2.

3.2.3 Vaginal histopathological analysis and epithelial thickness determination of mice

The vaginal tissue of the mice was fixed and preserved with 4% tissue fixative, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H & E). The HE staining pathology scoring standard is that a score of 0-5 is given according to the integrity of the tissue (clear structure and orderly cell arrangement), wherein 0 indicates that the tissue structure is perfect and 5 indicates that the structural tissue is completely destroyed. The epithelial layer thickness of random 3 sample sections was observed and measured under a section specimen microscope at 400 x magnification.

TABLE 2 mouse vaginal epithelial cell shedding and histological pathological results

Note that # represents significant differences from the model group

As can be seen from the results of Table 2, the vaginal epithelial cells of BV-infected mice are largely shed, the thickness of the vaginal epithelium is obviously reduced, and serious pathological conditions appear, while the intervention of the positive drug DM8909, the Lactobacillus gasseri M17C and the mixed strain obviously improves the pathological conditions of the mice, so that the shedding amount of the vaginal epithelial cells is obviously reduced, the pathological score is reduced, the thickness of the vaginal epithelium is obviously increased, and the health conditions of the vagina of the mice are improved. The intervention of lactobacillus gasseri M17B showed substantially no effect, and each test result was significantly different from Yu Geshi lactobacillus M17C.

Compared with lactobacillus gasseri M17C, the intervention of the mixed strain further obviously reduces the pathological score, even the improvement effect on the vaginal epithelial thickness is even better than that of positive medicine DM8909, and the health condition of the vagina of the mouse is obviously improved.

3.2.4 Determination of the sialidase Activity and cytokines of the mouse vaginal lavage fluid

The mice vaginal lavage fluid was centrifuged and 10ul of supernatant was taken for the experiment. Sialidase activity was determined by ELISA and OD ₄₅₀ _nm values were compared to standard curves.

A sample of mouse vaginal tissue (50 mg) was placed in 300ul of RIPA lysate containing 1% protease inhibitor cocktail and broken up with a high throughput tissue mill (70 Hz,30 s/time, 10 times) to give a vaginal tissue homogenate. Centrifugation was carried out at 14000rpm for 15 minutes at 4℃and the supernatant was assayed for Myeloperoxidase (MPO) activity and for the concentration of TNF- α, IL-1β, IL-6, IL-10 by ELISA.

TABLE 3 determination of MPO, inflammatory cytokines and sialidases in vaginal lavage fluid in mouse vaginal tissue

Note that # represents significant differences from the model group

From the results in Table 3, it can be seen that the vagina of BV-infected mice exhibited a significant inflammatory response, which is manifested by an increase in sialidase activity and MPO enzyme activity in the vagina, an increase in the levels of the pro-inflammatory factors TNF- α, IL-1 β, IL-6, and a decrease in the levels of the anti-inflammatory factor IL-10. The intervention of the positive medicine DM8909 obviously reduces sialidase activity and MPO enzyme activity in vagina, improves inflammatory reaction to a certain extent, and the intervention of the lactobacillus gasseri M17C obviously reduces the levels of pro-inflammatory factors TNF-alpha and IL-1 beta, obviously improves the level of anti-inflammatory factor IL-10, and reduces the levels of pro-inflammatory factors IL-6, sialidase activity and MPO enzyme activity. Meanwhile, the inflammatory reaction is improved to a great extent, wherein the reducing effect of the lactobacillus gasseri M17C on pro-inflammatory factors TNF-alpha and IL-1 beta and the improving effect on anti-inflammatory factors IL-10 are better than those of a positive medicament group. While the intervention of lactobacillus gasseri M17B showed substantially no effect, each test result was substantially identical to that of BV model group, significant difference Yu Geshi lactobacillus M17C.

Compared with lactobacillus gasseri M17C, the intervention of the mixed strain not only remarkably reduces sialidase activity and MPO enzyme activity, but also remarkably reduces the levels of pro-inflammatory factors TNF-alpha, IL-1 beta and IL-6, improves the level of anti-inflammatory factor IL-10 and greatly improves inflammatory response, and the reduction effect of the intervention of the mixed strain on the pro-inflammatory factors TNF-alpha, IL-1 beta and IL-6 and the improvement effect on the anti-inflammatory factors IL-10 are superior to those of a positive medicament group, even the levels of the pro-inflammatory factors IL-1 beta and IL-6 are lower than those of a blank control group. Thus, oral administration of Lactobacillus gasseri M17C and oral administration of the mixed strain (Lactobacillus crispatus X25B: lactobacillus crispatus M58D: lactobacillus gasseri M17C=1:1:1) had a better effect of improving the vaginal inflammation of mice caused by BV.

3.3 Remission of external Lactobacillus gasseri M17C and three Mixed probiotics on mouse vaginitis

3.3.1 Animal Experimental design

Bacterial vaginitis models were established 3 days before infection (day-3) and on the day of infection (day 0), and the remaining mice except the control group were subcutaneously injected with 100ul of estradiol benzoate (0.5 mg in 100ul of sesame oil). The viable bacteria count of the vaginal inoculation is 10 ⁹ CFU/mL, 20ul of gardnerella vaginalis suspension is continued for 5 days until the end of the vaginal gardnerella vaginalis inoculation on the 4 th day. After inoculation, the mice were inverted for 1 minute to prevent bacterial fluid from flowing out.

External experiments starting from day 5, the experimental mice were inoculated vaginally with 10 ⁸ CFU/mL of Lactobacillus bacteria solution 20ul. The mice were inverted, 20ul of the bacteria solution resuspended in PBS was pipetted into the vagina of the mice using a 200ul pipette tip, and the inverted posture was maintained for 1 minute to avoid bacterial solution outflow. The inoculation was performed once daily for 14 days. Mice in the placebo group were inoculated intravaginally with 20ul of PBS buffer once a day. After the last inoculation, all experimental mice were sacrificed using cervical dislocation.

3.3.2 Mouse vaginal epithelial shed cell assay

The mice were collected for vaginal lavage and 5ul of vaginal lavage was transferred to a slide prior to the last day of intervention, and gently smeared with a 10ul tip into a round shape of approximately 2 cm diameter. After fixation with an alcohol burner flame, gram staining was performed. The number of epithelial cells shed in 3 microscopic fields was randomly counted under a 40-fold microscope as shed cell count. And observing the morphology of the cells and the morphology and staining of the intracellular adherent bacteria under a 400-fold mirror and a 1000-fold mirror. The results are shown in Table 4.

3.3.3 Vaginal histopathological analysis and epithelial thickness determination in mice

TABLE 4 results of mouse vaginal epithelial cell shedding and histopathological conditions

Note that # represents significant differences from the model group

As can be seen from the results of Table 4, the vaginal epithelial cells of BV-infected mice are largely shed, the thickness of the vaginal epithelium is remarkably reduced, and serious pathological conditions appear, while the intervention of the positive drug strain DM890 remarkably improves the pathological conditions of the mice, so that the shedding amount of the vaginal epithelial cells is greatly reduced, the pathological scores are remarkably reduced, the thickness of the vaginal epithelium is remarkably increased, the intervention of the Lactobacillus gasseri M17C remarkably reduces the shedding amount of the vaginal epithelial cells, and meanwhile, the pathological scores are reduced, the thickness of the vaginal epithelium is increased, and the vaginal health condition of the mice is improved to a certain extent. The intervention of lactobacillus gasseri M17B showed substantially no effect, and each test result was significantly different from Yu Geshi lactobacillus M17C.

Compared with lactobacillus gasseri M17C, the intervention of the mixed strain significantly improves the pathological state of the mice, so that the vaginal epithelial cell shedding amount is significantly reduced, the pathological score is significantly reduced, the vaginal epithelial thickness is significantly increased, and the mixed strain is superior to the positive drug strain DM8909 in improving the epithelial cell thickness, and the vaginal health condition of the mice is significantly improved.

3.3.4 Determination of the sialidase Activity and cytokines of the mouse vaginal lavage fluid

A sample of mouse vaginal tissue (50 mg) was placed in 300ul of RIPA lysate containing 1% protease inhibitor cocktail and broken up with a high throughput tissue mill (70 Hz,30 s/time, 10 times) to give a vaginal tissue homogenate. Centrifugation was carried out at 14000rpm for 15 minutes at 4℃and the supernatant was assayed for Myeloperoxidase (MPO) activity and for the concentration of TNF- α, IL-1β, IL-6 by ELISA.

TABLE 5 determination of MPO, inflammatory cytokines and sialidases in vaginal lavage fluid in mouse vaginal tissue

Blank control group

BV model set

DM8909 group

M17B group

M17C group

Mixed strain group

MPO

26.9

34.4^*

30.6

33.5

28.6

27.8#

TNF-α

545.3

709.4^***

565.2^##

649.3

549.7^###

616.9

IL-1β

74.0

120.2^***

79.6^###

108.8

105.0

92.7##

IL-6

58.1

82.2^**

60.7^##

75.3

65.4^#

73.5

Sialidases

44.5

57.1^***

47.4^###

55.9

53.0

51.6#

Note that # represents significant differences from the model group

From the results in Table 5, it can be seen that the vagina of BV-infected mice exhibited a significant inflammatory response, which is manifested by an increase in sialidase activity and MPO enzyme activity in the vagina, an increase in the levels of the pro-inflammatory factors TNF- α, IL-1β, IL-6, an intervention of the positive drug DM8909 significantly reduced the sialidase activity and the levels of the pro-inflammatory factors TNF- α, IL-1β, IL-6 in the vagina, an improvement in inflammatory response to a certain extent, an intervention of Lactobacillus gasseri M17C significantly reduced the levels of the pro-inflammatory factors TNF- α and IL-6, and reduced the levels of sialidase activity, MPO enzyme activity, and pro-inflammatory factor IL-1β, and an improvement in inflammatory response to a certain extent, wherein the reduction of the pro-inflammatory factor TNF- α by Lactobacillus gasseri M17C was even superior to the positive drug group. The intervention of lactobacillus gasseri M17B showed substantially no effect, and each test result was significantly different from Yu Geshi lactobacillus M17C.

Compared with lactobacillus gasseri M17C, the intervention of the mixed strain significantly reduces the sialidase activity, MPO enzyme activity and the level of the pro-inflammatory factor IL-1 beta, improving the inflammatory response to some extent. Thus, the external lactobacillus gasseri M17C and the external mixed strain (lactobacillus crispatus X25B: lactobacillus crispatus M58D: lactobacillus gasseri m17c=1:1:1) have the effect of potentially improving the colpitis of mice caused by BV.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A lactobacillus gasseri (Lactobacillus gasseri) deposited with the cantonese collection of microorganism strains under accession number GDMCC No.63872.

2. The lactobacillus gasseri of claim 1 having one or more characteristics selected from the group consisting of:

(1) The lactobacillus gasseri has the ability to produce hydrogen peroxide of greater than 5umol/l (e.g., 5 to 8, 8 to 12, 12 to 15, 15 to 20 or more) under effective culture conditions;

(2) The lactobacillus gasseri is capable of lowering the pH of the female genital tract (e.g., lowering the pH by 1 or more);

(3) The lactobacillus gasseri is capable of inhibiting the growth of and/or inhibiting the biofilm formation of a strain of gardnerella;

(4) The lactobacillus gasseri is capable of reducing the level of a proinflammatory factor (e.g., TNF- α, IL-1β, IL-6);

(5) The lactobacillus gasseri is capable of increasing the level of an anti-inflammatory factor (e.g., IL-10);

(6) The colony of the lactobacillus gasseri is white, milky white and/or semitransparent;

(7) The lactobacillus gasseri is a gram positive bacterium.

3. A composition comprising the lactobacillus gasseri of claim 1 or 2;

Preferably, the composition further comprises a probiotic selected from bacteria, fungi (e.g., yeast), or any combination thereof;

preferably, the bacteria are selected from the group consisting of lactobacillus, bifidobacterium, bacillus, propionibacterium, streptococcus, lactococcus, pediococcus, enterococcus, staphylococcus, or any combination thereof;

Preferably, the bacterium of the genus Lactobacillus is selected from the group consisting of Lactobacillus paracasei (Lactobacillus paracasei), lactobacillus jensenii (Lactobacillus jensenii), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus rhamnosus (Lactobacillus rhamnosus), or any combination thereof;

Preferably, the Lactobacillus paracasei is Lactobacillus paracasei 207-27 with accession number GDMCC No. 60960;

Preferably, the composition comprises Lactobacillus crispatus having deposit No. GDMCC No.63705, lactobacillus crispatus having deposit No. GDMCC No.63707, and Lactobacillus gasseri having deposit No. GDMCC No. 63872;

preferably, the composition comprises Lactobacillus crispatus having deposit number GDMCC No.63705, lactobacillus crispatus having deposit number GDMCC No.63707, and Lactobacillus gasseri having deposit number GDMCC No.63872 in a ratio of 1-10:1-10:1-10 (e.g., 1:1:1,10-1:1, 1:10-1:1,1:1:10-1,10-1:10-1:1,10-1:1:10-1, 1:10-1:10-10-1).

4. A pharmaceutical composition comprising the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3;

Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or excipient, cryoprotectant, amino acid, vitamin, mineral, peptone, or any combination thereof;

Preferably, the pharmaceutically acceptable carrier and/or excipient comprises a filler, binder, lubricant, glidant, thickener, flavoring agent, edible oil, stabilizer, suspending agent, surfactant, 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-oligosaccharides, glutathione, or any combination thereof;

preferably, the pharmaceutical composition is formulated for oral administration or in vitro administration;

Preferably, the pharmaceutical composition comprises a formulation of lactobacillus gasseri;

preferably, the pharmaceutical composition is in the form of a pill, powder, capsule, tablet (e.g., effervescent tablet), oil drop, caplet, mouth-soluble granule, liquid, suppository, enema gel, and/or cream;

preferably, the pharmaceutical composition further comprises formulation excipients (e.g., excipients for the preparation of powders, tablets, capsules, oil drops);

Preferably, the pharmaceutical composition is used alone or in combination with other antifungal agents, antiviral agents, analgesic agents, anti-inflammatory agents, healing promoting agents and/or moisturizers;

Preferably, the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 is present in the pharmaceutical composition in an amount of 10 ⁶ to 10 ¹² CFU/dose (e.g., 10 ⁷ to 10 ⁹ CFU/ml);

Preferably, when the pharmaceutical composition is a solid, it comprises 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri according to claim 1 or 2 or the composition of claim 3 per gram (e.g., 10 ⁷CFU/g,10⁸CFU/g,10⁹ CFU/g);

Preferably, when the pharmaceutical composition is a liquid, it comprises 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri according to claim 1 or 2 or the composition according to claim 3 (e.g., 10 ⁷CFU/mL,10⁸CFU/mL,10⁹ CFU/mL) per milliliter;

Preferably, the lactobacillus crispatus and/or lactobacillus grignard in the pharmaceutical composition are present in a live form or in a dead form.

5. A culture comprising the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3;

Preferably, the culture further comprises a nutrient providing component (e.g., a solid or liquid medium, a feeder cell layer);

Preferably, the nutrient providing ingredient is selected from the group consisting of a protein (e.g., an enzyme), a carbohydrate, a fat, a vitamin, a mineral, a dietary fiber, an amino acid, or any combination thereof;

preferably, the culture further comprises a cell-free culture filtrate of lactobacillus crispatus and/or lactobacillus gasseri;

Preferably, the culture further comprises a derivative of Lactobacillus crispatus and/or Lactobacillus gasseri, wherein the derivative is selected from the group consisting of a metabolite, an enzyme, a cell structural component (e.g., a cell wall or component thereof), an extracellular polysaccharide, a bacteriocin, a compound comprising an immunogenic component, or any combination thereof.

6. A food product or dietary supplement or health food comprising the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 or the culture of claim 5;

Preferably, the food product is selected from a solid beverage, a liquid beverage, a tabletted candy, a puffed food, a protein bar or nut, or the food product is a dairy product (e.g., milk powder, milk flakes, yogurt, flavored fermented milk, lactobacillus beverage, cheese);

Preferably, the food product or dietary supplement or health food further comprises a prebiotic;

Preferably, the prebiotic is selected from fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, soy oligosaccharides, inulin, spirulina, arthrospira, coriolus versicolor polysaccharides, nitrogen-containing polysaccharides of carrot, or any combination thereof;

preferably, the food product or dietary supplement or health food further comprises an additional additive;

Preferably, the additional additive is selected from the group consisting of a protein (e.g., an enzyme), a carbohydrate, a fat, a vitamin, a mineral, a dietary fiber, an amino acid, or any combination thereof;

Preferably, the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 is present in the food product or dietary supplement or health food in an amount of 10 ⁶ to 10 ¹² CFU/dose (e.g., 10 ⁷ to 10 ⁹ CFU/ml);

Preferably, when the food product or dietary supplement or health food is a solid, comprising 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri according to claim 1 or 2 or the composition of claim 3 (e.g. 10 ⁷CFU/g,10⁸CFU/g,10⁹ CFU/g) per gram;

Preferably, when the food product or dietary supplement or health food product is a liquid comprising 10 ⁶ to 10 ¹² CFU of lactobacillus gasseri according to claim 1 or 2 or the composition according to claim 3 (e.g. 10 ⁷CFU/mL,10⁸CFU/mL,10⁹ CFU/mL) per mL;

preferably, the lactobacillus crispatus and/or lactobacillus grignard is present in the food product or dietary supplement or health food in a live or dead bacterial form.

7. A cleaning product comprising the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 or the culture of claim 5;

Preferably, the cleansing product is selected from the group consisting of solid soaps, liquid soaps, hand washes, shampoos, liquid conditioners, body cleansers, or any combination thereof;

Preferably, the cleansing product is a vaginal lavage, vaginal gel and/or vaginal cream.

8. Use of lactobacillus gasseri 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 for the manufacture of a medicament for the prevention and/or treatment of female genitourinary related diseases and/or symptoms in a female subject;

preferably, the female genitourinary tract-related disease is caused by an imbalance in the microbiota of the genital tract and/or wherein the pH of the genital tract is abnormal;

Preferably, the female genitourinary tract-related disease is related by one or more of the following:

(1) A reduction in the level of lactobacillus bacterial strains in the genital tract ecosystem;

(2) An increase in proliferation of at least one microorganism belonging to the group consisting of gardnerella vaginalis (GARDNERELLA VAGINALIS), candida albicans (Candida albicans), staphylococcus aureus (Streptococcus aureus), atobacter vaginalis (Atopobium vaginae), or any combination thereof;

(3) The female genitourinary tract-related disease is associated with an increase in proliferation of at least one microorganism belonging to the genus Prevotella (Prevolella spp.), pediococcus (Megasphaera spp.), sinapis (Sneathia spp.), agrobacter (Atopobium spp.), listeria (DIALISTER spp.), anaerobiosciens (Anaerococcus spp.), eggeria (EGGERTHELLA spp.), peptone bacterium (Peptoniphilus spp.), balloon (aerococus spp), acinetobacter (Mobiluncus spp), or any combination thereof, and/or

(4) An increase in pH in the genital tract environment;

Preferably, the medicament is for maintaining balance of microbiota of the female genital tract, lowering pH of the female genital tract, increasing levels of lactobacillus bacterial strains in the ecosystem of the female genital tract and/or lowering levels of gardnerella bacterial strains in the female genital tract;

preferably, the strain of gardnerella is gardnerella;

Preferably, the disorder is selected from infection of the female genitourinary tract, genital herpes, gonorrhea, inflammatory disorders associated with the female genitourinary tract (e.g., vaginitis, cervicitis), or any combination thereof;

Preferably, the symptom is selected from the group consisting of menstrual cycle changes, vaginal itching, vaginal redness, abnormal secretions, vaginal pain, or any combination thereof.

9. The use of claim 8, wherein the medicament is for preventing and/or treating inflammatory diseases associated with the female genitourinary tract by reducing the level of pro-inflammatory factors and/or increasing the level of anti-inflammatory factors;

Preferably, the medicament is used for preventing and/or treating inflammatory diseases related to female genitourinary tract by reducing sialidase activity and/or Myeloperoxidase (MPO) activity;

Preferably, the medicament is used for preventing and/or treating inflammatory diseases related to female genitourinary tract by increasing the level of hydrogen peroxide (H ₂O₂);

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 lactobacillus gasseri 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 product according to claim 6 or the cleaning product according to claim 7 for the preparation of a medicament for inhibiting gardnerella growth and/or reducing the amount thereof;

preferably, the agent inhibits the growth of a strain of gardnerella by increasing the level of hydrogen peroxide (H ₂O₂);

preferably, the agent inhibits the growth of a strain of gardnerella by lowering the level of the pH of the environment in which it is located;

Preferably, the medicament is for inhibiting biofilm formation of a strain of gardnerella, thereby inhibiting growth of a strain of gardnerella;

Preferably, the medicament is for inhibiting the growth and/or reducing the amount of gardnerella in a female or for inhibiting the growth and/or reducing the amount of gardnerella in an environment;

preferably, the female body comprises a mucous membrane and/or a genital tract;

Preferably, the environment comprises a home, a workplace, a laboratory, an industrial environment, an aquatic environment, medical equipment and/or gynecological examination equipment.

11. A method of inhibiting gardnerella growth and/or reducing the amount thereof in a female, the method comprising administering to a female subject in need thereof an effective amount of the lactobacillus gasseri 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 product of claim 6 or the cleansing product of claim 7;

Preferably, the method comprises:

(1) Implanting lactobacillus gasseri according to claim 1 or 2 or the composition according to claim 3 or the pharmaceutical composition according to claim 4 into the genital tract of a female subject;

(2) Applying (e.g. applying) the Lactobacillus gasseri according to claim 1 or 2 or the composition according to claim 3 or the pharmaceutical composition according to claim 4 or the cleaning product according to claim 7 to the genital tract and/or perineum of a female subject, or

(3) Orally (e.g., orally) administering the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 or the pharmaceutical composition of claim 4 or the food product or dietary supplement or health food of claim 6 to a female subject;

Preferably, the gardnerella comprises gardnerella (GARDNERELLA VAGINALIS);

preferably, the female body comprises a mucous membrane and/or a genital tract;

Preferably, the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 is administered to a subject in an amount of 10 ⁶ 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).

12. A method of inhibiting gardnerella growth and/or reducing the amount thereof in an environment, the method comprising administering to the environment an effective amount of the lactobacillus griseus of claim 1 or 2 or the composition of claim 3 or the pharmaceutical composition of claim 4 or the culture of claim 5;

preferably, the environment comprises a home, workplace, laboratory, industrial environment, aquatic environment, medical instrument and/or gynecological examination appliance;

Preferably, the lactobacillus gasseri of claim 1 or 2 or the composition of claim 3 is administered to the environment in an amount of 10 ⁶ 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).

13. A method of preventing and/or treating a female genitourinary tract-related disease and/or symptom, the method comprising administering to a female subject in need thereof an effective amount of lactobacillus gasseri according to claim 1 or 2 or a composition according to claim 3 or a pharmaceutical composition according to claim 4 or a culture according to claim 5 or a food product or dietary supplement or health food product according to claim 6 or a cleansing product according to claim 7;

preferably, the female genitourinary tract-related disease is associated with one or more of the following:

(2) An increase in proliferation of at least one microorganism belonging to the genus Gardnerella, polyrenia, streptococcus, anaerococcus, fingerd, prevotella, equisetum, staphylococcus, corynebacterium, or any combination thereof, and/or

(3) An increase in pH in the genital tract environment;

preferably, the strain of gardnerella is gardnerella;

Preferably, the symptom is selected from the group consisting of altered menstrual cycle, pruritus vulvae, redness of the vagina, burning of the vulva, abnormal secretions (e.g., abnormal leucorrhea), vaginal pain, or any combination thereof;

Preferably, the subject is a mammal;

Preferably, the subject is a human;