Lactobacillus gasseri and application thereof

Abstract

The invention provides lactobacillus gasseri which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on February 7, 2023, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.26504. The lactobacillus gasseri has the advantages that the lactobacillus gasseri can be used for preparing the lactobacillus gasseri; the lactobacillus gasseri has a relatively strong inhibition effect on pathogenic bacteria such as Gardneria vaginalis, Escherichia coli, staphylococcus aureus and two-way Pleuromycosis, and also has a certain inhibition effect on CA, so that the lactobacillus gasseri has the effects of killing pathogenic bacteria, improving the host mucosal immunity and resisting infection, and has a good application prospect in the field of animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry and animal husbandry. Meanwhile, growth and reproduction of harmful bacteria can be inhibited, vaginal infection is prevented, and inflammation is treated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a Lactobacillus gasseri and its uses. Background Art

[0002] With the economic development and the improvement of living standards, the disease spectrum of humans has changed greatly. The high incidence of female reproductive tract diseases has become increasingly prominent. Among them, the incidence of vaginitis is particularly common, which has seriously affected the quality of life, physical and mental health of women. Among the diseases related to vaginal infections, bacterial vaginosis (BV) is the most common. Epidemiological surveys show that the incidence of BV in China ranges from 4.96% to 36.00%. The main reason for this disease is the reduction of the dominant lactobacilli that maintain the normal acidic environment of the vagina, while the increase of various other anaerobic microorganisms. The dominant lactobacilli are replaced by Gardnerella vaginalis and mixed anaerobic bacteria. Gardnerella vaginalis is one of the most common pathogenic bacteria of BV in women. The typical clinical features of bacterial vaginosis are that the patient's vagina shows foul-smelling, watery, grayish secretions. The patient may also have many complications due to the infection of pathogens, such as chronic cervicitis, pelvic inflammatory disease, endometritis, and even infertility. Vulvovaginal candidiasis (VVC), commonly known as mycotic vaginitis, has an incidence second only to vaginal infections caused by BV. The dominant bacteria are mostly Candida albicans (white candida). Studies have found that about 78% of women have at least had VVC caused by Candida albicans infection once in their lifetime, 65% of women have 3 or fewer annual episodes, and 35% of women have 4 or more annual episodes. VVC usually presents as vulvar itching, burning, painful urination, and vaginal curd-like secretions, often resulting in a decline in the quality of life of patients and a serious national economic burden. The pathogenesis of VVC is relatively complex and is considered a multifactorial disease. Among them, vaginal microbial imbalance, estrogen level, host susceptibility factors, genetic susceptibility factors, and the morphology of Candida are all related to the occurrence of the disease. The imbalance of the vaginal microecosystem is closely related to the occurrence of these two vaginal infectious diseases.

[0003] The vaginal microbiota is different from the microbiota in other parts. The lower the diversity of the vaginal microbiota in healthy individuals, the more conducive it is to maintaining the healthy state of the vagina. The female vagina is a closed body cavity, and there are many bacteria in it. Since there is no air in the deep part of the vagina, the parasitic bacteria are mainly facultative anaerobes and anaerobes. More than 50 kinds of microorganisms parasitize in the vagina of healthy women. Under normal conditions, the ratio of anaerobes to aerobes is about 10:1, and the proportion of Lactobacillus is the highest, which plays an important role in maintaining the vaginal microecological balance. Under normal circumstances, Lactobacillus coexists with other microorganisms in the vagina and is in a state of microecological balance. Once affected by various factors, Lactobacillus loses its dominance, while the number of pathogenic bacteria and conditional pathogenic bacteria increases. At this time, the enzyme spectrum changes accordingly, breaking the microecological balance and leading to the occurrence of diseases. Lactobacillus maintains the stability of the vaginal microecology through multiple mechanisms: fermenting glycogen in the epithelium to produce lactic acid and maintaining the acidic pH value of the vagina; competing with pathogenic bacteria for adhesion to vaginal epithelial cells; producing broad-spectrum antibacterial factor H2O2; Lactobacillus and its metabolites stimulate the immune function of vaginal epithelial cells and inhibit the growth and reproduction of pathogenic bacteria.

[0004] Currently, the treatment of BV mainly relies on Western medicine, with the frequent use of antibacterial drugs such as antibiotics, including metronidazole, clindamycin, tinidazole, etc. However, the generation and persistent existence of pathogenic biofilms can lead to disease recurrence. According to the "Diagnosis and Treatment Guidelines for Bacterial Vaginosis (2021 Revised Edition)", the recurrence rate of BV is 20% one month after treatment, 40% three months after treatment, and as high as 60% twelve months after treatment. By the 12th month after treatment, 84% of patients have abnormal vaginal flora. In addition, the long-term and extensive use of antibiotics not only increases the drug resistance of pathogenic bacteria but also inhibits the reproduction of some vaginal flora, allowing the originally less numerous Candida albicans to multiply in large numbers, causing vaginal flora disorders. At present, the role of live bacteria preparations in the treatment of BV is still controversial. Relevant clinical studies show that the application of current live bacteria preparations mainly falls into two categories: the application of probiotics after conventional antibiotic treatment or the application of probiotics alone. Among them, the clinical trial results of multiple combined treatments show inconsistent results, and it is impossible to determine whether the combination of antibiotics and probiotics can be used for the treatment of bacterial vaginosis. In addition, some studies have shown that some patients did not achieve satisfactory efficacy when using probiotic preparations alone. Therefore, there is still a great deal of controversy about the effect of probiotics in the treatment of BV, and a large number of experiments are still needed for exploration and research. Currently, the only probiotic drug on the market for the treatment of bacterial vaginosis is Ding Junsheng (Live Lactobacillus Capsules for Vaginal Use) developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. For the treatment of VVC, the drugs commonly used in clinical practice for treating VVC are mainly azoles and polyenes. For example, compound metronidazole suppositories are often used to treat mycotic vaginitis. However, with the long-term use of clinical antifungal drugs, Candida albicans shows phenotypic changes, virulence factor mutations, and an increase in the infection of non-Candida albicans, challenging the therapeutic effect of traditional antibacterial drugs. In the treatment of various VVC patients, lactobacilli can be used as adjuvant treatment drugs in combination with antibacterial drugs such as azole drugs, with certain efficacy. However, compared with conventional antifungal drug treatment, there is currently not enough evidence to show that the use of lactobacillus preparations alone has a certain therapeutic effect on VVC.

[0005] Therefore, there is an urgent need for a lactobacillus with good cell adhesion and antibacterial ability, which can inhibit the growth of a variety of pathogenic bacteria. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent.

[0007] Therefore, the inventor isolated a strain from the vaginal secretions of a healthy reproductive-aged woman in Zhejiang Province. After identification, the strain was Lactobacillus gasseri (Lactobacillus gasseri HY1124). Through experiments, it was found that this strain can produce lactic acid and hydrogen peroxide (H2O2). Lactic acid belongs to antibacterial substances, which can compete with pathogenic bacteria for nutrients and adhesion sites, and improve the host mucosal immunity and anti-infection ability. H2O2 can directly kill pathogenic bacteria, and can also be catalytically oxidized by biological enzymes to form halides with stronger bactericidal effects. Therefore, this Lactobacillus gasseri has the potential to maintain a weakly acidic environment in the vagina, kill pathogenic bacteria, improve the host mucosal immunity and anti-infection ability, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0008] In view of this, in the first aspect of the present invention, the present invention provides a Lactobacillus gasseri (Lactobacillus gasseri HY1124). According to the embodiments of the present invention, this Lactobacillus gasseri was deposited with the China General Microbiological Culture Collection Center on February 7, 2023, with the deposit number CGMCC No. 26504, and the deposit address is: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. As mentioned above, the inventor isolated this strain from the vaginal secretions of a healthy reproductive-aged woman in Zhejiang Province. After further identifying the biochemical characteristics of this strain, it was found that this bacterium can produce lactic acid and hydrogen peroxide H2O2. Lactic acid belongs to antibacterial substances, which can compete with pathogenic bacteria for nutrients and adhesion sites, and improve the host mucosal immunity and anti-infection ability. H2O2 can directly kill pathogenic bacteria, and can also be catalytically oxidized by biological enzymes to form halides with stronger bactericidal effects. In addition, this bacterium has strong inhibitory effects on pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA) and Prevotella bivia (PB), and also has a certain inhibitory effect on Candida albicans (CA). Therefore, this Lactobacillus gasseri has the potential to maintain a weakly acidic environment in the vagina, kill pathogenic bacteria, improve the host mucosal immunity and anti-infection ability, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0009] According to the embodiments of the present invention, the 16S rDNA of the Lactobacillus gasseri has the nucleotide sequence shown in SEQ ID NO: 1.

[0010]

[0011] In the second aspect of the present invention, the present invention provides a Lactobacillus gasseri (Lactobacillus gasseri HY1124). According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus gasseri has one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO:1; (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO:1; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:1. The Lactobacillus gasseri of the present invention has strong inhibitory effects on pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA) and Prevotella bivia (PB), and also has a certain inhibitory effect on Candida albicans (CA). Therefore, the Lactobacillus gasseri has the potential to maintain a weakly acidic environment in the vagina, kill pathogenic bacteria, enhance the mucosal immunity and anti-infection ability of the host, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0012] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus gasseri contains the nucleotide sequence shown in SEQ ID NO:1.

[0013] According to an embodiment of the present invention, the Lactobacillus gasseri described in the first aspect or the second aspect further includes at least one of the following additional technical features:

[0014] According to an embodiment of the present invention, the gene sequence of the Lactobacillus gasseri further contains a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO:8; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:8; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4 or 5 nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:8. Therefore, the Lactobacillus gasseri of the present invention can be specifically identified through the above nucleotide sequences.

[0015] According to an embodiment of the present invention, the gene sequence of the Lactobacillus gasseri further contains the nucleotide sequence shown in SEQ ID NO:8.

[0016] According to a specific embodiment of the present invention, the Lactobacillus gasseri can produce at least 5.5 - 6.5 g / L of lactic acid under effective cultivation.

[0017] According to a specific embodiment of the present invention, the Lactobacillus gasseri can produce at least 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6.0 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, 6.4 g / L, 6.5 g / L of lactic acid under effective cultivation.

[0018] According to a specific embodiment of the present invention, the Lactobacillus gasseri can produce at least 1100 - 1400 μM of hydrogen peroxide under effective cultivation.

[0019] According to a specific embodiment of the present invention, the Lactobacillus gasseri can produce at least 1100 μM, 1120 μM, 1150 μM, 1170 μM, 1200 μM, 1220 μM, 1250 μM, 1270 μM, 1300 μM, 1320 μM, 1350 μM, 1370 μM, 1400 μM of hydrogen peroxide under effective cultivation.

[0020] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 85 - 90% against GV.

[0021] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 85%, 86%, 87%, 88%, 89%, 90% against GV.

[0022] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 98 - 100% against PB.

[0023] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 98%, 99%, 100% against PB.

[0024] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 98 - 100% against EC.

[0025] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 98%, 99%, 100% against EC.

[0026] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 95 - 100% against SA.

[0027] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 95%, 96%, 97%, 98%, 99%, 100% against SA.

[0028] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 55-60% against CA.

[0029] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an antibacterial rate of at least 55%, 56%, 57%, 58%, 59%, 60% against CA.

[0030] In the third aspect of the present invention, the present invention provides a primer set for detecting the Lactobacillus gasseri described in the first aspect or the second aspect. According to an embodiment of the present invention, the primer set includes a forward primer and a reverse primer; the forward primer has the nucleotide sequence shown in SEQ ID NO:2; the reverse primer has the nucleotide sequence shown in SEQ ID NO:3. Using the primer set of the present invention, it can highly match the DNA sequence of the Lactobacillus gasseri described in the first aspect or the second aspect, and only produce specific amplification in the Lactobacillus gasseri. This enables the primer to accurately and reliably identify the Lactobacillus gasseri and exclude the interference of other non-target strains.

[0031] In the fourth aspect of the present invention, the present invention provides a microbial preparation. According to an embodiment of the present invention, the microbial preparation contains the Lactobacillus gasseri described in the first aspect or the second aspect. As mentioned above, the inventors of the present invention isolated and obtained a new Lactobacillus gasseri, which has strong inhibitory effects on pathogenic bacteria GV, EC, SA and PB. In addition, it also has a certain inhibitory effect on CA. Therefore, the Lactobacillus gasseri can be made into a microbial preparation to facilitate patients' taking.

[0032] According to an embodiment of the present invention, the microbial preparation further includes a pharmaceutically acceptable carrier or excipient.

[0033] In the fifth aspect of the present invention, the present invention provides a single-dose preparation. According to an embodiment of the present invention, it includes 1×10 6 ~1×10 10 CFU of the Lactobacillus gasseri described in the first aspect or the second aspect as an active ingredient.

[0034] According to an embodiment of the present invention, it includes 10 6 CFU, 10 7 CFU, 10 8 CFU, 10 9 CFU, 10 10 CFU of the Lactobacillus gasseri described in the first aspect or the second aspect as an active ingredient.

[0035] According to an embodiment of the present invention, it includes 1×10 6 ~1×10 9 CFU of the Lactobacillus gasseri described in the first aspect or the second aspect as an active ingredient.

[0036] In the sixth aspect of the present invention, the present invention provides the use of the Lactobacillus gasseri described in the first aspect or the second aspect, the microbial preparation described in the fourth aspect, or the single-dose preparation described in the fifth aspect in the preparation of a drug for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

[0037] In the seventh aspect of the present invention, the present invention provides the use of the Lactobacillus gasseri described in the first aspect or the second aspect, the microbial preparation described in the fourth aspect, or the single-dose preparation described in the fifth aspect in the preparation of a drug for inhibiting vaginal pathogenic bacteria.

[0038] According to an embodiment of the present invention, the use described in the sixth aspect or the seventh aspect further includes at least one of the following additional technical features:

[0039] According to an embodiment of the present invention, the vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA), Prevotella bivia (PB), and Candida albicans (CA).

[0040] According to an embodiment of the present invention, the vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA), and Prevotella bivia (PB).

[0041] According to an embodiment of the present invention, the related diseases caused by vaginal pathogenic bacteria infection include bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, trichomonal vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, or pelvic inflammatory disease.

[0042] In the eighth aspect of the present invention, the present invention provides the application of the Lactobacillus gasseri described in the first aspect or the second aspect, the microbial preparation described in the fourth aspect, or the single-dose preparation described in the fifth aspect in the preparation of a product for regulating the balance of vaginal flora.

[0043] According to an embodiment of the present invention, the product includes at least one of a drug and an external health care product.

[0044] In a ninth aspect of the present invention, the present invention provides a molecular marker of Lactobacillus gasseri described in the first or second aspect. According to an embodiment of the present invention, the molecular marker comprises a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO:8; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:8; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:8.

[0045] According to an embodiment of the present invention, the molecular marker has the nucleotide sequence shown in SEQ ID NO:8.

[0046] ACCTGCAGAAGAAACTGCGACAACAGAAAGCAACAAACCAAAATCACGTAGAAAACGTGCTCTAGATGCAAACGATCAGAACCGATCAGGTGATGCAGCTACAACAGATGGAGATGATGATTCAGTTGTCAACGAAAAGGATCTACATGTGGTAAACCCATATTTTGAAGATAACGGAAAACCTACTACTGCTGAAAAATGGAAAGTTATTAATGCTTTTGACTTGATTGGATGGAAGCCGATCAATCCTAATCAAAAAAAAGTAGTTATTGCAAATGGTAAAGTAACAGAGTATGGAGGTTACGGATCTGTTGTAAATCAAACTCATCCATATAGTATTCCGTTGGCACTATATAAAAAAGCTGGTGATCGCACTAGTAAGAGTGATCAGTTTGATGGGGTGTATCAAGATATTGATGTAATTCCTGGACAAGAGATTGTAATTACTCAAAATACAGGTACTTTTGGACCGATTGGATCAAAAGACAATAGAACTATACTAACAGTATCTTACCCAGAAAGAGGGAATGAGGTTCAAGGAAAGATTGTTTGGAGATCACTTATGACACCATATAATGGTGTAGTGACAGTACCTAAAGGGATAACTAAACTTCGTGTACGTCTTGAGGTAGATCCAGATTCAAATGTAGCACATAAAGATAATGGTAAAATTGAAATTGATGGAGAAACATATTACCTTGGAGCAATGGTATCTAATCTATCGATAACTACCGGAGCTCACGTTGTAGCTAAACCATCAACTGTAACATACAACGAAGTATCACCATCAGCGACAGCAA(SEQ ID NO:8)

[0047] According to an embodiment of the present invention, the primers of the molecular marker include the primer group described in the third aspect.

[0048] In the tenth aspect of the present invention, the present invention provides a method for detecting Lactobacillus gasseri described in the first aspect or the second aspect. According to an embodiment of the present invention, the method includes: amplifying the DNA of the strain to be detected using the primer set described in the third aspect, and screening to obtain Lactobacillus gasseri described in the first aspect or the second aspect. As described above, the primer set described in the third aspect is a specific primer for Lactobacillus gasseri of the present invention, which can highly match the DNA sequence of Lactobacillus gasseri described in the first aspect or the second aspect, and only produce specific amplification in the Lactobacillus gasseri. Using the method of the present invention, the Lactobacillus gasseri of the present invention can be efficiently and simply identified and detected.

[0049] According to an embodiment of the present invention, the product of the amplification treatment has the nucleotide sequence shown in SEQ ID NO:8, which is an indication that the strain to be detected is Lactobacillus gasseri described in the first aspect or the second aspect. Using the primer set described in the third aspect can amplify the nucleotide sequence shown in SEQ ID NO:8. The nucleotide sequence shown in SEQ ID NO:8 is a specific nucleotide sequence marker for Lactobacillus gasseri of the present invention, providing a reliable tool and basis for identifying and detecting Lactobacillus gasseri of the present invention.

[0050] According to an embodiment of the present invention, the method for detecting Lactobacillus gasseri described in the first aspect or the second aspect includes the following steps: extracting a DNA sample from the strain to be detected, performing PCR amplification on the DNA sample using the primer set described in the third aspect, and then comparing the amplification result with the molecular marker described in the ninth aspect to identify whether the strain to be detected is Lactobacillus gasseri described in the first aspect or the second aspect. Using the method of the present invention, the Lactobacillus gasseri of the present invention can be efficiently and simply identified and detected.

[0051] In the eleventh aspect of the present invention, the present invention provides the use of the molecular marker described in the ninth aspect in identifying and detecting Lactobacillus gasseri described in the first aspect or the second aspect.

[0052] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0054] Figure 1 is a colony morphology diagram of Bacillus gasserii HY1124 according to an embodiment of the present invention;

[0055] Figure 2It is the morphological diagram of Lactobacillus gasseri HY1124 according to an embodiment of the present invention;

[0056] Figure 3 It is the PCR amplification electrophoresis pattern of primer 1-p1 for 4 Lactobacillus gasseri strains according to an embodiment of the present invention, where M is Marker, 1 uses the supernatant after lysis of HY1124 as the template, 2 uses the supernatant after lysis of HY02772 as the template, 3 uses the supernatant after lysis of HY09235 as the template, and 4 uses the supernatant after lysis of HY11276 as the template;

[0057] Figure 4 It is the PCR amplification electrophoresis pattern of primer 2-p1 for 4 Lactobacillus gasseri strains according to an embodiment of the present invention, where M is Marker, 1 uses the supernatant after lysis of HY1124 as the template, 2 uses the supernatant after lysis of HY02772 as the template, 3 uses the supernatant after lysis of HY09235 as the template, and 4 uses the supernatant after lysis of HY11276 as the template;

[0058] Figure 5 It is the hemolysis test result diagram of Lactobacillus gasseri HY1124 according to an embodiment of the present invention;

[0059] Figure 6 It is the BV pharmacodynamic animal experiment result diagram according to an embodiment of the present invention;

[0060] Figure 7 It is the VVC pharmacodynamic animal experiment result diagram according to an embodiment of the present invention. Detailed implementation manners

[0061] The following specific embodiments illustrate the implementation manners of the invention of this application. Those skilled in the art can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0062] Term definitions

[0063] In this application, the term "Lactobacillus gasseri" or "L. gasseri" generally refers to a species of the genus Lactobacillus. This species is usually distinguished from other Lactobacillus based on the polynucleotide sequence of the ribosomal 16S rDNA gene.

[0064] In this application, the term "effective culture conditions" generally refers to the environment in which Lactobacillus gasseri is placed or exposed to promote the growth of the bacterium. Therefore, this term generally refers to the culture medium, temperature, atmospheric conditions, substrate, stirring conditions, etc. that can affect the growth of the bacterium.

[0065] In the present application, the term "antibiotic-sensitive" means that bacteria have low resistance to antibiotics, and under the condition of administering a small amount of the drug, the normal growth of the bacteria can be affected. According to an embodiment of the present invention, Lactobacillus gasseri HY1124 is moderately sensitive to clindamycin and ciprofloxacin and sensitive to cefuroxime.

[0066] In the present application, the term "vagina" generally refers to the vaginal region or division or the surrounding area, including the labia, vulva, cervix, uterus, fallopian tubes, ovaries, urethra, bladder, anus, and rectum, including their mucosal tissues.

[0067] In the present application, the terms "disease" or "disorder" are used interchangeably and generally refer to any change in the state of the body or certain organs that impedes or disrupts the performance of functions and / or causes symptoms such as discomfort, dysfunction, pain, or even death in a person suffering from or in contact with the disease.

[0068] In the present application, the term "pathogenic" (e.g., "pathogenic bacterium") generally refers to a substance, microorganism, or condition that is capable of causing a disease. In certain contexts, pathogens also include microorganisms (e.g., bacteria) that are associated with a disease or disorder but for which a causal relationship (e.g., a direct causal relationship) has not yet been established or remains to be established. In some embodiments, microorganisms that are not pathogens and can be symbionts can cause or be associated with a disease or dysbiosis, depending on various factors (e.g., the immune status of the site, the abundance of the microbial taxon, etc.). Such microorganisms are referred to as "pathogenic organisms".

[0069] In the present application, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms that colonize the vagina.

[0070] In the present application, the term "inhibit" generally refers to the process of inhibiting or impeding the growth, reproduction, and activity of bacteria.

[0071] In the present application, the term "isolated", when applied to a nucleic acid or a protein, generally means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in its natural state. For example, it can be in a homogeneous state and can be in a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0072] The term "isolated", when applied to bacteria, generally refers to bacteria that have been (1) separated from at least some of the components with which they were associated at the time of their initial production (whether in nature or in an experimental setting), and / or (2) bacteria that have been produced, prepared, purified, and / or manufactured artificially, such as by using artificial culture conditions, such as (but not limited to) culturing on plates and / or in fermenters. Isolated bacteria include those that are cultured, even if such cultures are not pure cultures. Isolated bacteria can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were initially associated. In embodiments, the isolated bacteria are greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater than about 99%. According to embodiments of the present invention, the bacterial populations provided in this application include isolated bacteria. According to embodiments of the present invention, the compositions provided in this application include isolated bacteria. According to embodiments of the present invention, the administered bacteria are isolated bacteria.

[0073] In this application, the term "pharmaceutically acceptable carrier" generally refers to a substance that aids in the administration of an active agent to a subject and is absorbed by the subject and that can be included in the compositions of this application without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline solution, lactated Ringer's solution, common sucrose, common glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and dyes, etc. Such products can be sterilized, and those skilled in the art will recognize that other pharmaceutical carriers can also be used in this application.

[0074] In this application, the term "CFU (Colony-Forming Units)" generally refers to the total number of microbial communities such as bacteria, fungi, yeast, etc. in a product, and is usually used for calculating the number of viable bacteria.

[0075] The term "CFU / dose" means the amount of bacteria present in the composition / food or dietary supplement / drug provided to the subject per day or per administration. For example, in certain embodiments, Lactobacillus gasseri in the food or dietary supplement is present in an amount of 10 6 to 10 10 CFU / dose (e.g., 10 6 to 10 9CFU / dose). In such an embodiment, if Lactobacillus gasseri is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject daily or each time may contain about 10 6 to 10 10 CFU of Lactobacillus gasseri. Of course, alternatively, the amount of this bacterium can be divided into multiple administrations, as long as the total amount of Lactobacillus gasseri received by the subject within any specific time period (e.g., every 24-hour period) is from about 10 6 to about 10 10 CFU of the bacterium, that is, the Lactobacillus gasseri in the above-mentioned food product or dietary supplement is present in an amount of 10 6 to 10 10 CFU / dose (e.g., 10 6 to 10 9 CFU / dose).

[0076] In the present application, the term "treatment and / or prevention" not only includes treating and / or preventing a disease, but generally includes preventing the onset of the disease, slowing down or reversing the disease process, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing a further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to treating the patient. The compositions of the present application do not need to achieve complete cure or eradication of any symptoms or manifestations of the disease to form viable therapeutic agents. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a prophylactic administration of a treatment does not need to be completely effective in preventing the onset of a disorder. Simply reducing the impact of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by enhancing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of the disease occurring or worsening is sufficient.

[0077] In the present application, the term "about" generally refers to a variation within a range of about 0.5% - 10% above or below the specified value, e.g., within a range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value.

[0078] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the field or according to the product instructions are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0079] Example 1 Screening and isolation of Lactobacillus gasseri

[0080] (1) Collection of samples

[0081] The sample sources are natural fermented foods with regional characteristics, vaginal secretion samples of volunteers collected by hospital gynecologists meeting the inclusion criteria, and human samples (feces and breast milk).

[0082] (2) Strain isolation

[0083] The vaginal secretions of healthy women of childbearing age were serially diluted tenfold with physiological saline. The dilution solution of an appropriate dilution gradient was spread on an anaerobic blood agar plate (purchased from Huankai Microorganisms), and cultured at 37 °C in an anaerobic workstation for 48 - 72 h. Single colonies with different morphologies were picked and streaked and purified on the blood agar plate, and then continued to be cultured. The pure culture was picked for strain identification (16S rDNA sequencing). Through identification and screening, a strain of Lactobacillus gasseri was obtained and named Lactobacillus gasseri HY1124.

[0084] After clarifying the strain species, the pure culture (i.e., Lactobacillus gasseri HY1124) was inoculated into MRS broth liquid medium for amplification culture. When the strain grew to an appropriate concentration, a sterile 50% glycerol solution was mixed evenly with the same volume of liquid culture and placed in a -80 °C strain library for preservation.

[0085] Example 2 Identification of Lactobacillus gasseri

[0086] (1) Colony characteristics

[0087] A inoculation loop was used to dip the bacterial liquid in the strain tube and streaked and inoculated on the MRS plate, and cultured anaerobically at 37 °C for 48 h, and the colony morphology on the plate was observed. As Figure 1 shown, the strain formed round, rough-edged, grayish-white colonies after culturing on the MRS plate.

[0088] (2) Staining and microscopic examination

[0089] A inoculation loop was used to pick 1 loop of sterilized distilled water onto a clean glass slide, and a single colony was picked from the MRS plate and mixed evenly with the distilled water and spread on the glass slide. Staining was carried out according to the instructions of the Gram staining solution kit (purchased from Qingdao Haibo), and then the cell morphology was observed under an electron microscope. As Figure 2As shown, after the strain is stained and observed under an optical microscope, it can be determined that the bacteria are Gram-positive, rod-shaped, presenting short rods and long rods.

[0090] (3) Biochemical identification and analysis

[0091] ① Lactobacillus culture: Inoculate the target strain into MRS broth medium (purchased from Qingdao Haibo), place it in an anaerobic incubator, and culture it at 37°C for 24 h.

[0092] ② Preparation of bacterial suspension: Centrifuge the strain fermentation broth at 4000 rpm for 5 min in a centrifuge, discard the supernatant, wash the bacterial pellet with normal saline, then centrifuge at 4000 rpm for 5 min, discard the supernatant, wash the bacterial pellet with normal saline again, centrifuge at 4000 rpm for 5 min to discard the supernatant, and add normal saline and the bacterial pellet and mix evenly for standby.

[0093] ③ 1% sodium hippurate identification experiment: Pipette 50 μL of the bacterial suspension, add it to a 1% sodium hippurate identification tube, seal it with a sealing film, and culture it in a 37°C water bath for 2 h. Then slowly add 200 μL of ninhydrin solution (3.5% ninhydrin solution: 0.175 g of hydrindantin, 2.5 mL of acetone, 2.5 mL of butanol) along the tube wall without shaking, and place it in a 37°C water bath for 10 min and then read the result.

[0094] ④ Other identification experiments: Pipette 50 μL of the bacterial suspension, add it to a biochemical identification tube (Note: After adding the bacterial solution to the esculin identification tube, the liquid surface needs to be covered with sterile liquid paraffin), seal it with a sealing film, place it in an anaerobic incubator, and culture it at 37°C for 48 h and then read the result. The read results are shown in Table 1:

[0095] Table 1. Interpretation results of Lactobacillus gasseri identification tubes

[0096]

[0097] Note: + represents positive; - represents negative; +w represents weakly positive.

[0098] The results of biochemical identification show that Lactobacillus gasseri can utilize 9 other carbon sources (esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin, lactose) except raffinose and 1% sodium hippurate.

[0099] (4) 16S rDNA identification

[0100] The above-mentioned Lactobacillus gasseri was amplified and sequenced by 16S rDNA. After obtaining the sequence, BLAST alignment was performed in the NCBI database. The 16S rDNA alignment result showed that the above strain was Lactobacillus gasseri, named Lactobacillus gasseri HY1124. The gene sequence determined by 16S rDNA is as follows:

[0101] GTCGAGCGAGCTTGCCTAGATGAATTTGGTGCTTGCACCAGATGAAACTAGATACAAGCGAGC

[0102] GGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAAGAGACTGGGATAACACCTGGAAACAGAT

[0103] GCTAATACCGGATAACAACACTAGACGCATGTCTAGAGTTTAAAAGATGGTTCTGCTATCACTCTTG

[0104] GATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAATGATGCATAGCCG

[0105] AGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAG

[0106] TAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTC

[0107] GGCTCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAA

[0108] TTACTTAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGT

[0109] CCGGATTTATTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTTTGGCTC

[0110] AACCGGAGAATTGCATCAGAAACTGTTGAACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTG

[0111] TAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACT

[0112] GACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAAC

[0113] GATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGC

[0114] CTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGA

[0115] GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCAGTGCAAACCTAA

[0116] GAGATTAGGTGTTCCCTTCGGGGACGCTGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGT

[0117] GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTAAGTTGGGC

[0118] ACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCC

[0119] TTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGAAGCGAACCTGCGAAGGCAA

[0120] GCGGATCTCTGAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGCTGGAAT

[0121] CGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC

[0122] ACACCATGAGAGTCTGTAACACCCAAAGCCGGTGGGATAACCTTTATAGGAGTCAGCCGT(SEQ IDNO:1).

[0123] (5) Specific nucleotide sequence molecular marker

[0124] ① Specific primer design

[0125] a. Screening of specific nucleotide sequences: The whole genome of strain HY1124 was sequenced and genome analysis was performed. It was compared and analyzed with the genome sequences of Lactobacillus gasseri strains deposited in the NCBI database to screen out the specific nucleotide sequences of Lactobacillus gasseri HY1124.

[0126] b. Primer design: Primers were designed for the specific nucleotide sequences screened in step a. A total of primer fragments with predicted product lengths of approximately 200 to 800 bp were designed for the screened specific nucleotide sequences, and 2 pairs of primers were designed for other nucleotide sequences on the whole genome as controls. The primer sequences are shown in Table 2:

[0127] Table 2 Primer list

[0128]

[0129] c. Primer screening: Lactobacillus gasseri HY1124 and 3 strains of the same species (specific strain information is shown in Table 3) were used as control groups to prepare templates. Single colonies were picked into a 50 μL lysis solution (TaKaRa) respectively. After simple centrifugation, they were lysed at 80 °C for 15 min and centrifuged at 4000 rpm for 5 min. The supernatant was used as the template. Through primer screening, 1-p1 was selected for PCR amplification experiment. The PCR amplification system was 12.5 μL of Taq enzyme, 1 μL of F, 1 μL of R, 1.5 μL of template, and supplemented with ddH2O to 25 μL; the PCR reaction conditions are shown in Table 4 below, and PCR experiments were carried out. After the PCR experiment, 1.5 g of agarose was added to 100 mL of 1×TAE buffer, heated and melted thoroughly, 5 μL of Gel Red dye was added, and then poured into a gel plate to solidify for use. 4 μL of the PCR product was taken for agarose gel electrophoresis. The electrophoresis conditions were as follows: voltage 130 V, current 400 mA, and time 35 min.

[0130] Table 3 Strain information

[0131] Strain number Identified name HY1124 Lactobacillus gasseri HY02772 Lactobacillus gasseri HY09235 Lactobacillus gasseri HY11276 Lactobacillus gasseri

[0132] Note: The strains with strain numbers HY02772, HY09235, and HY11276 in Table 3 are Lactobacillus gasseri strains screened from the samples collected by the inventors according to Example 1. In order to verify the specificity of the above-mentioned specific nucleotide sequence for Lactobacillus gasseri HY1124, the above primers were used to perform PCR amplification on HY1124 and 3 strains of the same species respectively.

[0133] Table 4 PCR reaction conditions

[0134]

[0135] Among the primers designed for the specific nucleotide sequence, when using 1-p1 as the primer for PCR amplification experiment, only HY1124 had a specific amplification band, and the band was clear and had a high concentration, with relatively few primer dimers, while HY02772, HY09235, and HY11276 had no amplification bands. The agarose gel electrophoresis detection pattern is shown in Figure 3 . When using primers designed with other nucleotide sequences for PCR amplification experiment, there was a situation where bands were also amplified in the control bacteria group (taking 2-p1 as an example, the agarose gel electrophoresis detection pattern is shown in Figure 4 ). Therefore, it shows that primer 1-p1 is the molecular marker primer for HY1124, and the amplification product generated by this primer is the molecular marker of Lactobacillus gasseri HY1124. The size of the amplification product of primer 1-p1 is 800 bp, and the nucleotide sequence is as shown in SEQ ID NO:8. Thus, it is determined that the nucleotide sequence shown in SEQ ID NO:8 is a part of the specific nucleotide sequence screened in step a, further verifying that primer 1-p1 can be used as the specific primer for Lactobacillus gasseri HY1124, and the nucleotide shown in SEQ ID NO:8 is the specific nucleotide sequence marker of Lactobacillus gasseri HY1124, which provides a reliable tool and basis for identifying and detecting Lactobacillus gasseri HY1124.

[0136] ACCTGCAGAAGAAACTGCGACAACAGAAAGCAACAAACCAAAATCACGTAGAAAACGTGCTCTAGATGCAAACGATCAGAACCGATCAGGTGATGCAGCTACAACAGATGGAGATGATGATTCAGTTGTCAACGAAAAGGATCTACATGTGGTAAACCCATATTTTGAAGATAACGGAAAACCTACTACTGCTGAAAAATGGAAAGTTATTAATGCTTTTGACTTGATTGGATGGAAGCCGATCAATCCTAATCAAAAAAAAGTAGTTATTGCAAATGGTAAAGTAACAGAGTATGGAGGTTACGGATCTGTTGTAAATCAAACTCATCCATATAGTATTCCGTTGGCACTATATAAAAAAGCTGGTGATCGCACTAGTAAGAGTGATCAGTTTGATGGGGTGTATCAAGATATTGATGTAATTCCTGGACAAGAGATTGTAATTACTCAAAATACAGGTACTTTTGGACCGATTGGATCAAAAGACAATAGAACTATACTAACAGTATCTTACCCAGAAAGAGGGAATGAGGTTCAAGGAAAGATTGTTTGGAGATCACTTATGACACCATATAATGGTGTAGTGACAGTACCTAAAGGGATAACTAAACTTCGTGTACGTCTTGAGGTAGATCCAGATTCAAATGTAGCACATAAAGATAATGGTAAAATTGAAATTGATGGAGAAACATATTACCTTGGAGCAATGGTATCTAATCTATCGATAACTACCGGAGCTCACGTTGTAGCTAAACCATCAACTGTAACATACAACGAAGTATCACCATCAGCGACAGCAA(SEQ ID NO:8).

[0137] d. During the experiment, the inventors found that when using the 1-p1 primer to sequence multiple Lactobacillus gasseri HY1124 samples, there were differences in individual bases between the amplified product sequence and the nucleotide sequence shown in SEQ ID NO:8. However, compared with the control bacteria, there were specific amplification bands, and the bands were clear and had a high concentration, with relatively few primer dimers.

[0138] In an experiment, it was found that the nucleotide sequence of the product amplified from the Lactobacillus gasseri HY1124 sample using the 1-p1 primer was as shown in SEQ ID NO:9, and the result of alignment with the nucleotide sequence shown in SEQ ID NO:8 was 99.87%.

[0139] CACAGAAGCAACAAACCAAAATCACGTAGAAAACGTGCTCTAGATGCAAACGATCAGAACCGATCAGGTGATGCAGCTACAACAGATGGAGATGATGATTCAGTTGTCAACGAAAAGGATCTACATGTGGTAAACCCATATTTTGAAGATAACGGAAAACCTACTACTGCTGAAAAATGGAAAGTTATTAATGCTTTTGACTTGATTGGATGGAAGCCGATCAATCCTAATCAAAAAAAAGTAGTTATTGCAAATGGTAAAGTAACAGAGTATGGAGGTTACGGATCTGTTGTAAATCAAACTCATCCATATAGTATTCCGTTGGCACTATATAAAAAAGCTGGTGATCGCACTAGTAAGAGTGATCAGTTTGATGGGGTGTATCAAGATATTGATGTAATTCCTGGACAAGAGATTGTAATTACTCAAAATACAGGTACTTTTGGACCGATTGGATCAAAAGACAATAGAACTATACTAACAGTATCTTACCCAGAAAGAGGGAATGAGGTTCAAGGAAAGATTGTTTGGAGATCACTTATGACACCATATAATGGTGTAGTGACAGTACCTAAAGGGATAACTAAACTTCGTGTACGTCTTGAGGTAGATCCAGATTCAAATGTAGCACATAAAGATAATGGTAAAATTGAAATTGATGGAGAAACATATTACCTTGGAGCAATGGTATCTAATCTATCGATAACTACCGGAGCTCACGTTGTAGCTAAACCATCAACTGTAACAT(SEQID NO:9)

[0140] Therefore, after multiple sequencing and alignment, the homology range between the molecular marker of Lactobacillus gasseri HY1124 and the nucleotide sequence shown in SEQ ID NO:8 was determined. That is, the molecular marker of Lactobacillus gasseri HY1124 has a nucleotide sequence with at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:8.

[0141] Example 3 Strain Characteristics

[0142] (1) Antibiotic Sensitivity Test

[0143] Lactobacillus gasseri HY1124 was cultured in MRS broth, and then the bacterial solution was evenly spread on an MRS plate. After the bacterial solution was absorbed and dried, antibiotic susceptibility test discs were applied, and the plate was anaerobically cultured at 37°C for 48 h. The diameter of the inhibition zone was measured using a vernier caliper. The antibiotic sensitivity of the strain was judged by the diameter of the inhibition zone around the antibiotic susceptibility test disc. The results are shown in Table 5. The strain was resistant to metronidazole, norfloxacin, ofloxacin, and kanamycin, moderately sensitive to clindamycin and ciprofloxacin, and sensitive to cefuroxime.

[0144] Table 5 Results of Antibiotic Sensitivity Test

[0145]

[0146] Note: S: Sensitive (15 - 20 mm, highly sensitive; >20 mm, extremely sensitive) I: Intermediate (10 - 14 mm, moderately sensitive) R: Resistant (<10 mm, insensitive)

[0147] (2) Toxicity Test

[0148] ① Hemolysis Experiment

[0149] The bacterial solution in the strain cryopreservation tube was dipped and streaked on an anaerobic blood agar plate, and anaerobically cultured at 37°C for 48 h. The change in the color of the blood agar plate around the colony was observed. The results of the hemolysis experiment are as Figure 5 shown. Grayish-white small colonies appeared in the medium around the colony, and no hemolysis ring appeared around the colony, indicating that Lactobacillus gasseri HY1124 was γ-hemolytic, that is, non-hemolytic.

[0150] ② Mouse Toxicity Experiment

[0151] Five mice weighing 18 - 22 g were used. Each mouse was orally gavaged with 0.5 ml of fresh bacterial solution (not less than 1.0×10 9 CFU / 0.5 ml) once a day for 3 consecutive days. From the first day of gavage, the mice were continuously observed until the 7th day, and the survival and body weight of the mice were observed. The experimental results showed that after oral gavage of the fresh bacterial solution of Lactobacillus gasseri HY1124, all the mice survived healthily and their body weights increased.

[0152] (3) Determination of metabolite content

[0153] ① Detection of D-lactic acid

[0154] Use a D-lactic acid detection kit (purchased from Sigma-Aldrich) to detect the D-lactic acid production in the supernatant of Lactobacillus. The method adopted by this kit is as follows: D-lactic acid is oxidized by a specific D-lactic acid dehydrogenase to produce a color reaction, which has a proportional relationship with the D-lactic acid concentration, and measure the absorbance at 450 nm.

[0155] ② Detection of L-lactic acid

[0156] Filter the supernatant with a 0.22 μm sterile filter membrane. Use a biosensor to determine the L-lactic acid concentration.

[0157] ③ Detection of hydrogen peroxide

[0158] Prepare 100 μl of hydrogen peroxide standard solutions with concentrations of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mM using PBS solution, add 200 μl of enzyme reagent (0.5 mM / L 4-aminoantipyrine (4-AA), 0.2 mM / L peroxidase), add 200 μl of phenol solution (19.66 mM / L), mix well, incubate at 37 °C for 20 min, evenly take 200 μL and place it in a 96-well plate, and measure the absorbance of each well at a wavelength of 505 nm. Establish a standard curve for the hydrogen peroxide solution based on the detected results.

[0159] Inoculate Lactobacillus into MRS medium at an inoculation amount of 0.5%, and culture it in an anaerobic chamber at 37 °C for 24 h. When the Lactobacillus is cultured for 24 h, centrifuge at 4000 rpm for 20 min at 4 °C, discard the supernatant, wash it 1-2 times with PBS buffer, and resuspend the cells with 0.5% glucose solution until OD 600 = 1.0. Incubate at 37 °C and 170 rpm for 5 hours, centrifuge at 10,000 rpm for 5 min, collect the supernatant, filter and sterilize it with a 0.22 μm filter for standby. Take 100 μl of the supernatant, add 200 μL of enzyme reagent and phenol reagent respectively, mix well, incubate at 37 °C for 20 min. Evenly take 200 μL and place it in a 96-well plate, and measure the absorbance of each well at a wavelength of 505 nm.

[0160] Lactobacillus gasseri HY1124 was cultured in MRS broth for 24 h. The detection results of D-lactic acid, L-lactic acid and hydrogen peroxide are shown in Table 6. Organic acids such as D-lactic acid and L-lactic acid belong to bacteriostatic substances, which can compete with pathogenic bacteria for nutrients and adhesion sites, and improve the mucosal immunity and anti-infection ability of the host. Among them, the content of D-lactic acid in the metabolites of Lactobacillus gasseri HY1124 was 2.07 g / L, the content of L-lactic acid was 3.9 g / L, and the total acid production of Lactobacillus gasseri HY1124 was 5.97 g / L, while the total acid production of Lactobacillus delbrueckii subsp. bulgaricus DJS was only 5.48 g / L. The acid production of Lactobacillus gasseri HY1124 was higher than that of Lactobacillus delbrueckii subsp. bulgaricus DJS; H2O2 can directly kill pathogenic bacteria, and can also be catalytically oxidized by biological enzymes to form halides with stronger bactericidal effects. As can be seen from Table 6, Lactobacillus gasseri HY1124 can produce 1240 μM hydrogen peroxide, while the amount of hydrogen peroxide produced by Lactobacillus delbrueckii subsp. bulgaricus DJS was only 80.67 μM. The ability of Lactobacillus gasseri HY1124 to produce hydrogen peroxide was much higher than that of Lactobacillus delbrueckii subsp. bulgaricus DJS. Therefore, Lactobacillus gasseri HY1124 has the potential to kill pathogenic bacteria, improve the mucosal immunity and anti-infection of the host, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0161] Table 6 Determination results of metabolite contents

[0162] Metabolite Lactobacillus gasseri HY1124 Lactobacillus delbrueckii subsp. bulgaricus DJS D-lactic acid 2.07 g / L 5.24 g / L L-lactic acid 3.9 g / L 0.24 g / L Hydrogen peroxide 1240 μM 80.67 μM

[0163] Example 4 Application function analysis

[0164] (1) Bacteriostatic experiment

[0165] ① Preparation of working bacterial solution: Lactobacillus was inoculated into MM medium (modified MRS broth, with the composition of peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) at an inoculation amount of 0.5%, and cultured in an anaerobic workstation. Six pathogenic bacteria were cultured using a suitable medium (Gardnerella vaginalis ATCC14018, abbreviated as GV; Escherichia coli ATCC 25922, abbreviated as EC; Staphylococcus aureus ATCC25923, abbreviated as SA; Prevotella bivia NCTC 11156, abbreviated as PB; Canidia albicans ATCC 10231, abbreviated as CA). After culturing the target strains, the supernatant was obtained by centrifugation, and the cell-free supernatant was obtained by filtration through a 0.22 μm filter, and was used immediately or stored in a -80 °C refrigerator. After culturing the pathogenic bacteria, the OD 600 value was measured, and it was diluted to an OD 600 value of about 0.005 (the viable count was maintained at 5.0×10 5 CFU / mL to 5.0×10 6 CFU / mL) (WS / T 650—2019 Antibacterial and bacteriostatic effect evaluation method).

[0166] ② Interaction: The same volume of supernatant and pathogenic bacteria solution were taken and mixed evenly. Immediately, 100 μL of the evenly mixed bacterial solution was taken and put into a blank 96-well plate to measure the OD 600 ; the remaining culture solution was placed at 37 °C and cultured anaerobically or aerobically according to the culture conditions of the pathogenic bacteria. After culturing for 48 h, 100 μL of the evenly mixed bacterial solution was taken and put into a blank 96-well plate to measure the OD 600 . A blank control group was set up, with 2 replicates for each sample. According to the following formula, the bacteriostatic rate of Lactobacillus against pathogenic bacteria was calculated.

[0167] Bacteriostatic rate = (A - B) / A * 100%

[0168] A: The OD 600 value increased within 48 h in the positive control group (i.e., blank medium);

[0169] B: The OD 600 value increased within 48 h in the experimental group.

[0170] Three different strains of the same bacterial species and the strain in the positive drug Ding Junsheng were selected, and the antibacterial performance was detected with the screened Lactobacillus gasseri HY1124. The results are shown in Table 7. This strain has strong antibacterial effects on GV, PB, EC, SA, and CA bacteria. Among them, the antibacterial effects on GV, PB, and CA are significantly better than those of the control group of the same bacterial species and the positive control group.

[0171] Table 7 Results of antibacterial experiments

[0172]

[0173] Note: The strains with the strain numbers HY02772, HY01880, and HY02172 in Table 7 are Lactobacillus gasseri strains screened from the samples collected by the inventors according to Example 1 to verify the inhibition rates of Lactobacillus gasseri HY1124 and three strains of the same bacterial species against different pathogenic bacteria.

[0174] (2) Co-culture antibacterial experiment

[0175] 1) Co-culture experiment of Lactobacillus and CA

[0176] Cultivation of Lactobacillus: Take the glycerol tube of Lactobacillus gasseri and inoculate it into the MM medium (modified MRS broth, the composition of the components is: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L). Concentrate or dilute the cultured bacterial liquid to about 1×10 9 CFU / mL as the working bacterial liquid of Lactobacillus.

[0177] Cultivation of CA: Take the CA glycerol tube and inoculate it into the Sabouraud medium, and culture it aerobically at 37°C. Centrifuge the cultured bacterial liquid to remove the supernatant, and then adjust the bacterial liquid concentration to about 1.0×10 7 CFU / mL with fresh Sabouraud medium as the working bacterial liquid of CA.

[0178] Take 400 μL of the working bacterial liquids of Lactobacillus and CA and inoculate them into 40 ml of MRS liquid medium respectively. CA is inoculated alone as the blank control group, with 2 replicates in each group. Gently shake the bacterial liquid and culture it in an anaerobic workstation at 37°C. Sampling is carried out 20 h after cultivation, and the viable count of CA is performed using Candida chromogenic medium (purchased from bioMérieux, France). The experimental results are shown in Table 8. After co-culturing Lactobacillus gasseri and CA for 20 h, the antibacterial rate reaches 31.2%, indicating that this strain has a certain inhibitory effect on the growth of CA.

[0179] Table 8 Results of co-culture antibacterial experiment of Lactobacillus and CA

[0180] Strain Viable count of CA in 20 h bacterial solution (cfu / ml) Inhibitory rate of Lactobacillus on CA in 20 h CA blank control 4.17E+06 0％ Lactobacillus gasseri HY1124 2.87E+06 31.2％

[0181] 2) Co-culture experiment of Lactobacillus and GV

[0182] Cultivation of Lactobacillus: Take the glycerol tube of Lactobacillus gasseri and inoculate it into MM medium (modified MRS broth, the composition of the components is: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L), and concentrate or dilute the cultured bacterial liquid to about 1×10 9 CFU / mL concentration as the working bacterial liquid of Lactobacillus.

[0183] Cultivation of GV: Take the GV glycerol tube and inoculate it into BHI liquid medium containing 10% fetal bovine serum, and culture it anaerobically at 37°C. Centrifuge the cultured bacterial liquid to remove the supernatant, and then adjust the bacterial liquid concentration to about 1.0×10 7 CFU / mL as the working bacterial liquid of GV.

[0184] Take 400 μL of the working bacterial liquid of Lactobacillus and GV respectively and inoculate them into 40 ml of BHI liquid medium containing 10% fetal bovine serum. GV inoculated alone is used as the blank control group, with 2 replicates in each group. Culture them in an anaerobic workstation and sample at 27 h after culture. Use GV-specific probe primers to detect the viable count of GV by fluorescence quantitative qPCR method. Detect the viable count of GV after co-culturing Lactobacillus gasseri and GV for 27 h. The experimental results are shown in Table 9. After co-culturing Lactobacillus gasseri HY1124 and GV for 27 h, the antibacterial rate reaches 94%, indicating that this strain has a strong inhibitory effect on the growth of GV.

[0185] Table 9 Results of antibacterial experiment of co-culture of Lactobacillus and GV

[0186] Strain Viable count of GV in 27 h bacterial solution (cfu / ml) Inhibitory rate of Lactobacillus on GV in 27 h GV blank control 4.06E+08 0 Lactobacillus gasseri HY1124 1.37E+07 94％

[0187] (3) Biofilm removal experiment

[0188] 1) Experiment on the removal of GV biofilm by Lactobacillus

[0189] Adjust the cultured bacterial liquid of GV to 1.0×10 7CFU / mL, inoculated into 96-well plates, with a total of 2 groups set up, and 4 replicates in each group. The two groups were respectively added with the same volume of blank MM liquid medium (modified MRS broth medium, the composition of which is: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and the supernatant of Lactobacillus gasseri HY1124. Incubated in an anaerobic workstation at 37 °C for 24 h.

[0190] After the incubation, the microplate crystal violet staining method was used to determine the inhibitory effect of Lactobacillus gasseri HY1124 on the formation of GV biofilm. The experimental results are shown in Table 10. The supernatant of Lactobacillus gasseri HY1124 had a strong inhibitory effect on the formation of GV biofilm, reaching 89.6%. Therefore, it can effectively inhibit the growth of GV.

[0191] Table 10 Results of the experiment on the clearance of GV biofilm by Lactobacillus

[0192] Strain Inhibitory rate of Lactobacillus supernatant on GV biofilm formation Lactobacillus gasseri HY1124 89.6％±1.7％

[0193] 2) Experiment on the clearance of CA biofilm by Lactobacillus

[0194] Inhibitory biofilm clearance experiment: The cultured CA bacterial solution was adjusted to 1.0×10 7 CFU / mL, inoculated into 96-well plates, with a total of 2 groups set up, and 4 replicates in each group. The two groups were respectively added with the same volume of Sabouraud dextrose liquid medium and the supernatant of Lactobacillus. Incubated in an anaerobic workstation at 37 °C for 24 h. After the incubation, the microplate crystal violet staining method was used to determine the inhibitory effect of Lactobacillus on the formation of CA biofilm.

[0195] Biofilm clearance experiment: The cultured CA bacterial solution was adjusted to 1.0×10 7 CFU / mL, inoculated into 96-well plates, with a total of 2 groups set up, and 4 replicates in each group. Incubated in an anaerobic workstation at 37 °C for 24 h. After 24 h of incubation, the mixture in the wells was discarded and washed with sterile PBS. Then, the same volume of MM medium (modified MRS broth medium, the composition of which is: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and the supernatant of Lactobacillus were added to each group and incubated at 37 °C for 24 h. After the incubation, the microplate crystal violet staining method was used to determine the clearance effect of the Lactobacillus supernatant on the mature CA biofilm.

[0196] The experimental results are shown in Table 11. Compared with the ability to remove the formed biofilm, this Lactobacillus gasseri has a stronger inhibitory effect on the formation of CA biofilm, and the inhibition rate reaches 51.2%.

[0197] Table 11 Experimental results of Lactobacillus on the removal of CA biofilm

[0198] Strain Inhibitory rate of Lactobacillus supernatant on CA biofilm formation Removal rate of Lactobacillus supernatant on CA biofilm Lactobacillus gasseri HY1124 51.2％±5.6％ 8.4％±16.8％

[0199] (4) Cell adhesion experiment

[0200] ① Preparation of working bacterial solution: Culture Lactobacillus and detect the OD 600 value. Centrifuge the cultured Lactobacillus culture solution at 4°C and 4000 rpm for 5 min, discard the supernatant, wash it three times repeatedly with PBS, and finally resuspend Lactobacillus with complete MEM medium (purchased from Zhongqiao Xinzhou). Take an appropriate amount of the resuspended solution and detect the viable cell count by the method of plate coating, which is L1.

[0201] ② Cell culture: Seed Hela cells in a 24-well plate at a cell density of 1.5×10 5 cells / mL and culture for 16 h until the confluence rate reaches 90%. Wash the HeLa cells cultured in the 24-well plate with serum-free MEM medium (purchased from Zhongqiao Xinzhou), and then count them, denoted as C1.

[0202] ③ Interaction: Inoculate Lactobacillus into the cells at a ratio of 100:1, and incubate in an environment of 37°C and 5% CO2 for 1.5 h. Add the centrifuged supernatant to the cells as a blank control group. After 1.5 h, collect the culture solution in the wells, wash the cells in the wells with MEM medium, then add trypsin to each well for digestion, and then add complete MEM medium to terminate the reaction. Collect the suspension, take a part to count the cell number C2, and use the plate coating method to detect the viable cell count L2 of Lactobacillus adhering to the cells.

[0203] ④ Calculate the adhesion number and adhesion rate: The average cell adhesion number of Lactobacillus = L2 / C2.

[0204] The single-cell adhesion number of Lactobacillus gasseri HY1124 to Hela cells is 12.31 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epidermal cells.

[0205] (5) Efficacy test of BV (bacterial vaginitis) animal model

[0206] Healthy SPF-grade Balb / c mice, female, 6 - 8 weeks old, were used for modeling. After adaptive cultivation of the animals, they were randomly divided into groups of 8 each, including a model group (M), an experimental group (Lactobacillus gasseri HY1124), a control group of the same strain (Lactobacillus gasseri HY02172), and a positive control group (DJS). Before inoculating the pathogenic bacteria in each group of animals, estradiol benzoate injection was subcutaneously injected, and then GV of the same concentration (20 μl) was vaginally administered to establish a BV pathogenic bacteria model. After successful modeling, the experimental group and the control group of the same strain were continuously given the corresponding lactobacillus solution (1×10 10 CFU / mL, 20 μL) vaginally for 5 days, the positive control group was continuously given an equal amount of Lactobacillus delbrueckii solution of the test product (the strain in Ding Junsheng, 1×10 10 CFU / mL, 20 μL) vaginally for 5 days, and the model group was given an equal volume of normal saline for treatment. After the treatment ended, 50 μL / time of PBS was used for each animal, 4 times per animal, to lavage the vagina, the liquid was collected, placed in a 1.5 mL Ep tube, and stored at -80 °C. The qPCR method was used to detect the load of GV in the lavage fluid, and then the differences in the load of GV in the lavage fluid among the groups were compared.

[0207] The experimental results were as Figure 6 shown. The experimental results showed that the use of Lactobacillus gasseri HY1124 to treat the BV-infected animal model significantly reduced the content of GV in its vagina, and the treatment effect was significantly better than that of the positive drug (DJS) and slightly better than that of the same strain HY02172, indicating that this strain has a good treatment effect on BV in mice.

[0208] (6) Pharmacodynamic test of the animal model of VVC (vulvovaginal candidiasis)

[0209] Healthy SPF-grade Balb / c mice, female, 6 - 8 weeks old, were used for modeling. After adaptive cultivation of the animals, they were randomly divided into groups of 8 each, including a model group (M), an experimental group (Lactobacillus gasseri HY1124), and a positive control group (Shuangzuotai suppository). Each group was pretreated by subcutaneous injection of estradiol benzoate injection; CA (15 μL / animal) was continuously given from D0 to D2, and from D3 - D7, the experimental group was given Lactobacillus gasseri HY1124 (5×10 9 CFU / mL, 20 μL) vaginally every day, the positive control group was given Shuangzuotai suppository (30 mg), and the model group was given an equal volume of normal saline (20 μL). On D8, the vagina was lavaged, the lavage fluid was diluted and spread on a Candida albicans identification medium (chromogenic medium), and the number of green colonies on the culture dish was observed and counted after culturing at 37 °C for 48 h. One-way ANOVA was performed using GraphPad Prism 5 software, and P < 0.05 was considered statistically significant.

[0210] The experimental results were asFigure 7 As shown, compared with the model group, the content of pathogenic bacteria in the vaginal lavage fluid of the Lactobacillus gasseri HY1124 group was significantly reduced after treatment, which was equivalent to the therapeutic effect of the positive drug Metronidazole and Clotrimazole Suppositories, indicating that this strain has a good therapeutic effect on VVC in mice.

[0211] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection 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 a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0212] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Lactobacillus gasseri, characterized in that, It was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 7, 2023, with the deposit number CGMCC No. 26504.

2. A Lactobacillus gasseri, characterized in that, The 16S rDNA of the Lactobacillus gasseri has one of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO:1; (2) A nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO:1; (3) A nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:1; Preferably, the 16S rDNA gene sequence of the Lactobacillus gasseri comprises the nucleotide sequence shown in SEQ ID NO:

1.

3. A primer set for detecting Lactobacillus gasseri according to any one of claims 1 or 2, characterized in that, It includes a forward primer and a reverse primer; The forward primer has the nucleotide sequence shown in SEQ ID NO:2; The reverse primer has the nucleotide sequence shown in SEQ ID NO:

3.

4. A microbial preparation, characterized in that, It contains the Lactobacillus gasseri as claimed in claim 1 or 2.

5. The microbial preparation according to claim 4, characterized in that, It further includes a pharmaceutically acceptable carrier or excipient.

6. A single-dose preparation, characterized in that, including 1×10 6 ~1×10 10 CFU Lactobacillus gasseri according to claim 1 as an active ingredient.

7. Use of the Lactobacillus gasseri as claimed in claim 1 or 2, the microbial preparation as claimed in claim 4 or 5, or the single-dose preparation as claimed in claim 6 in the preparation of a drug for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

8. Use of the Lactobacillus gasseri as claimed in claim 1 or 2, the microbial preparation as claimed in claim 4 or 5, or the single-dose preparation as claimed in claim 6 in the preparation of a drug for inhibiting vaginal pathogenic bacteria.

9. Use according to claim 7 or 8, characterized in that, The pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Prevotella bivia and Candida albicans.

10. Application of the Lactobacillus gasseri as claimed in claim 1 or 2, the microbial preparation as claimed in claim 4 or 5, or the single-dose preparation as claimed in claim 6 in the preparation of a product for regulating vaginal flora balance.

11. A molecular marker of Lactobacillus gasseri according to claim 1 or 2, characterized in that, The molecular marker comprises a nucleotide sequence selected from one of the following: (1) The nucleotide sequence shown in SEQ ID NO:8; (2) A nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:8; (3) A nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:

8.

12. The molecular marker according to claim 11, wherein The molecular marker has the nucleotide sequence shown in SEQ ID NO:

8.

13. The molecular marker according to claim 11, wherein The primer of the molecular marker comprises the primer group as claimed in claim 3.

14. A method for detecting Lactobacillus gasseri according to claim 1 or 2, characterized in that, It includes: Using the primer group as claimed in claim 3 to perform amplification treatment on the DNA of the strain to be detected, so as to obtain the Lactobacillus gasseri as claimed in claim 1 or 2.

15. The method according to claim 14, wherein The product of the amplification treatment has the nucleotide sequence shown in SEQ ID NO:8, which is an indication that the strain to be detected is the Lactobacillus gasseri as claimed in claim 1 or 2.

16. The method according to claim 14, wherein It includes the following steps: extracting a DNA sample from the strain to be detected, performing PCR amplification on the DNA sample using the primer set described in claim 3, and then comparing the amplification result with the molecular marker described in claim 11, so as to identify whether the strain to be detected is Lactobacillus gasseri described in claim 1 or 2.

17. Use of the molecular marker described in claim 11 for identifying and detecting Lactobacillus gasseri described in claim 1 or 2.

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