Lactobacillus crispatus VB250 and use thereof

Abstract

Provided is a Lactobacillus crispatus VB250, which is deposited in the China General Microbiological Culture Collection Center (CGMCC) on 20 February 2023, with the deposit number of CGMCC No.26574. The Lactobacillus crispatus provided in the present invention has good growth activity and multiple pathogenic bacteria inhibition activity in a low pH environment, and can be used for preventing and / or relieving and / or treating related diseases caused by vaginal flora disorder, such as bacterial vaginitis, colpitis mycotica, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and vaginitis caused by virus infection. Moreover, the Lactobacillus crispatus has great application prospects in preparation of products such as food, health-care food, drugs, and hygienic products.

Description

Lactobacillus crispatus VB250 and its application Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a Lactobacillus crispatus VB250 having good gastrointestinal tolerance and inhibitory activity against multiple pathogens and its application. More specifically, the present invention relates to a Lactobacillus crispatus, a fermentation supernatant, a microbial agent, a food, a health food, a medicine or a sanitary product and their uses. Background Art

[0002] Vaginitis is a common gynecological condition that can be caused by various pathogens and is also related to external stimuli and hormone levels. Particularly in menopausal women, due to declining ovarian function and declining estrogen levels, the vaginal wall atrophies, epithelial cell glycogen levels decrease, and the vaginal pH increases. This reduces local resistance, susceptibility to overgrowth of other pathogens, primarily aerobic bacteria, leading to vaginitis. Treatment for vaginitis often involves topical antibiotics to inhibit bacterial growth and boost resistance. However, with increasing antibiotic resistance, recurrence rates are increasing. Therefore, researchers are exploring new treatments to improve the treatment of vaginitis caused by dysbiosis. Studies have shown that using probiotics to increase the number of lactic acid bacteria in the vagina can inhibit the growth of pathogenic microorganisms, thereby effectively treating vaginitis.

[0003] Therefore, there is an urgent need to develop a new strain of bacteria that can prevent and treat bacterial vaginosis.

[0004] Summary of the Invention

[0005] 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 a Lactobacillus crispatus strain having good growth activity and multi-pathogen inhibitory activity in a low pH environment, and can be used to prevent and / or alleviate and / or treat diseases associated with vaginal flora disorders.

[0006] The present invention is accomplished based on the following findings of the inventors:

[0007] Currently, there are two vaginal probiotic preparations in China for the treatment of bacterial vaginosis caused by bacterial flora imbalance. One is the "Wanze Shuangqi Dingjunsheng" vaginal lactobacillus capsules produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., whose active ingredient is Lactobacillus delbrueckii; the other is the "Yanhua" lactobacillus vaginal capsules produced by Xi'an Zhenghao Biopharmaceutical Co., Ltd., whose active ingredient is live Streptococcus enterica. However, neither of these strains is a dominant native bacterial species in the vagina and cannot be used for prevention.

[0008] However, Lactobacillus crispatus is one of the dominant strains in the female vagina in my country. It can maintain the normal pH value in the vagina and prevent the occurrence of bacterial vaginosis.

[0009] Based on this, a first object of the present invention is to provide a Lactobacillus crispatus VB250. According to an embodiment of the present invention, the Lactobacillus crispatus VB250 has a deposit number of CGMCC No. 26574 and a deposit date of February 20, 2023. The Lactobacillus crispatus of the present invention has the advantages of low pH tolerance, high lactic acid production, antibacterial and antibiotic resistance, and strong vaginal cell adhesion. In addition, the Lactobacillus crispatus has a short growth start-up time, a high activation rate, and strong growth vigor.

[0010] A second object of the present invention is to provide a fermentation supernatant comprising: the aforementioned Lactobacillus crispatus VB250; and / or metabolites of the aforementioned Lactobacillus crispatus VB250. The Lactobacillus crispatus of the present invention has the advantages of low pH tolerance, high lactic acid production, antibacterial and antibiotic resistance, and strong vaginal cell adhesion. Furthermore, the Lactobacillus crispatus has a short growth initiation time, a high activation rate, and strong growth vigor.

[0011] A third object of the present invention is to provide a microbial agent comprising the aforementioned Lactobacillus crispatus VB250 and / or the aforementioned fermentation supernatant. The microbial agent of the present invention is used to prevent and / or treat diseases related to vaginal flora disorders.

[0012] As a specific embodiment, the Lactobacillus crispatus VB250 exists in the form of living cells and / or non-living cells.

[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 fourth object of the present invention is to provide a use of the aforementioned Lactobacillus crispatus VB250, the aforementioned fermentation supernatant or the aforementioned microbial agent in preparing a product, wherein the product is used to prevent and / or treat related diseases caused by vaginal flora disorders.

[0015] As a specific embodiment, the related disease caused by vaginal flora disorder is vaginitis.

[0016] As a specific embodiment, the vaginitis includes but is not limited to bacterial vaginitis, candidal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis and vaginitis caused by viral infection.

[0017] As a specific embodiment, the product is food, health food, medicine or sanitary product. Beneficial effects:

[0018] (1) Compared with other Lactobacillus crispatus, the Lactobacillus crispatus VB250 screened and obtained in the present invention has a faster growth start-up, a higher slope in the logarithmic growth phase, and an 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).

[0019] (2) The Lactobacillus crispatus VB250 obtained by screening in the present invention has a stronger ability to produce lactic acid than other Lactobacillus crispatus. The different Lactobacillus crispatus fermentation broths cultured for 48 hours in Example 1 were cultured for 48 hours. Compared with other Lactobacillus crispatus, VB250 had higher L-lactic acid and D-lactic acid production, indicating that this strain is more beneficial to maintaining human vaginal health.

[0020] (3) Compared with other Lactobacillus crispatus, the Lactobacillus crispatus VB250 of the present invention has better digestive tract tolerance and is more conducive to its oral migration through the intestine to the vagina.

[0021] (4) Compared with other Lactobacillus crispatus, the Lactobacillus crispatus VB250 of the present invention can reduce pH faster and reach acid-base balance at pH 4, which indicates that the strain can create and maintain a healthy vaginal pH environment and has a stronger ability to regulate pH.

[0022] (5) The Lactobacillus crispatus VB250 of the present invention has inhibitory activity against a variety of pathogenic bacteria, and can inhibit the growth of Escherichia coli, Staphylococcus aureus, Candida albicans, Gardnerella vaginalis and Clostridium perfringens, etc., and can protect the gastrointestinal tract, female vagina, breast tissue or other organs from the invasion of pathogenic bacteria. Therefore, it can be used alone or in combination with other probiotics for the auxiliary treatment of various diseases, such as gynecological diseases, gastrointestinal diseases, mastitis, etc.

[0023] (6) The Lactobacillus crispatus VB250 of the present invention has high drug resistance to kanamycin, ofloxacin, nystatin, metronidazole, norfloxacin and fluconazole, and can be used in combination with clinical drugs to treat bacterial vaginosis and fungal vaginitis.

[0024] (7) The Lactobacillus crispatus VB250 of the present invention has a strong ability to adhere to vaginal cells, is more likely to colonize in the vagina, can inhibit the growth of pathogenic microorganisms, and regulate the balance of vaginal flora.

[0025] (8) Continuous administration of Lactobacillus crispatus VB250 for 10 days can significantly reduce the number of Staphylococcus aureus in the vagina of infected mice and reduce the level of inflammatory factor IL-6 in plasma, thus having the effect of treating vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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:

[0027] FIG1 is a scanning electron microscope photograph of Lactobacillus crispatus VB250 in Example 1 of the present invention;

[0028] FIG2 is a curve showing the change of OD600 over time at pH = 4.0 for Lactobacillus crispatus VB250 and other strains in Example 2 of the present invention;

[0029] 3 is a curve showing changes in pH value of fermentation broth of Lactobacillus crispatus VB250 and other strains over time in Example 3 of the present invention;

[0030] FIG4 shows the adhesion of Lactobacillus crispatus VB250 and other strains to HeLa cells in Example 8 of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The present invention provides a Lactobacillus crispatus, a fermentation supernatant, a microbial agent, a food, a health food, a medicine or a sanitary product and uses thereof, which will be described in detail below.

[0038] strains

[0039] In one aspect, the present invention provides Lactobacillus crispatus VB250. According to an embodiment of the present invention, the Lactobacillus crispatus VB250 has the deposit number CGMCC No. 26574. The Lactobacillus crispatus VB250 has the advantages of low pH tolerance, high lactic acid production, antibacterial and antibiotic resistance, and strong vaginal cell adhesion. Furthermore, the Lactobacillus crispatus VB250 exhibits a short growth initiation time, a high activation rate, and strong growth vigor.

[0040] As used herein, "Lactobacillus crispatus VB250" and "Lactobacillus crispatus VB250" are synonymous.

[0041] Fermentation supernatant

[0042] 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 crispatus VB250; and / or metabolites of the aforementioned Lactobacillus crispatus VB250. The Lactobacillus crispatus of the present invention has advantages such as low pH tolerance, high lactic acid production, antibacterial and antibiotic resistance, and strong vaginal cell adhesion. Furthermore, the Lactobacillus crispatus exhibits a short growth initiation time, a high activation rate, and strong growth vigor.

[0043] It should be noted that “fermentation supernatant” refers to the supernatant after centrifugation of the fermentation broth, which may contain Lactobacillus crispatus VB250, or metabolites of Lactobacillus crispatus VB250, or both Lactobacillus crispatus VB250 and its metabolites.

[0044] microbial agents

[0045] In another aspect, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent comprises the aforementioned Lactobacillus crispatus VB250 and / or the aforementioned fermentation supernatant. The microbial agent of the present invention is used to prevent and / or treat diseases related to vaginal flora disorders.

[0046] 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.

[0047] According to an embodiment of the present invention, the Lactobacillus crispatus VB250 exists in the form of living cells and / or non-living cells.

[0048] In this context, "living cells" refer to Lactobacillus crispatus VB250 cells that have the ability to metabolize, reproduce or replicate.

[0049] For example, the living cells may be immobilized cells. Herein, "immobilized cells" refer to Lactobacillus crispatus VB250 fixed on a carrier, which can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0050] 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.

[0051] As a specific embodiment, the Lactobacillus crispatus VB250 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0052] As a specific embodiment, the dry bacteria are obtained by freeze-drying the Lactobacillus crispatus VB250.

[0053] As a specific embodiment, the microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 treatment or pharmaceutical compositions is encompassed.

[0058] 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 crispatus VB250 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 contemplated by the present disclosure.

[0059] use

[0060] In another aspect of the present invention, the present invention provides a use of the aforementioned Lactobacillus crispatus VB250, the aforementioned fermentation supernatant or the aforementioned microbial agent in preparing a product, wherein the product is used to prevent and / or treat related diseases caused by vaginal flora disorders.

[0061] As a specific embodiment, the related disease caused by vaginal flora disorder is vaginitis.

[0062] As a specific embodiment, the vaginitis includes but is not limited to bacterial vaginitis, candidal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis and vaginitis caused by viral infection.

[0063] As a specific embodiment, the product is food, health food, medicine or sanitary product.

[0064] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0065] In the following examples of the present invention, Lactobacillus crispatus ATCC55221 was purchased from Beijing Biobowei Biotechnology Co., Ltd., which is the vaginal Lactobacillus crispatus in the patent publication CN109234298A.

[0066] Example 1: Strain collection and identification

[0067] 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 176 strains of Lactobacillus crispatus were obtained and numbered #1-#176. A strain of Lactobacillus crispatus ATCC55221 was purchased from Beijing Biobo Biotechnology Co., Ltd. as a control strain. After physiological and biochemical identification, growth activity in low pH environment, lactic acid production ability, digestive tract stability, pH regulation ability, antagonism against five vaginal pathogens, HeLa cell adhesion ability, and antibiotic sensitivity tests were carried out simultaneously with the above 176 strains of Lactobacillus crispatus, the best performance strain Lactobacillus crispatus #66 was screened and named VB250. It was deposited in the General Microbiology Center of China Culture Collection Administration on February 20, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.26574.

[0068] Table 1: Physiological and biochemical characteristics of different Lactobacillus crispatus strains

[0069] Note: + represents positive, - represents negative.

[0070] As can be seen from Table 1, the strain VB250 screened in this example can decompose and utilize milk, as well as various carbohydrates such as glucose semisolid, arabinose, raffinose, D-ribose, melezitose, galactose, mannose, melebiose, and turanose, compared with other Lactobacillus crispatus.

[0071] The 16S rDNA sequencing result of Lactobacillus crispatus VB250 is shown in SEQ ID NO: 1.

[0072] Example 2: Growth activity experiment of Lactobacillus crispatus VB250 in low pH environment

[0073] After activation, different Lactobacillus crispatus were inoculated with 1% of the culture medium and cultured anaerobically at 37°C in MRS broth at pH 4.0 for 48 h. The OD of the fermentation liquid was measured every 4 h. 600 Figure 2 shows the OD of VB250 and other strains at pH = 4.0 600 As can be seen from Figure 2, the OD of VB250 was 600Compared with the initial value, the OD value of VB250 increased by 44.8%, while that of strains ATCC55221 and #13 increased by only 15.5% and 0%, respectively. 600 Compared with the initial value, it increased by 137.5%, while strains ATCC55221 and #13 only increased by 56.0% and 12.0%, respectively. In addition, the slope of VB250 in the logarithmic growth phase was higher, and the OD 600 The values ​​are higher, which indicates that strain VB250 starts growth faster, has a higher activation rate and stronger growth activity in a low pH environment.

[0074] Example 3: Lactic acid production capacity

[0075] The fermentation broths of different Lactobacillus crispatus cultured for 48 h in Example 2 were centrifuged and filtered. The L-lactic acid content in the fermentation filtrate 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.

[0076] Studies have found that lactic acid produced by the metabolism of lactobacilli in the vagina can inhibit the growth of vaginal pathogens. As can be seen from Table 2, the Lactobacillus crispatus VB250 obtained in the present invention has higher L-lactic acid and D-lactic acid production than other Lactobacillus crispatus, indicating that the Lactobacillus crispatus VB250 of the present invention is more conducive to maintaining human vaginal health.

[0077] Table 2: Lactic acid content in 48h fermentation broth of different Lactobacillus crispatus

[0078] Example 4: Gastrointestinal stability

[0079] The fermentation broths of different Lactobacillus crispatus cultured for 48 hours in Example 2 were centrifuged and filtered to obtain bacterial cells, which were then placed in artificial gastric juice and intestinal juice, and the number of viable bacteria was measured every 2 hours.

[0080] Table 3: Stability of different Lactobacillus crispatus strains in artificial gastric juice (pH = 2.5) and intestinal juice (pH = 6.8)

[0081] As can be seen from Table 3, the VB250 strain obtained by the present invention has a 4-hour survival rate of approximately 90% in artificial gastric fluid (pH = 2.5) and artificial intestinal fluid (pH = 6.8), which is much higher than that of other Lactobacillus crispatus, indicating that the strain has good digestive tract tolerance, which will be more conducive to its oral migration through the intestine to the vagina and maintain vaginal health.

[0082] Example 5: pH Adjustment Capability

[0083] 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.

[0084] After activation, different Lactobacillus crispatus (Lactobacillus crispatus VB250, #13, and ATCC55221 obtained in Example 1 of the present invention) were inoculated at a 1% inoculum and anaerobically cultured at 37°C in MRS broth at pH 6.5 for 48 h. The pH of the fermentation broth was measured every 4 h. The test results are shown in Figure 3.

[0085] Therefore, it can be seen from FIG3 that the Lactobacillus crispatus VB250 obtained in the present invention can reduce the pH faster than other Lactobacillus crispatus and reach acid-base balance at pH 4, which indicates that the strain can create and maintain a healthy vaginal pH environment.

[0086] Example 6: Antagonism against five common vaginal pathogens

[0087] The antibacterial activity of different strains of Lactobacillus crispatus against five common vaginal pathogens was determined using a double-layer plate method. The specific method is as follows:

[0088] After activating different Lactobacillus crispatus (Lactobacillus crispatus VB250, #13, ATCC55221 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.

[0089] The activated indicator bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli 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 crispatus after culturing 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 crispatus. The test results are shown in Table 4.

[0090] As shown in Table 4, the diameters of the inhibition zones of Lactobacillus crispatus VB250 against Escherichia coli, Staphylococcus aureus, Candida albicans, Gardnerella vaginalis and Clostridium perfringens were all greater than 7 mm, indicating that the Lactobacillus crispatus VB250 had an antibacterial effect.

[0091] Table 4: Inhibition zone test results of different Lactobacillus crispatus against 5 pathogenic bacteria

[0092] Example 7: Antibiotic sensitivity test

[0093] The susceptibility of different Lactobacillus crispatus strains (Lactobacillus crispatus VB250, #13, and ATCC 55221 obtained in Example 1 of the present invention) to kanamycin, gentamicin, chloramphenicol, clindamycin, vancomycin, amoxicillin, cefaclor, ofloxacin, nystatin, metronidazole, norfloxacin, penicillin sodium, azithromycin, fluconazole, and imipenem was determined by broth microdilution method. The test results are shown in Table 5.

[0094] The results in Table 5 show that the Lactobacillus crispatus VB250 obtained by screening in the present invention has high resistance to kanamycin, ofloxacin, nystatin, metronidazole, norfloxacin and fluconazole, indicating that Lactobacillus crispatus VB250 can be used in combination with clinical drugs to treat bacterial vaginosis and candidal vaginitis.

[0095] Table 5: Antibiotic resistance of different Lactobacillus crispatus strains

[0096] Example 8: Adhesion to HeLa cells assay

[0097] Different Lactobacillus crispatus (Lactobacillus crispatus VB250, #13, ATCC55221 obtained in Example 1 of the present invention) were activated and inoculated into MRS broth culture 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, 100 μL of bacterial suspension was pipetted 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 remove free lactobacilli, added 25μL of trypsin solution to each well of a six-well cell culture plate containing HeLa cells, and placed in a 37°C incubator to digest the cells. After the HeLa cells were digested into spheres, 75μL of complete culture medium was added to each well and repeatedly pipetted evenly. After complete digestion, 20μL of the suspension was aspirated and diluted tenfold with sterile PBS. Appropriate dilution gradients were selected for coating and viable bacteria counts. The results are shown in Figure 4.

[0098] As shown in FIG4 , the Lactobacillus crispatus VB250 obtained by screening of the present invention has a higher number of bacteria adhering to each HeLa cell than other Lactobacillus crispatus strains, which indicates that the strain is more likely to colonize in the vagina.

[0099] Example 9: Therapeutic effect on bacterial vaginosis in mice

[0100] Bacterial vaginosis mouse infection model: 50 ICR mice were selected and subcutaneously injected with 0.1 mL of estradiol benzoate solution (1 mg / mL, prepared in PBS, sterilized by filtration with a 0.22 μm filter) once every two days for a total of three times to induce estrus in the mice. Six days later, a No. 5 scalp needle silicone tube was coated with sterile paraffin oil and then slowly inserted into the mouse vagina about 1.5 cm. The mouse vagina was irrigated with sterile PBS solution three times. Escherichia coli (1×10 9 CFU / mL)+ Staphylococcus aureus (1×10 9 CFU / mL) mixed with 20 μL of bacterial solution; 4 days after inoculation, when the mouse vagina showed obvious congestion, redness and swelling with a large amount of purulent secretions, a vaginal secretion smear was collected and Gram-stained for microscopic examination to examine the degree of bacterial infection; the blank control group (10 mice) used the same operation method, and the mixed bacterial solution was replaced with sterile PBS. The mice in the blank control group should not show symptoms of obvious vaginal congestion, redness and swelling with a large amount of purulent secretions. If the above symptoms appear, it indicates that the model is contaminated.

[0101] Administration: Successfully infected mice were randomly divided according to body weight into 8 mice per group: the model group, treatment group 1, and treatment group 2. Healthy mice served as the control group. 20 μL of the drug was administered intravaginally once daily for 10 consecutive days. Information on the drugs or solvents administered to the model, treatment groups 1, treatment groups 2, and control groups is reproduced in Table 6.

[0102] Testing: Four days after the end of administration, 10% aqueous solution of ulexol was injected intraperitoneally. Using a No. 5 scalp needle silicone tube, 200 μL of sterile PBS was perfused into the vagina. 100 μL of the lavage fluid was diluted to different proportions and spread on the Staphylococcus selective culture medium BAT. The bacterial count was calculated. The test results are shown in Table 5. Blood was collected from the eyeballs and placed in anticoagulant tubes. After thorough mixing, the blood was centrifuged at 1500 rpm, 4°C, for 15 minutes. The plasma IL-6 level was detected using a kit. The test results are shown in Table 8.

[0103] From the results in Table 7 and Table 8, it can be seen that the bacterial suspension of Lactobacillus crispatus VB250 (the fermentation broth was centrifuged, the bottom bacterial residue was left, washed twice with PBS, and the bacterial suspension was resuspended with PBS, 1×10 8 Continuous vaginal administration of 100 mg / mL (500 mg / mL) for 10 days can significantly reduce the number of Staphylococcus aureus in the vagina of infected mice and reduce the level of inflammatory factor IL-6 in plasma, indicating that Lactobacillus crispatus VB250 has the effect of treating bacterial vaginosis.

[0104] Table 6: Different group information

[0105] Table 7: Staphylococcus aureus content in vaginal lavage fluid

[0106] Table 8: IL-6 levels in plasma

[0107] 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.

[0108] 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 crispatus VB250, characterized in that The preservation number of the Lactobacillus crispatus in the China General Microbiological Culture Collection Center is CGMCC No.26574, and the preservation date is February 20, 2023.

2. A fermentation supernatant, characterized in that: include: The Lactobacillus crispatus VB250 of claim 1; and / or The metabolite of Lactobacillus crispatus VB250 according to claim 1.

3. A microbial agent, characterized in that: include: The Lactobacillus crispatus VB250 according to claim 1, and / or the fermentation supernatant according to claim 2.

4. The microbial agent according to claim 3, characterized in that: The Lactobacillus crispatus VB250 exists in the form of living cells and / or non-living cells.

5. The microbial agent according to claim 3 or 4, characterized in that: The microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

6. A food, health food, medicine or sanitary product, characterized in that: include: The Lactobacillus crispatus VB250 according to claim 1, the fermentation supernatant according to claim 2, or the microbial agent according to any one of claims 3 to 5.

7. Use of Lactobacillus crispatus VB250 according to claim 1, the fermentation supernatant according to claim 2 or the microbial agent according to any one of claims 3 to 5 in preparing a product for preventing and / or treating related diseases caused by vaginal flora disorders.

8. The use according to claim 7, characterized in that The related disease caused by the vaginal flora disorder is vaginitis.

9. The use according to claim 8, characterized in that The vaginitis includes at least one of bacterial vaginitis, candidal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis and vaginitis caused by viral infection.

10. The use according to any one of claims 7 to 9, characterized in that: The product is food, health food, medicine or sanitary product.

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