Lactobacillus gasseri VB247 and use thereof

Abstract

Provided is Lactobacillus gasseri VB247, which was deposited at the China General Microbiological Culture Collection Center on 20 February 2023 with a deposit number of CGMCC No. 26575. The Lactobacillus gasseri VB247 can significantly improve the AQP2 mRNA expression level in female cervical and vaginal epithelial cells, and can be used for preventing and / or alleviating and / or treating vaginal lubrication disorders caused by perimenopause, premature ovarian failure, etc. In addition, the Lactobacillus gasseri VB247 has application prospects in the preparation of products such as foods, health-care foods, medicines, and hygiene products.

Description

Lactobacillus gasseri VB247 and its application Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a Lactobacillus gasseri VB247 capable of significantly increasing the expression level of AQP2 mRNA in female cervicovaginal epithelial cells and its application. More specifically, the present invention relates to a Lactobacillus gasseri VB247, a fermentation supernatant, a fermentation broth, a microbial agent, a food, a health food, a medicine or a sanitary product, and their uses. Background Art

[0002] Female sexual dysfunction (FSD) can have numerous adverse effects on women's reproductive health, including dyspareunia, difficulty conceiving, anxiety and depression, disruptions to their partner's sexual function, and disharmony in family life. The incidence of FSD in my country is as high as 37.6%, and globally it ranges from 25% to 63%. Vaginal lubrication disorder is the most common, accounting for 97.9% of the total incidence. Currently, treatment for vaginal lubrication disorder mostly involves oral or topical vaginal estrogen administration, which can effectively alleviate vaginal lubrication disorders caused by estrogen deficiency during perimenopause and premature ovarian failure. However, excessive estrogen intake can have certain side effects on the body, such as nausea, vomiting, headaches, changes in menstrual flow, and the risk of breast cancer.

[0003] Studies have found that aquaporin (AQP) is an important substance that regulates water balance within cells, between cells and the entire body. In the aquaporin family, aquaporin 2 (AQP2) is mainly expressed in vaginal epithelial cells. Healthy women are in an open channel state during sexual excitement, while women with vaginal lubrication disorders have low AQP2 mRNA expression in vaginal epithelial cells.

[0004] Therefore, there is an urgent need to develop a bacterial strain that can effectively increase the expression of AQP2 mRNA without affecting the vaginal flora environment.

[0005] Summary of the Invention

[0006] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides, for the first time, a strain of Lactobacillus gasseri VB247, derived from the vaginas of healthy women of childbearing age in my country. This strain exhibits advantages such as low pH tolerance, high lactic acid production, resistance to five common vaginal pathogens, and strong vaginal cell adhesion. Furthermore, it can significantly increase AQP2 mRNA expression levels in female cervicovaginal epithelial cells, and can be used to prevent and / or alleviate and / or treat vaginal lubrication disorders caused by perimenopause, premature ovarian failure, and other causes.

[0007] Based on this, the first object of the present invention is to provide a Lactobacillus gasseri VB247. According to an embodiment of the present invention, the Lactobacillus gasseri VB247 is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, with a deposit number of CGMCC No. 26575, a deposit date of February 20, 2023, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus gasseri VB247 of the present invention has the advantages of low pH resistance, regulation of vaginal pH environment, high lactic acid production, resistance to 5 common vaginal pathogens, strong vaginal cell adhesion ability, and can significantly increase the AQP2 mRNA expression level in female cervical vaginal epithelial cells.

[0008] The second object of the present invention is to provide a fermentation supernatant comprising: the aforementioned Lactobacillus gasseri VB247; and / or the metabolites of the aforementioned Lactobacillus gasseri VB247.

[0009] The third object of the present invention is to provide a fermentation broth, which comprises the aforementioned Lactobacillus gasseri VB247.

[0010] A fourth object of the present invention is to provide a microbial agent comprising the aforementioned Lactobacillus gasseri VB247, and / or the aforementioned fermentation supernatant, and / or the aforementioned fermentation broth. As previously mentioned, Lactobacillus gasseri VB247 exhibits advantages such as low pH tolerance, vaginal pH regulation, high lactic acid production, resistance to five common vaginal pathogens, and strong vaginal cell adhesion. The microbial agent of the present invention exhibits similar advantages and can be used to prevent and / or alleviate and / or treat vaginal lubrication disorders caused by perimenopause, premature ovarian failure, and other causes.

[0011] As a specific embodiment, the Lactobacillus gasseri VB247 exists in the form of living cells and / or non-living cells.

[0012] As a specific embodiment, the microbial agent may further contain at least one strain acceptable to food, medicine, health food and feed.

[0013] As a specific embodiment, the microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

[0014] A fifth object of the present invention is to provide a food, health food or sanitary product, characterized in that it contains at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth and the aforementioned microbial agent.

[0015] A sixth object of the present invention is to provide a use of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth or the aforementioned microbial agent in the preparation of a medicament, wherein the medicament has at least one of the following uses: for preventing and / or alleviating and / or treating vaginal lubrication disorders caused by perimenopause, premature ovarian failure, etc.; for preventing and / or alleviating and / or treating vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection; for increasing the expression level of AQP2 mRNA in vaginal epithelial cells; for regulating the vaginal pH environment.

[0016] As a specific embodiment, the pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0017] A seventh object of the present invention is to provide a medicine comprising at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, and the aforementioned microbial agent.

[0018] As a specific embodiment, the drug further contains an excipient and / or a carrier.

[0019] As a specific embodiment, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

[0020] As a specific embodiment, the carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

[0021] As a specific embodiment, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

[0022] An eighth object of the present invention is to provide the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent or the aforementioned medicine having at least one of the following uses: preventing and / or alleviating and / or treating vaginal lubrication disorders; preventing and / or alleviating and / or treating vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection; increasing the expression level of AQP2 mRNA in vaginal epithelial cells; and regulating the vaginal pH environment.

[0023] According to an embodiment of the present application, the pathogenic bacteria include at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0024] A ninth object of the present invention is to provide a method for inhibiting the growth of pathogenic bacteria. According to an embodiment of the present application, the method comprises: contacting the pathogenic bacteria with at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned drug.

[0025] According to an embodiment of the present application, the pathogenic bacteria include at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0026] A tenth object of the present invention is to provide a method for preventing and / or alleviating and / or treating vaginal lubrication disorders. According to an embodiment of the present application, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned drug.

[0027] An eleventh object of the present invention is to provide a method for preventing and / or alleviating and / or treating vaginal pathogen infection or diseases related to vaginal pathogen infection. According to an embodiment of the present application, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned drug.

[0028] According to an embodiment of the present application, the pathogenic bacteria include at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens. Beneficial effects:

[0029] (1) Compared with other Lactobacillus gasseri isolates, the Lactobacillus gasseri VB247 screened by the present invention has a faster growth start, a higher slope in the logarithmic growth phase, and a higher OD value in the stable growth phase. 600 It is higher, indicating that its growth starts faster, the activation rate is higher, and the growth activity is stronger in a low pH environment (pH=4.0).

[0030] (2) Compared with other Lactobacillus gasseri isolates, the lactic acid (L-lactic acid and D-lactic acid) content in the fermentation supernatant of Lactobacillus gasseri VB247 obtained by the present invention is higher, indicating that the strain has a high lactic acid production.

[0031] (3) The Lactobacillus gasseri VB247 of the present invention can reduce pH faster than other Lactobacillus gasseri isolates, which indicates that the strain has a stronger ability to regulate pH and can create and maintain a healthy vaginal pH environment.

[0032] (4) The Lactobacillus gasseri VB247 of the present invention has inhibitory activity against a variety of pathogenic bacteria and can inhibit the growth of vaginal pathogens such as Escherichia coli, Staphylococcus aureus, Candida albicans, Gardnerella vaginalis and Clostridium perfringens. Therefore, it can be used alone or in combination with other probiotics for the adjuvant treatment of a variety of gynecological diseases, such as vaginitis and vaginal lubrication disorders.

[0033] (5) The Lactobacillus gasseri VB247 of the present invention has a strong ability to adhere to vaginal cells and is more likely to colonize in the vagina.

[0034] (6) The Lactobacillus gasseri VB247 of the present invention can significantly increase the expression level of AQP2 mRNA in female cervical vaginal epithelial cells, thereby improving vaginal dryness, and can be used to prevent and / or alleviate and / or treat vaginal lubrication disorders caused by perimenopause, premature ovarian failure, etc.

[0035] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0037] FIG1 is a scanning electron microscope photograph of Lactobacillus gasseri VB247 in Example 1 of the present invention;

[0038] FIG2 is the OD of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 at pH = 4.0 in Example 2 of the present invention 600 Curves of change over time;

[0039] 3 is a curve showing changes in pH value of the fermentation broth of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 over time in Example 4 of the present invention;

[0040] FIG4 shows the adhesion of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 to HeLa cells in Example 6 of the present invention;

[0041] FIG5 shows the effects of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 on AQP2 mRNA expression in VK2 / E6E7 human vaginal epithelial cells in Example 7 of the present invention. DETAILED DESCRIPTION

[0042] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0044] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0045] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0046] As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," Lactobacillus muciniphila ferments or a product containing the same can be administered to a subject at risk for developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0047] As used herein, the terms "treat" and "alleviate" refer to methods used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0048] The present invention provides a Lactobacillus gasseri VB247, a fermentation supernatant, a fermentation broth, a microbial agent, a food, a health food, a medicine or a sanitary product and uses thereof, which will be described in detail below.

[0049] strains

[0050] In one aspect, the present invention provides Lactobacillus gasseri VB247. According to an embodiment of the present invention, the deposit number of Lactobacillus gasseri VB247 is CGMCC No. 26575. Lactobacillus gasseri VB247 exhibits advantages such as low pH tolerance, vaginal pH regulation, high lactic acid production, resistance to five common vaginal pathogens, and strong vaginal cell adhesion. Furthermore, it can significantly increase the expression level of AQP2 mRNA in female cervicovaginal epithelial cells.

[0051] As used herein, "Lactobacillus gasseri VB247" and "Lactobacillus gasseri VB247" are synonymous.

[0052] Fermentation supernatant

[0053] In another aspect, the present invention provides a fermentation supernatant. According to an embodiment of the present invention, the fermentation supernatant comprises: the aforementioned Lactobacillus gasseri VB247; and / or a metabolite of the aforementioned Lactobacillus gasseri VB247.

[0054] It should be noted that the “fermentation supernatant” refers to the supernatant after the fermentation broth is centrifuged and filtered, which mainly contains the metabolites of Lactobacillus gasseri VB247.

[0055] fermentation broth

[0056] In another aspect of the present invention, a fermentation broth is provided. According to an embodiment of the present invention, the fermentation broth comprises the aforementioned Lactobacillus gasseri VB247.

[0057] It should be noted that the "fermentation broth" mentioned in the present invention refers to the solution obtained after culturing Lactobacillus gasseri VB247 for a period of time.

[0058] microbial agents

[0059] In yet another aspect, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent comprises the aforementioned Lactobacillus gasseri VB247, and / or the aforementioned fermentation supernatant, and / or the aforementioned fermentation broth. The microbial agent of the present invention is used to prevent and / or treat diseases associated with vaginal flora disorders.

[0060] It should be noted that the microbial agent of the present invention can be a liquid microbial agent, including but not limited to fermentation broth, etc.; it can also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0061] According to an embodiment of the present invention, the Lactobacillus gasseri VB247 exists in the form of living cells and / or non-living cells.

[0062] As used herein, "living cells" refer to Lactobacillus gasseri VB247 cells that have the ability to metabolize, reproduce or replicate.

[0063] For example, the living cells may be immobilized cells. Herein, "immobilized cells" refer to Lactobacillus gasseri VB247 that are fixed on a carrier and can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0064] In this article, "non-viable cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dried bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0065] As a specific embodiment, the Lactobacillus gasseri VB247 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0066] As a specific embodiment, the dry bacteria are obtained by freeze-drying the Lactobacillus gasseri VB247.

[0067] As a specific embodiment, the microbial agent may further contain at least one strain acceptable to food, medicine, health food and feed.

[0068] As a specific embodiment, the microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

[0069] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to food requirements in different countries.

[0070] In this article, "acceptable in health foods" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health food requirements of different countries.

[0071] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0072] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0073] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the Lactobacillus gasseri VB247 of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0074] use

[0075] In another aspect of the present invention, the present invention provides a use of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth or the aforementioned microbial agent in the preparation of a medicament, wherein the medicament has at least one of the following uses: for preventing and / or alleviating and / or treating vaginal lubrication disorders; for preventing and / or alleviating and / or treating vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection; for increasing the expression level of AQP2 mRNA in vaginal epithelial cells; for regulating the vaginal pH environment.

[0076] In another aspect of the present invention, the present invention proposes that the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent or the aforementioned medicine has at least one of the following uses: preventing and / or alleviating and / or treating vaginal lubrication disorders; preventing and / or alleviating and / or treating vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection; increasing the expression level of AQP2 mRNA in vaginal epithelial cells; regulating the vaginal pH environment.

[0077] As a specific embodiment, the vaginal lubrication disorder includes but is not limited to vaginal lubrication disorder caused by estrogen deficiency such as perimenopause and premature ovarian failure.

[0078] As a specific embodiment, the pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0079] Food, health food or hygiene products

[0080] In another aspect of the present invention, the present invention provides a food, a health food or a sanitary product, which comprises at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth and the aforementioned microbial agent.

[0081] drug

[0082] In another aspect of the present invention, the present invention provides a medicine comprising at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, and the aforementioned microbial agent.

[0083] As a specific embodiment, the drug further contains an excipient and / or a carrier.

[0084] As a specific embodiment, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

[0085] As a specific embodiment, the carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

[0086] As a specific embodiment, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

[0087] method

[0088] In another aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria. According to an embodiment of the present application, the method comprises: contacting the pathogenic bacteria with at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned drug.

[0089] In another aspect, the present invention provides a method for preventing and / or alleviating and / or treating vaginal lubrication disorders. According to an embodiment of the present application, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned drug.

[0090] In another aspect, the present invention provides a method for preventing and / or alleviating and / or treating vaginal pathogen infection or diseases caused by vaginal pathogen infection. According to an embodiment of the present application, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned drug.

[0091] According to an embodiment of the present application, the pathogenic bacteria include at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0092] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0093] Example 1: Strain collection and identification

[0094] Vaginal secretions were collected from 68 women of childbearing age who had undergone health examinations (Hangzhou, China) using disposable sterile cotton swabs. The samples were spread on MRS solid medium and incubated anaerobically at 37°C for 48 hours. Single colonies suspected to be Lactobacillus were picked and amplified and sequenced using the bacterial 16S rDNA. The sequencing results were compared by BLAST. Based on the comparison results, the strains were analyzed and stored in glycerol tubes. A total of 157 strains of Lactobacillus gasseri were obtained and numbered #1-#157. After testing the 157 strains for low-pH growth viability, lactic acid production, pH regulation, antagonism against five vaginal pathogens, HeLa cell adhesion, and antibiotic susceptibility, the best performing strain, Lactobacillus gasseri #18, was identified and designated Lactobacillus gasseri VB247. It was deposited with the General Microbiology Center of the China Culture Collection Administration on February 20, 2023, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, under the accession number CGMCC No. 26575. A scanning electron micrograph of Lactobacillus gasseri VB247 is shown in Figure 1, showing its well-defined cylindrical morphology.

[0095] The 16S rDNA sequencing result of Lactobacillus gasseri VB247 is shown in SEQ ID NO: 1.

[0096] The 16S rDNA sequencing results of Lactobacillus gasseri isolate #86 are shown in SEQ ID NO: 2.

[0097] Physiological and biochemical characteristics of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 were analyzed. The specific experiments were performed with reference to Bergey's Handbook of Bacterial Identification. The test results are shown in Table 1. Compared with Lactobacillus gasseri isolate #86, the strain VB247 obtained by screening in this example can utilize milk and multiple carbohydrates such as L-rhamnose, mannitol, and sorbitol.

[0098] Table 1: Physiological and biochemical characteristics of different Lactobacillus gasseri

[0099] Note: + represents positive, - represents negative.

[0100] Example 2: Growth activity experiment under low pH environment

[0101] After activation, different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) were inoculated at a 1% inoculum into MRS broth at pH = 4.0 and anaerobically cultured at 37°C for 48 h. The OD of the fermentation broth was measured every 4 h. 600 Figure 2 shows the OD values ​​of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 at pH = 4.0 600 As can be seen from Figure 2, the OD of Lactobacillus gasseri VB247 was 600 Compared with the initial value, it increased by 20.9%, while Lactobacillus gasseri isolate #86 only increased by 1.2%; after 8 h of cultivation, the OD of Lactobacillus gasseri VB247 600 Compared with the initial value, it increased by 101.2%, while Lactobacillus gasseri isolate #86 only increased by 18.5%. In addition, the slope of Lactobacillus gasseri VB247 in the logarithmic phase of growth was higher, and the OD 600 The values ​​are higher, which indicates that Lactobacillus gasseri VB247 starts to grow faster, has a higher activation rate and stronger growth activity in a low pH environment.

[0102] Example 3: Lactic acid production capacity

[0103] The fermentation broths of different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) after culturing for 48 h in Example 2 were centrifuged to obtain fermentation supernatants. The L-lactic acid content in the fermentation supernatants was measured using a bioprocess analyzer, and the D-lactic acid content was measured using a D-lactic acid detection kit. The results are listed in Table 2.

[0104] Studies have found that lactic acid produced by the metabolism of lactobacilli in the vagina can inhibit the growth of vaginal pathogens and maintain vaginal health. As can be seen from Table 2, the amount of L-lactic acid and D-lactic acid produced by the Lactobacillus gasseri VB247 obtained by the present invention is nearly twice that of the Lactobacillus gasseri isolate #86. Lactobacillus gasseri VB247 can produce more L-lactic acid and D-lactic acid, indicating that the Lactobacillus gasseri VB247 of the present invention is more conducive to maintaining human vaginal health.

[0105] Table 2: Lactic acid content in 48h fermentation broth of different Lactobacillus gasseri

[0106] Example 4: pH Adjustment Capability

[0107] Studies have found that the pH value in the normal female vaginal environment is between 3.8 and 4.4. Vaginal flora disorder is usually accompanied by an increase in vaginal pH, which is caused by the excessive reproduction of other pathogenic bacteria, mainly aerobic bacteria.

[0108] After activation, different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) were inoculated at a 1% inoculum into MRS broth medium at pH = 6.5 and anaerobically cultured at 37° C. for 48 h. The pH of the fermentation broth was measured every 4 h. The test results are shown in FIG3 .

[0109] As can be seen from Figure 3, the Lactobacillus gasseri VB247 obtained in the present invention can reduce the pH faster than the Lactobacillus gasseri isolate #86 and reach acid-base balance at pH 4, which indicates that the strain can create and maintain a healthy vaginal pH environment.

[0110] Example 5: Antagonism against five common vaginal pathogens

[0111] The antibacterial activity of different Lactobacillus gasseri strains (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) against five common vaginal pathogens was determined using a double-layer plate method. The specific method is as follows:

[0112] After activating different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention), 2 μL of each was spotted on an MRS solid plate and anaerobically cultured at 37° C. for 48 h for later use.

[0113] The activated indicator bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8099, Candida albicans ATCC 10231, Gardnerella vaginalis ATCC 14018 and Clostridium perfringens ATCC 13124) were mixed with the molten agar medium cooled to 40-45°C after sterilization in a certain proportion. The concentration of indicator bacteria was 10 6 CFU / mL, poured into the plate of Lactobacillus gasseri cultured for 48 hours, placed in an environment suitable for indicator bacteria and cultured for 16-18 hours, observed and recorded the size of the inhibition zone to compare the antibacterial activity of different Lactobacillus gasseri. The test results are shown in Table 3.

[0114] As shown in Table 3, the diameters of the inhibition zones of Lactobacillus gasseri VB247 against Escherichia coli, Staphylococcus aureus, Candida albicans, Gardnerella vaginalis and Clostridium perfringens were all greater than 7 mm, indicating that it had an antibacterial effect. In addition, compared with Lactobacillus gasseri isolate #86, Lactobacillus gasseri VB247 had a stronger antibacterial effect against all five bacteria.

[0115] Table 3: Inhibition zone test results of different Lactobacillus gasseri against 5 pathogenic bacteria

[0116] Example 6: Adhesion to HeLa cells assay

[0117] After activation, different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) were inoculated into MRS broth at a pH of 6.5, cultured anaerobically at 37°C overnight, washed twice with PBS by centrifugation, and resuspended in PBS to 10 8 CFU / mL, and obtain bacterial suspensions containing different Lactobacillus gasseri. Pipette 100 μL of bacterial suspensions containing different Lactobacillus gasseri into a six-well cell culture plate containing HeLa cells (3×10 5 / well), incubated at 37°C for 2h, washed twice with sterile PBS to wash away free lactobacilli, and 25μL of trypsin solution was added to each well of a six-well cell culture plate containing HeLa cells. The plate was placed in a 37°C incubator to digest the cells. After the HeLa cells were digested into spheres, 75μL of DMEM medium (containing 10% fetal bovine serum) was added to each well and the cells were repeatedly pipetted evenly. After complete digestion, 20μL of the suspension was aspirated and diluted tenfold with sterile PBS. An appropriate dilution gradient was selected for coating and viable bacteria count. The results are shown in Figure 4.

[0118] As shown in FIG4 , the Lactobacillus gasseri VB247 obtained by screening of the present invention has a higher number of bacteria adhering to each HeLa cell than the Lactobacillus gasseri isolate #86, which indicates that the strain is more likely to colonize in the vagina.

[0119] Example 7: Effect on AQP2 mRNA expression in vaginal epithelial cells

[0120] VK2 / E6E7 human vaginal epithelial cells were purchased from Shanghai Yaji Biotechnology Co., Ltd., revived and passaged according to the product instructions, and seeded into three 6-well plates. The 6-well plates were then divided into three groups, each with three replicates. Two wells were added with 100 μL of the fermentation supernatant of Lactobacillus gasseri VB247 cultured for 48 h in Example 2, and the cells were harvested for 24 and 48 h. Two wells were added with 100 μL of the fermentation supernatant of Lactobacillus gasseri isolate #86 cultured for 48 h in Example 2, and the cells were harvested for 24 and 48 h. Two wells were added with 100 μL of progesterone to a final concentration of 10 nmol / L, and the cells were harvested for 24 and 48 h as a positive control. Among them, progesterone is an estrogen that can alleviate vaginal lubrication disorders during menopause. There are reports that progesterone can promote the expression of AQP2 mRNA (Zhang Jing et al. Effects of vaginal microenvironment-related factors on the expression of aquaporin 2 mRNA in vaginal epithelial cells [J]. Chinese Journal of Sexology, 2021, 30(4):4.). Real-time PCR technology was used to observe the expression of AQP2 mRNA in vaginal epithelial cells. The results are shown in Figure 5.

[0121] As shown in FIG5 , the Lactobacillus gasseri VB247 obtained by screening in the present invention can significantly increase the AQP2 mRNA expression level in female cervicovaginal epithelial cells compared with the Lactobacillus gasseri isolate #86.

[0122] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0123] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A Lactobacillus gasseri VB247, characterized in that The Lactobacillus gasseri VB247 has a deposit number of CGMCC No. 26575 at the General Microbiology Center of China Culture Collection Administration, and a deposit date of February 20, 2023.

2. A fermentation supernatant, characterized in that: include: The Lactobacillus gasseri VB247 according to claim 1; and / or The metabolite of Lactobacillus gasseri VB247 according to claim 1.

3. A fermentation broth, characterized in that: The invention comprises the Lactobacillus gasseri VB247 as claimed in claim 1.

4. A microbial agent, characterized in that: The method comprises at least one of the Lactobacillus gasseri VB247 according to claim 1, the fermentation supernatant according to claim 2 and the fermentation broth according to claim 3.

5. The microbial agent according to claim 4, characterized in that: The Lactobacillus gasseri VB247 exists in the form of living cells and / or non-living cells.

6. The microbial agent according to claim 4, characterized in that: It further includes pharmaceutically, food or health food acceptable auxiliary materials or carriers.

7. A food, health food or sanitary product, characterized in that: The method comprises at least one of the Lactobacillus gasseri VB247 according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3 and the microbial agent according to any one of claims 4 to 6.

8. Use of the Lactobacillus gasseri VB247 according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3 or the microbial agent according to any one of claims 4 to 6 in the preparation of a medicament, wherein the medicament has at least one of the following uses: Preventing and / or alleviating and / or treating vaginal lubrication disorders; Prevent and / or alleviate and / or treat vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection; Increase the expression of AQP2 mRNA in vaginal epithelial cells; Regulates the vaginal pH environment.

9. The use according to claim 8, characterized in that The pathogenic bacteria is selected from at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

10. A drug, characterized in that The drug comprises at least one of the Lactobacillus gasseri VB247 according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3 and the microbial agent according to any one of claims 4 to 6.

11. The drug according to claim 10, characterized in that The medicament further contains excipients and / or carriers.

12. The drug according to claim 11, characterized in that The excipient comprises at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

13. The drug according to claim 11, characterized in that The carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

14. The drug according to claim 10, characterized in that The dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

15. The Lactobacillus gasseri VB247 according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, or the drug according to claim 10 has at least one of the following uses: Preventing and / or alleviating and / or treating vaginal lubrication disorders; Prevent and / or alleviate and / or treat vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection; Increase the expression of AQP2 mRNA in vaginal epithelial cells; Regulates the vaginal pH environment.

16. A method for inhibiting the growth of pathogenic bacteria, characterized in that: include: The pathogenic bacteria are contacted with at least one of the Lactobacillus gasseri VB247 according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, or the drug according to claim 10.

17. The use according to claim 15 or the method according to claim 16, characterized in that The pathogenic bacteria include at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

Images