Lactobacillus gasseri VB247 and application thereof

Abstract

The invention discloses lactobacillus gasseri VB247 which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on February 20, 2023, and the preservation number of the lactobacillus gasseri VB247 is CGMCC No. 26575. The lactobacillus gasseri VB247 can obviously improve the expression level of AQP2mRNA in female cervix and vagina epithelial cells, and can be used for preventing and / or relieving and / or treating vagina lubrication disorder caused by perimenopausal period, premature ovarian failure and the like; in addition, the lactobacillus gasseri VB247 disclosed by the invention has a huge application prospect in preparation of products such as foods, health-care foods, medicines, hygienic products and the like.

Description

[0001] Case information

[0002] This application is a divisional application of the patent application filed with the State Intellectual Property Office of China on September 20, 2023, with patent application number 202311216998.4 and patent name "A Lactobacillus gasseri VB247 and its application". Technical Field

[0003] The present invention belongs to the field of microbial technology, and in particular relates to a Lactobacillus gasseri VB247 capable of significantly improving the expression level of AQP2 mRNA in female cervical vaginal epithelial cells and an application thereof. 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 uses thereof. Background Art

[0004] Currently, the treatment for vaginal lubrication disorders is mostly oral or topical estrogen administration, which can effectively alleviate vaginal lubrication disorders caused by estrogen deficiency during perimenopause and premature ovarian failure. However, excessive intake of estrogen will have certain side effects on the body, such as nausea, vomiting, headaches, changes in menstrual volume, and the risk of breast cancer.

[0005] Studies have found that aquaporin (AQP) is an important substance that regulates water balance within cells, between cells and in the entire organism. In the aquaporin family, aquaporin 2 (AQP2) is mainly expressed in vaginal epithelial cells. Healthy women are in an open channel state when they are sexually excited, while women with vaginal lubrication disorders have low AQP2 mRNA expression in vaginal epithelial cells.

[0006] Therefore, it is urgent to develop a bacterial strain that can effectively increase the expression of AQP2 mRNA without affecting the vaginal flora environment. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a Lactobacillus gasseri VB247 for the first time, which is derived from the vagina of healthy women of childbearing age in my country, has the advantages of low pH resistance, high lactic acid production, resistance to 5 common vaginal pathogens, strong vaginal cell adhesion ability, etc., and can significantly increase the AQP2 mRNA expression level in female cervical vaginal epithelial cells, and can be used to prevent and / or alleviate and / or treat vaginal lubrication disorders caused by perimenopause, premature ovarian failure, etc.

[0008] 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 Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No. 26575, a deposit date of February 20, 2023, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus gasseri VB247 of the present invention has the advantages of low pH resistance, regulating vaginal pH environment, high lactic acid production, resistance to 5 common vaginal pathogens, strong vaginal cell adhesion ability, etc., and can significantly increase the AQP2mRNA expression level in female cervical vaginal epithelial cells.

[0009] The second object of the present invention is to provide a fermentation supernatant, which comprises: the aforementioned Lactobacillus gasseri VB247; and / or the metabolites of the aforementioned Lactobacillus gasseri VB247.

[0010] The third object of the present invention is to provide a fermentation broth, which comprises the aforementioned Lactobacillus gasseri VB247.

[0011] The fourth object of the present invention is to provide a microbial agent, which contains the aforementioned Lactobacillus gasseri VB247, and / or the aforementioned fermentation supernatant, and / or the aforementioned fermentation broth. As mentioned above, Lactobacillus gasseri VB247 has the advantages of low pH resistance, regulating vaginal pH environment, high lactic acid production, resistance to 5 common vaginal pathogens, and strong vaginal cell adhesion ability. The microbial agent of the present invention also has the same advantages and can be used to prevent and / or alleviate and / or treat vaginal lubrication disorders caused by perimenopause, premature ovarian failure, etc.

[0012] As a specific embodiment, the Lactobacillus gasseri VB247 exists in the form of living cells and / or non-living cells.

[0013] As a specific implementation, the microbial agent may also contain at least one strain acceptable to food, medicine, health food and feed.

[0014] As a specific embodiment, the microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

[0015] A fifth object of the present invention is to provide a food, a health food or a 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.

[0016] The 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 drug, wherein the drug 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.

[0017] As a specific embodiment, the pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0018] The seventh object of the present invention is to provide a medicine, which comprises at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth and the aforementioned microbial agent.

[0019] As a specific embodiment, the drug further contains an excipient and / or a carrier.

[0020] 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.

[0021] 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 hydrolyzed cereal solids.

[0022] As a specific embodiment, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

[0023] Beneficial effects:

[0024] (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 Higher, indicating that its growth initiation is faster, the activation rate is higher, and the growth activity is stronger in a low pH environment (pH=4.0).

[0025] (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 screened and obtained in the present invention is higher, indicating that the strain has a high lactic acid production.

[0026] (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.

[0027] (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 pathogenic bacteria 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 auxiliary treatment of a variety of gynecological diseases, such as vaginitis, vaginal lubrication disorders, etc.

[0028] (5) The Lactobacillus gasseri VB247 of the present invention has a stronger ability to adhere to vaginal cells and is more likely to colonize in the vagina.

[0029] (6) The Lactobacillus gasseri VB247 of the present invention can significantly increase the AQP2 mRNA expression level 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.

[0030] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0032] Figure 1 is a scanning electron microscope photograph of Lactobacillus gasseri VB247 in Example 1 of the present invention;

[0033] Figure 2 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;

[0034] Figure 3 The curve showing the change of pH value of the fermentation broth of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 over time in Example 4 of the present invention;

[0035] Figure 4 The adhesion of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 to HeLa cells in Example 6 of the present invention;

[0036] Figure 5This is the effect of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 on the expression of AQP2 mRNA in VK2 / E6E7 human vaginal epithelial cells in Example 7 of the present invention. DETAILED DESCRIPTION

[0037] 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.

[0038] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0039] In this document, the terms “contain”, “include” or “comprise” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0040] 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.

[0041] As used herein, "prevention" and "prevention" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," fermented Lactobacillus muciniphila or a product containing it may be administered to a subject at risk of 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 been made.

[0042] As used herein, the terms "treatment" and "alleviation" both refer to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be 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 an individual who is susceptible to the disease but has 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 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 an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0043] 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.

[0044] Bacteria

[0045] In one aspect of the present invention, the present invention provides a Lactobacillus gasseri VB247. According to an embodiment of the present invention, the deposit number of the Lactobacillus gasseri VB247 is CGMCC No. 26575. The Lactobacillus gasseri VB247 of the present invention has the advantages of low pH resistance, regulating vaginal pH environment, high lactic acid production, resistance to 5 common vaginal pathogens, strong vaginal cell adhesion ability, etc., and can significantly increase the AQP2mRNA expression level in female cervical vaginal epithelial cells.

[0046] As used herein, "Lactobacillus gasseri VB247" and "Lactobacillus gasseri VB247" are synonymous.

[0047] Fermentation supernatant

[0048] In another aspect of the present invention, 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.

[0049] 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.

[0050] Fermentation Broth

[0051] In another aspect of the present invention, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth includes the aforementioned Lactobacillus gasseri VB247.

[0052] It should be noted that the "fermentation liquid" mentioned in the present invention refers to the solution obtained after culturing Lactobacillus gasseri VB247 for a period of time.

[0053] Microbial Agents

[0054] In another aspect of the present invention, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent contains 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 related diseases caused by vaginal flora disorders.

[0055] It should be noted that the microbial agent of the present invention may be a liquid microbial agent, including but not limited to fermentation broth, etc.; it may also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0056] According to an embodiment of the present invention, the Lactobacillus gasseri VB247 exists in the form of living cells and / or non-living cells.

[0057] In this context, "living cells" refer to Lactobacillus gasseri VB247 having the ability to metabolize, reproduce or replicate.

[0058] Exemplarily, the living cells may be immobilized cells. Herein, "immobilized cells" refer to Lactobacillus gasseri VB247 being immobilized on a carrier and being able to carry out life activities such as growth, development, reproduction, inheritance and metabolism in a certain spatial range.

[0059] In this article, "non-living cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0060] As a specific embodiment, the Lactobacillus gasseri VB247 exists in the form of living cells, dry bacteria, immobilized cells or any other forms.

[0061] As a specific embodiment, the dry bacteria are obtained by freeze-drying the Lactobacillus gasseri VB247.

[0062] As a specific implementation, the microbial agent may also contain at least one strain acceptable to food, medicine, health food and feed.

[0063] As a specific embodiment, the microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

[0064] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the food requirements of different countries.

[0065] As used herein, "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.

[0066] In this article, "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 mammals treated therewith. Preferably, "pharmaceutically acceptable" as described in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, particularly humans.

[0067] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, drug 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 treatment or pharmaceutical compositions is covered.

[0068] In this article, the term "pharmaceutically acceptable excipient" may include any solvent suitable for a particular target dosage form. Except for any conventional excipients that are incompatible with the Lactobacillus gasseri VB247 of the present disclosure, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0069] use

[0070] In another aspect of the present invention, the present invention proposes 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 drug, wherein the drug 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.

[0071] 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.

[0072] As a specific embodiment, the pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Candida albicans, Gardnerella vaginalis and Clostridium perfringens.

[0073] Food, health food or hygiene products

[0074] 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.

[0075] drug

[0076] In another aspect of the present invention, the present invention provides a medicine, which includes at least one of the aforementioned Lactobacillus gasseri VB247, the aforementioned fermentation supernatant, the aforementioned fermentation broth and the aforementioned microbial agent.

[0077] As a specific embodiment, the drug further contains an excipient and / or a carrier.

[0078] 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.

[0079] 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 hydrolyzed cereal solids.

[0080] As a specific embodiment, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

[0081] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0082] Example 1: Strain collection and identification

[0083] Vaginal secretion samples were collected from 68 women of childbearing age who had passed health examinations (Hangzhou, China) using disposable sterile cotton swabs. The samples were spread on MRS solid culture medium and cultured anaerobically at 37°C for 48 hours. Single colonies suspected to be lactobacilli were picked and amplified and sequenced by bacterial 16S rDNA. The sequencing results were compared by BLAST. The strains were analyzed based on the comparison results and preserved in glycerol tubes, respectively. A total of 157 strains of Lactobacillus gasseri were obtained and numbered #1-#157. After testing the growth activity in a low pH environment, lactic acid production, pH regulation, antagonism to five vaginal pathogens, HeLa cell adhesion, and antibiotic sensitivity of the above 157 strains of Lactobacillus gasseri, the best performing strain #18 was screened and named Lactobacillus gasseri VB247. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection on February 20, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.26575. The scanning electron microscope image of Lactobacillus gasseri VB247 is shown below. Figure 1 As shown, it can be seen that the bacteria have a good appearance and are all cylindrical.

[0084] The 16S rDNA sequencing result of Lactobacillus gasseri VB247 is shown in SEQ ID NO:1.

[0085]

[0086] The 16S rDNA sequencing results of Lactobacillus gasseri isolate #86 are shown in SEQ ID NO:2.

[0087]

[0088] The 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 Bacterial Identification Manual. The test results are shown in Table 1. Compared with Lactobacillus gasseri isolate #86, the strain VB247 screened in this example can utilize milk, as well as multiple carbohydrates such as L-rhamnose, mannitol and sorbitol.

[0089] Table 1: Physiological and biochemical characteristics of different Lactobacillus gasseri

[0090]

[0091] Identification Project VB247 #86 Inulin + + lactose + + glucose + + D-Ribose - - Melezitose - - Galactose + + Gluconate - - Mannose + + fructose + + Trehalose + + Xylose + + Melbiose + + turanose + + Methyl Red (MR) - + VP determination - + Gelatin liquefaction - - Starch hydrolysis - -

[0092] Note: + represents positive, - represents negative.

[0093] Example 2: Growth activity experiment under low pH environment

[0094] After activation, different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) were inoculated at 1% in MRS broth medium with pH = 4.0 and cultured anaerobically at 37° C. for 48 h. The OD of the fermentation broth was measured every 4 h. 600 . Figure 2 OD of Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 at pH = 4.0 600 The curve of change over time, from Figure 2 It can be seen that after 4 hours of cultivation, the OD of Lactobacillus gasseri VB247 600 Compared with the initial value, it increased by 20.9%, while Lactobacillus gasseri isolate #86 only increased by 1.2%; at 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 growing faster, has a higher activation rate and stronger growth activity in a low pH environment.

[0095] Example 3: Lactic acid production capacity

[0096] The fermentation broth 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 was centrifuged to obtain a fermentation supernatant. The L-lactic acid content in the fermentation supernatant was detected using a bioprocess analyzer, and the D-lactic acid content was determined using a D-lactic acid detection kit. The results are listed in Table 2.

[0097] 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, and 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.

[0098] Table 2: Lactic acid content in 48h fermentation broth of different Lactobacillus gasseri

[0099] Lactobacillus gasseri VB247 #86 L-lactic acid content (g / L) 6.35 3.74 D-lactic acid content (g / L) 12.60 6.51

[0100] Example 4: pH Adjustment Capability

[0101] Studies have found that the pH value of a normal female vaginal environment is between 3.8 and 4.4. Vaginal flora disorders are usually accompanied by an increase in vaginal pH, which is caused by the excessive reproduction of other pathogenic bacteria, mainly aerobic bacteria.

[0102] After activation, different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) were inoculated at 1% in MRS broth medium with pH = 6.5 and cultured anaerobically at 37° C. for 48 h. The pH of the fermentation broth was measured every 4 h. The test results are shown in Figure 3 .

[0103] from Figure 3 It can be seen that the Lactobacillus gasseri VB247 obtained by the present invention can reduce 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.

[0104] Example 5: Antagonism against five common vaginal pathogens

[0105] The antibacterial activity of different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) against five common vaginal pathogens was determined by a double-layer plate method. The specific method is as follows:

[0106] 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.

[0107] 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°C-45°C after sterilization in a certain proportion. 6 CFU / mL, poured into the plate of Lactobacillus gasseri after 48h of culture, placed in an environment suitable for indicator bacteria for 16-18h, 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.

[0108] As shown in Table 3, the diameters of the inhibition rings of Lactobacillus gasseri VB247 against Escherichia coli, Staphylococcus aureus, Candida albicans, Gardnerella vaginalis and Clostridium perfringens are all greater than 7 mm, indicating that it has an antibacterial effect. Moreover, compared with Lactobacillus gasseri isolate #86, Lactobacillus gasseri VB247 has a stronger antibacterial effect against the five bacteria.

[0109] Table 3: Inhibition ring test results of different Lactobacillus gasseri against 5 pathogenic bacteria

[0110]

[0111] Example 6: Adhesion to HeLa cells assay

[0112] After activation, different Lactobacillus gasseri (Lactobacillus gasseri VB247 and Lactobacillus gasseri isolate #86 obtained in Example 1 of the present invention) were inoculated in MRS broth medium with a pH value 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), incubate at 37℃ for 2h, wash twice with sterile PBS to wash away free lactobacilli, add 25μL trypsin solution to each well of the six-well cell culture plate containing Hela cells, place in a 37℃ constant temperature box to digest the cells, and after the HeLa cells are digested into spheres, add 75μL DMEM culture medium (containing 10% fetal bovine serum) to each well, repeatedly blow and beat evenly, and after complete digestion, draw 20μL of the suspension, dilute it tenfold with sterile PBS, select appropriate dilution gradient to spread and count the viable bacteria, and the results are shown in the table. Figure 4 .

[0113] like Figure 4As shown, the number of bacteria adhering to a unit HeLa cell of Lactobacillus gasseri VB247 obtained by screening of the present invention is higher than that of Lactobacillus gasseri isolate #86, which indicates that the strain is more likely to colonize in the vagina.

[0114] Example 7: Effect on AQP2 mRNA expression in vaginal epithelial cells

[0115] VK2 / E6E7 human vaginal epithelial cells were purchased from Shanghai Yaji Biotechnology Co., Ltd., revived and passaged according to the product instructions, and seeded in three 6-well plates. Then they were divided into three groups in the 6-well plate, with 3 parallels in each group, 100 μL of the fermentation supernatant of Lactobacillus gasseri VB247 cultured for 48 hours in Example 2 was added to two wells, cultured for 24 hours and 48 hours, and cells were collected; 100 μL of the fermentation supernatant of Lactobacillus gasseri isolate #86 cultured for 48 hours in Example 2 was added to two wells, cultured for 24 hours and 48 hours, and cells were collected; 100 μL of progesterone was added to two wells to a final concentration of 10 nmol / L, cultured for 24 hours and 48 hours, and cells were collected as positive controls. Among them, progesterone is an estrogen that can relieve vaginal lubrication disorders during menopause. There are literature 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 .

[0116] like Figure 5 As shown, the Lactobacillus gasseri VB247 screened and obtained in the present invention can significantly increase the AQP2 mRNA expression level in female cervical vaginal epithelial cells compared with the Lactobacillus gasseri isolate #86.

[0117] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0118] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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.

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