Lactobacillus gasseri, molecular marker thereof and use thereof

Abstract

Provided are a Lactobacillus gasseri strain, a molecular marker thereof and the use thereof. The molecular marker has a nucleotide sequence selected from one of the following nucleotide sequences: (1) a nucleotide sequence shown as SEQ ID NO: 8; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown as SEQ ID NO: 8; and (3) a nucleotide sequence obtained by means of one or more, for example, 1, 2, 3, 4, 5 or a plurality of nucleotide substitutions, deletions or insertions on the nucleotide sequence shown as SEQ ID NO: 8.

Description

Lactobacillus gasseri and its molecular markers and uses Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to Lactobacillus gasseri and its molecular marker and use. Background Art

[0002] With economic development and rising living standards, the human disease landscape has undergone significant changes. The prevalence of female reproductive tract diseases has become increasingly prominent, with vaginitis being particularly prevalent, severely impacting women's quality of life and physical and mental health. Among vaginal infections, bacterial vaginosis (BV) is the most common. Epidemiological surveys show that the incidence of BV in my country ranges from 4.96% to 36.00%. The primary cause of this disease is a decrease in the dominant Lactobacillus bacteria that maintain the normal acidic vaginal environment, while increasing the abundance of various anaerobic microorganisms. The dominant Lactobacillus bacteria are replaced by Gardnerella vaginalis and mixed anaerobic bacteria. Gardnerella vaginalis is one of the most common pathogens of BV in women. The typical clinical feature of bacterial vaginosis is a foul-smelling, watery, gray vaginal discharge. Patients may also develop numerous complications due to the infection, including chronic cervicitis, pelvic inflammatory disease, endometritis, and even infertility. Vulvovaginal candidiasis (VVC), commonly known as candidal vaginitis, is a vaginal infection second only to bacterial vaginosis in incidence. The dominant organism is often Candida albicans. Studies have found that approximately 78% of women experience at least one episode of VVC in their lifetime, 65% experience three or fewer annual episodes, and 35% experience four or more annual episodes. VVC typically presents with vulvar itching, burning, painful urination, and a dreg-like vaginal discharge, often leading to a decline in patients' quality of life and a significant economic burden. The pathogenesis of VVC is complex and is considered a multifactorial disease, with vaginal microbial imbalance, estrogen levels, host susceptibility factors, genetic predisposition, and Candida morphology all contributing to its development. Imbalances in the vaginal microbiome are closely linked to the development of these two vaginal infections.

[0003] The vaginal microbiome differs from the microbiome of other parts of the body. The lower the diversity of a healthy vaginal microbiome, the better it maintains vaginal health. The female vagina is a closed body cavity, home to numerous bacteria. Because air is absent deep within the vagina, parasites are primarily facultative anaerobes and anaerobic bacteria. Over 50 species of microorganisms in a healthy woman's vagina are parasitic. Normally, the ratio of anaerobic to aerobic bacteria is approximately 10:1, with Lactobacillus species having the highest proportion, playing a crucial role in maintaining vaginal microecological balance. Under normal circumstances, Lactobacillus species coexist with other microorganisms in a state of microecological equilibrium. However, if various factors cause Lactobacillus species to lose their dominance and pathogenic and opportunistic pathogens to increase, corresponding changes in the enzyme profile disrupt the microecological balance, leading to disease. Lactobacillus species maintain vaginal microecological stability through multiple mechanisms: fermentation of intraepithelial glycogen to produce lactic acid, maintaining an acidic vaginal pH; competition with pathogens for adhesion to vaginal epithelial cells; production of the broad-spectrum antimicrobial factor H2O2; and stimulation of the immune system by Lactobacillus species and their metabolites, which inhibit the growth and reproduction of pathogens.

[0004] Currently, the treatment of BV is mainly based on Western medicine, with the use of antibacterial drugs such as antibiotics, including metronidazole, clindamycin, and tinidazole. However, the production and persistence of pathogenic biofilms can cause recurrence of the disease. According to the "Guidelines for the Diagnosis and Treatment of 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% 12 months after treatment. By the 12th month after treatment, 84% of patients had abnormal vaginal flora. In addition, long-term and excessive use of antibiotics not only increases the resistance of pathogens but also inhibits the reproduction of some vaginal flora, allowing the previously small number of Candida albicans to proliferate, causing vaginal flora disorder. Currently, the role of live bacterial preparations in the treatment of BV is still controversial. Relevant clinical studies have shown that the current application of live bacterial preparations is mainly divided into two types: the use of probiotics after conventional antibiotic treatment or the use of probiotics alone. The results of multiple clinical trials of combination therapy have shown inconsistent results, and it is unclear whether antibiotics combined with probiotics can be used to treat bacterial vaginosis. Other studies have shown that some patients fail to achieve satisfactory therapeutic effects using probiotic preparations alone. Therefore, there is still considerable controversy regarding the effectiveness of probiotics in treating BV, and extensive research is still needed. Currently, the only marketed probiotic for the treatment of bacterial vaginosis is the Lactobacillus Live Vaginal Capsule (Dingjunsheng) developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. For the treatment of VVC, the most commonly used clinical medications are azoles and polyenes, such as diazepam suppositories, which are often used to treat candidal vaginosis. However, with the long-term use of clinical antifungal drugs, Candida albicans undergoes phenotypic changes, virulence factor mutations, and an increase in non-albicans Candida infections, posing challenges to the therapeutic efficacy of traditional antifungal drugs. In the treatment of various types of VVC patients, Lactobacillus can be used as an adjunctive therapy in combination with antibacterial drugs such as azoles, with some efficacy. However, compared with conventional antifungal treatment, there is currently insufficient evidence to demonstrate that the use of Lactobacillus preparations alone is effective in treating VVC.

[0005] Therefore, there is an urgent need for a lactobacillus that has good cell adhesion and the ability to inhibit the growth of multiple pathogens and inhibit biofilm formation, so as to be able to treat vaginal infections. Summary of the Invention

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

[0007] Therefore, the inventors isolated a strain from the vaginal secretions of a healthy woman of childbearing age in Zhejiang Province. The strain was identified as Lactobacillus gasseri (Lactobacillus gasseri HY1124). Experiments found that the strain can produce lactic acid and hydrogen peroxide (H2O2). Lactic acid is an antibacterial substance that can compete with pathogenic bacteria for nutrients and adhesion sites, thereby improving the host mucosal immunity and anti-infection ability. H2O2 can directly kill pathogens and can also be 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 pathogens, improve host mucosal immunity and anti-infection, 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 proposes a molecular marker for Lactobacillus gasseri. According to an embodiment of the present invention, the molecular marker has a nucleotide sequence selected from one of the following: (1) a nucleotide sequence as 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. Therefore, the molecular marker of the present invention can not only quickly and easily screen for the target Lactobacillus gasseri, but also can highly specifically identify the target Lactobacillus gasseri, eliminating the interference of other strains.

[0009] According to an embodiment of the present invention, the molecular marker has a nucleotide sequence as shown in SEQ ID NO:8.

[0010] In a second aspect of the present invention, the present invention provides a Lactobacillus gasseri. According to an embodiment of the present invention, the Lactobacillus gasseri comprises the molecular marker described in the first aspect.

[0011] According to an embodiment of the present invention, the Lactobacillus gasseri was deposited in the General Microbiology Center of the China Culture Collection Administration on February 7, 2023, with a deposit number of CGMCC No. 26504.

[0012] According to an embodiment of the present invention, the 16S rDNA of Lactobacillus gasseri has a nucleotide sequence of one of the following: (1) a nucleotide sequence as 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, for example, 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 1.

[0013] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus gasseri has the nucleotide sequence shown in SEQ ID NO: 1.

[0014] In a third aspect of the present invention, the present invention provides a Lactobacillus gasseri (Lactobacillus gasseri HY1124). According to an embodiment of the present invention, the Lactobacillus gasseri was deposited on February 7, 2023 at the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 26504, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. As mentioned above, the inventors isolated the strain from the vaginal secretions of a healthy woman of childbearing age in Zhejiang Province. After further identification of the biochemical characteristics of the strain, it was found that the bacteria can produce lactic acid and hydrogen peroxide H2O2. Lactic acid is an antibacterial substance that can compete with pathogenic bacteria for nutrients and adhesion sites, thereby improving the host mucosal immunity and anti-infection ability. H2O2 has the ability to directly kill pathogens and can also be oxidized by biological enzymes to form halides with stronger bactericidal effects. In addition, the bacteria has a strong inhibitory effect on the pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA) and Prevotella bivia (PB), and also has a strong inhibitory effect on Candida albicans. albicans (CA) also has a certain inhibitory effect. Therefore, this Lactobacillus gasseri has the potential to maintain a weakly acidic environment in the vagina, kill pathogens, enhance host mucosal immunity and anti-infection, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infection and treat inflammation.

[0015] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus gasseri has the nucleotide sequence shown in SEQ ID NO: 1.

[0016] In a fourth 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 a nucleotide sequence that is one of the following: (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, for example, 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 a strong inhibitory effect 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 pathogens, enhance host mucosal immunity and anti-infection, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infection and treat inflammation.

[0017] According to an embodiment of the present invention, the 16S rDNA of the Lactobacillus gasseri comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0018] According to an embodiment of the present invention, the Lactobacillus gasseri described in the second aspect, the third aspect or the fourth aspect further includes at least one of the following additional technical features:

[0019] According to an embodiment of the present invention, the gene sequence of the Lactobacillus gasseri further comprises a nucleotide sequence selected from the following: (1) a nucleotide sequence as shown in SEQ ID NO: 8; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence as shown in SEQ ID NO: 8; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4 or 5 nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 8. Therefore, the Lactobacillus gasseri of the present invention can be specifically identified by the above nucleotide sequence.

[0020] According to an embodiment of the present invention, the gene sequence of Lactobacillus gasseri further comprises a nucleotide sequence as shown in SEQ ID NO:8.

[0021] 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 culture.

[0022] 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, and 6.5 g / L of lactic acid under effective culture.

[0023] According to a specific embodiment of the present invention, the Lactobacillus gasseri is capable of producing at least 1100-1400 μM hydrogen peroxide under effective culture.

[0024] 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, and 1400 μM hydrogen peroxide under effective culture.

[0025] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 85-90% against GV.

[0026] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 85%, 86%, 87%, 88%, 89%, or 90% against GV.

[0027] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 98-100% against PB.

[0028] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 98%, 99%, or 100% against PB.

[0029] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 98-100% against EC.

[0030] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 98%, 99%, or 100% against EC.

[0031] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 95-100% against SA.

[0032] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 95%, 96%, 97%, 98%, 99%, or 100% against SA.

[0033] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 55-60% against CA.

[0034] According to a specific embodiment of the present invention, the Lactobacillus gasseri has an inhibition rate of at least 55%, 56%, 57%, 58%, 59%, or 60% against CA.

[0035] In the fifth aspect of the present invention, the present invention proposes a primer set for detecting the Lactobacillus gasseri described in the second aspect, the third aspect, or the fourth 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. The primer set described in the present invention can be highly matched with the DNA sequence of the Lactobacillus gasseri described in the second aspect, the third aspect, or the fourth aspect, and can only produce specific amplification in the Lactobacillus gasseri. This enables the primers to accurately and reliably identify the Lactobacillus gasseri, eliminating interference from other non-target strains.

[0036] In a sixth aspect, the present invention provides a microbial preparation. According to embodiments of the present invention, the microbial preparation comprises the Lactobacillus gasseri described in the second, third, or fourth aspects. As previously mentioned, the inventors of the present invention have isolated a new Lactobacillus gasseri that exhibits strong inhibitory effects against pathogenic bacteria GV, EC, SA, and PB, and also exhibits a certain inhibitory effect against CA. Therefore, this Lactobacillus gasseri can be formulated into a microbial preparation, making it convenient for patients to take.

[0037] According to an embodiment of the present invention, the microbial preparation further includes a pharmaceutically acceptable carrier or excipient.

[0038] In the seventh aspect of the present invention, the present invention provides a single-dose formulation. According to an embodiment of the present invention, comprising 1×10 6 ~1×10 10 The Lactobacillus gasseri described in the second, third or fourth aspects of CFU is used as an active ingredient.

[0039] According to an embodiment of the present invention, comprising 10 6 CFU, 10 7 CFU, 10 8 CFU, 10 9 CFU, 10 10 The Lactobacillus gasseri described in the second, third or fourth aspects of CFU is used as an active ingredient.

[0040] According to an embodiment of the present invention, including 1×10 6 ~1×109 The Lactobacillus gasseri described in the second, third or fourth aspects of CFU is used as an active ingredient.

[0041] In the eighth aspect of the present invention, the present invention proposes the use of the Lactobacillus gasseri described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the preparation of a medicament for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

[0042] In the ninth aspect of the present invention, the present invention proposes the use of the Lactobacillus gasseri described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the preparation of a medicament for inhibiting vaginal pathogens.

[0043] According to an embodiment of the present invention, the use described in the eighth aspect or the ninth aspect further includes at least one of the following additional technical features:

[0044] According to an embodiment of the present invention, the vaginal pathogenic bacteria is selected from at least one of Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA), Prevotella bivia (PB), and Candida albicans (CA).

[0045] According to an embodiment of the present invention, the vaginal pathogenic bacteria is selected from at least one of Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA), and Prevotella bivia (PB).

[0046] According to an embodiment of the present invention, the related diseases caused by vaginal bacterial infection include bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, Trichomonas vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection or pelvic inflammatory disease.

[0047] In the tenth aspect of the present invention, the present invention proposes the use of the Lactobacillus gasseri described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the preparation of a product for regulating the balance of vaginal flora.

[0048] According to an embodiment of the present invention, the product includes at least one of a medicine and an external health care product.

[0049] In an eleventh aspect of the present invention, the present invention provides a molecular marker for the Lactobacillus gasseri of the third aspect or the fourth aspect. According to an embodiment of the present invention, the molecular marker comprises a nucleotide sequence selected from one of the following: (1) a nucleotide sequence as shown in SEQ ID NO: 8; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence as shown in SEQ ID NO: 8; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 8.

[0050] According to an embodiment of the present invention, the molecular marker has a nucleotide sequence as shown in SEQ ID NO:8.

[0051] According to an embodiment of the present invention, the primers for molecular markers include the primer set described in the fifth aspect.

[0052] In a twelfth aspect of the present invention, a method for detecting the Lactobacillus gasseri described in the second, third, or fourth aspects is provided. According to an embodiment of the present invention, the method comprises: using the primer set described in the fifth aspect to amplify the DNA of the strain to be detected, and screening to obtain the Lactobacillus gasseri described in the second, third, or fourth aspects. As described above, the primer set described in the fifth aspect is a specific primer for the Lactobacillus gasseri described in the present invention, can highly match the DNA sequence of the Lactobacillus gasseri described in the second, third, or fourth aspects, and can only produce specific amplification in the Lactobacillus gasseri strain. Using the method of the present invention, the Lactobacillus gasseri of the present invention can be efficiently and conveniently identified and detected.

[0053] According to an embodiment of the present invention, the product of the amplification treatment comprises the molecular marker described in the first or eleventh aspect, indicating that the strain to be detected is the Lactobacillus gasseri described in the second, third, or fourth aspect. The nucleotide sequence of the molecular marker described in the first or eleventh aspect can be amplified using the primer set described in the fifth aspect. The molecular marker described in the first or eleventh aspect is a specific nucleotide sequence marker for the Lactobacillus gasseri of the present invention, providing a reliable tool and basis for identifying and detecting the Lactobacillus gasseri of the present invention.

[0054] According to an embodiment of the present invention, the method for detecting the Lactobacillus gasseri described in the second, third, or fourth aspects comprises 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 fifth aspect, and then comparing the amplification result with the molecular marker described in the first or eleventh aspect, thereby identifying whether the strain to be detected is the Lactobacillus gasseri described in the second, third, or fourth aspects. The method of the present invention can efficiently and conveniently identify and detect the Lactobacillus gasseri of the present invention.

[0055] In the thirteenth aspect of the present invention, the present invention proposes use of the molecular marker described in the first aspect or the eleventh aspect in identifying and detecting the Lactobacillus gasseri described in the second aspect, the third aspect or the fourth aspect.

[0056] In the fourteenth aspect of the present invention, the present invention proposes the use of the Lactobacillus gasseri described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in inhibiting the growth of vaginal pathogens.

[0057] In the fifteenth aspect of the present invention, the present invention proposes the use of the Lactobacillus gasseri described in the second, third or fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

[0058] According to an embodiment of the present invention, the vaginal pathogenic bacteria is selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Prevotella diversifolia, and Candida albicans.

[0059] According to an embodiment of the present invention, the related diseases caused by vaginal pathogenic bacteria infection include at least one of bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, Trichomonas vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, and pelvic inflammatory disease.

[0060] In a sixteenth aspect, the present invention provides a method for inhibiting the growth of vaginal pathogens. According to an embodiment of the present invention, the method comprises: contacting the vaginal pathogens with at least one of the Lactobacillus gasseri described in the second, third, or fourth aspects, the microbial preparation described in the sixth aspect, or the single-dose preparation described in the seventh aspect.

[0061] In its seventeenth aspect, the present invention provides a method for treating and / or preventing vaginal pathogen infection or diseases caused by vaginal pathogen infection. According to an embodiment of the present invention, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the Lactobacillus gasseri described in the second, third, or fourth aspects, the microbial preparation described in the sixth aspect, or the single-dose preparation described in the seventh aspect.

[0062] According to an embodiment of the present invention, the vaginal pathogenic bacteria is selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Prevotella diversifolia, and Candida albicans.

[0063] According to an embodiment of the present invention, the related diseases caused by vaginal pathogenic bacteria infection include at least one of bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, Trichomonas vaginitis, mixed vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, and pelvic inflammatory disease.

[0064] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] 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:

[0066] FIG1 is a colony morphology diagram of Bacillus subtilis HY1124 according to an embodiment of the present invention;

[0067] FIG2 is a bacterial morphology diagram of Lactobacillus gasseri HY1124 according to an embodiment of the present invention;

[0068] FIG3 is an electrophoretic pattern of PCR amplification of four Lactobacillus gasseri by primer 1-p1 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY1124 as a template, 2 uses the supernatant after lysis of HY02772 as a template, 3 uses the supernatant after lysis of HY09235 as a template, and 4 uses the supernatant after lysis of HY11276 as a template;

[0069] FIG4 is an electrophoretic pattern of PCR amplification of four Lactobacillus gasseri by primer 2-p1 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY1124 as a template, 2 uses the supernatant after lysis of HY02772 as a template, 3 uses the supernatant after lysis of HY09235 as a template, and 4 uses the supernatant after lysis of HY11276 as a template;

[0070] FIG5 is a graph showing the results of a hemolysis experiment on Lactobacillus gasseri HY1124 according to an embodiment of the present invention;

[0071] FIG6 is a diagram showing the results of a BV efficacy animal experiment according to an embodiment of the present invention;

[0072] FIG. 7 is a diagram showing the results of an animal experiment on the efficacy of VVC according to an embodiment of the present invention. DETAILED DESCRIPTION

[0073] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0074] Definition of terms

[0075] In the present invention, the term "Lactobacillus gasseri" or "L. gasseri" generally refers to a species of the genus Lactobacillus. This species is generally distinguished from other lactobacilli based on the polynucleotide sequence of the ribosomal 16S rDNA gene.

[0076] In the present invention, the term "effective culture conditions" generally refers to the environment in which Lactobacillus gasseri is placed or exposed to promote the growth of the bacteria. Therefore, the term generally refers to the culture medium, temperature, atmospheric conditions, substrate, agitation conditions, etc. that can affect the growth of the bacteria.

[0077] In the present invention, the term "antibiotic-sensitive" refers to a bacterium's weak resistance to antibiotics, where a small amount of antibiotics can affect its normal growth. According to an embodiment of the present invention, the Lactobacillus gasseri HY1124 is moderately sensitive to clindamycin and ciprofloxacin, and sensitive to cefuroxime.

[0078] In the present invention, the term "vagina" generally refers to the vaginal area or surrounding area, including the labia, vulva, cervix, uterus, fallopian tubes, ovaries, urethra, bladder, anus and rectum, including their mucosal tissues.

[0079] In the present invention, the terms "disease" or "disorder" are used interchangeably and generally refer to any change in the state of the body or certain organs that prevents or disrupts the performance of functions and / or causes symptoms such as discomfort, dysfunction, suffering or even death in the person suffering from the disease or being exposed to it.

[0080] In the present invention, the term "pathogenic" (e.g., "pathogenic bacteria") generally refers to substances, microorganisms, or conditions that can cause disease. In some situations, pathogens also include microorganisms (e.g., bacteria) that are associated with a disease or condition, but for which a causal relationship (e.g., a direct causal relationship) has not yet been established or is yet to be established. In some embodiments, microorganisms that are not pathogens and can be symbionts can cause or be associated with disease or dysbiosis, depending on various factors (e.g., the immune status of the site, the abundance of microbial taxa, etc.). Such microorganisms are referred to as "pathogenic organisms."

[0081] In the present invention, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms that colonize the vagina.

[0082] In the present invention, the term "inhibit" generally refers to the process of inhibiting or hindering the growth and reproduction of bacteria and their activity.

[0083] In the present invention, the term "isolated" as applied to a nucleic acid or protein generally means that the nucleic acid or protein is substantially free of other cellular components with which it is naturally associated. For example, it can be in a homogeneous state and can be in dry solution or in aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0084] In the present invention, the term "isolated" as applied to bacteria generally refers to bacteria that have been (1) separated from at least some of the components with which they were associated when originally produced (whether in nature or in an experimental setting), and / or (2) artificially produced, prepared, purified and / or manufactured, for example, using artificial culture conditions, such as (but not limited to) culturing on plates and / or in fermenters. Isolated bacteria include those that have been cultured, even if such cultures are not monocultures. 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 originally 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 an embodiment of the present invention, the bacterial population provided by the present invention includes isolated bacteria.

[0085] In the present invention, the term "pharmaceutically acceptable carrier" generally refers to a substance that facilitates administration of an active agent to a subject and is absorbed by the subject and that can be included in the Lactobacillus gasseri of the present invention without causing significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline solution, lactated Ringer's solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, spices, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and dyes. Such preparations can be sterilized, and those skilled in the art will recognize that other pharmaceutical carriers can also be used in the present invention.

[0086] In the present invention, the term "CFU (Colony-Forming Units)" generally refers to the total number of microorganisms such as bacteria, fungi, yeast, etc. in a product, and is usually used for calculation of viable bacteria.

[0087] In the present invention, the term "CFU / dose" means the amount of bacteria present in a food or dietary supplement / drug provided to a subject each day or each time. For example, in certain embodiments, the amount of Lactobacillus gasseri present in the food or dietary supplement is 10 6 to 10 10 The amount of CFU / dose present (e.g. 10 6 to 10 9 In this embodiment, if Lactobacillus gasseri is administered in a food product (e.g., a solid beverage, yogurt), the food product (e.g., a solid beverage, yogurt) provided to the subject each day or each time may contain about 10 CFU / dose. 6 to 10 10 Alternatively, of course, this amount of bacteria can be divided into multiple administrations, so long as the total amount of Lactobacillus gasseri received by the subject in any particular time (e.g., per 24-hour period) is from about 10 6 to about 10 10 CFU of bacteria, that is, Lactobacillus gasseri in food products or dietary supplements that meet the above requirements, is 10 6 to 10 10 The amount of CFU / dose present (e.g. 10 6 to 10 9 CFU / dose).

[0088] In the present invention, the term "treating and / or preventing" includes not only treating and / or preventing a disease, but also generally includes preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing 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 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 the patient being treated. It is not necessary for the Lactobacillus gasseri of the present invention to achieve a complete cure or eradication of any symptoms or manifestations of the disease in order for the Lactobacillus gasseri to constitute a viable therapeutic agent. As recognized in the relevant art, drugs used as therapeutic agents can reduce the severity of a given disease state, but do 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 the condition in order for it to constitute a viable preventative agent. It is sufficient to simply reduce the effects of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of the disease occurring or worsening.

[0089] In the present invention, the term "about" generally refers to a variation within the range of about 0.5%-10% above or below the specified value, for example, a variation within the 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.

[0090] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0091] Example 1 Screening and Isolation of Lactobacillus gasseri

[0092] (1) Collect samples

[0093] The sources of samples are naturally fermented foods with regional characteristics, vaginal secretion samples collected by hospital gynecologists from volunteers who meet the inclusion criteria, and human samples (feces and breast milk).

[0094] (2) Strain isolation

[0095] The sample was serially diluted tenfold with physiological saline, and appropriate dilution gradients were plated onto anaerobic blood plates (purchased from Huankai Microbiology). The plates were then incubated at 37°C in an anaerobic workstation for 48-72 hours. Individual colonies of varying morphology were streaked onto the blood plates for purification. Further incubation was performed, and pure cultures were selected for strain identification (16S rDNA sequencing). Through identification and screening, a strain of Lactobacillus gasseri was isolated from the vaginal secretion sample and named Lactobacillus gasseri HY1124.

[0096] After the strain species was determined, the pure culture (i.e., Lactobacillus gasseri HY1124) was inoculated into MRS broth liquid culture medium for expansion. When the strain grew to an appropriate concentration, sterile 50% glycerol solution was used to mix the liquid culture with the same volume and stored in a -80°C strain bank.

[0097] Example 2 Identification of Lactobacillus gasseri

[0098] (1) Colony characteristics

[0099] Dip the inoculating loop into the culture tube and streak onto an MRS plate. Incubate anaerobically at 37°C for 48 hours and observe the colony morphology. As shown in Figure 1, the strain forms round, off-white colonies with rough edges after incubation on the MRS plate.

[0100] (2) Staining microscopy

[0101] Use an inoculating loop to transfer one loop of sterile distilled water to a clean glass slide. Pick a single colony from the MRS plate, mix it with distilled water, and evenly spread it on the slide. Stain according to the instructions of the Gram stain kit (purchased from Qingdao Haibo), and then observe the bacterial morphology under an electron microscope. As shown in Figure 2, the stained strain was observed under an optical microscope and was Gram-positive. The bacteria were rod-shaped, with short and long rods.

[0102] (3) Biochemical identification and analysis

[0103] ① Lactobacillus culture: The target strain was inoculated into MRS broth medium (purchased from Qingdao Haibo), placed in an anaerobic incubator, and cultured at 37°C for 24 hours.

[0104] ② Preparation of bacterial suspension: centrifuge the fermentation liquid of the strain at 4000 rpm for 5 min in a centrifuge, remove the supernatant, wash the bacterial mud with physiological saline, centrifuge at 4000 rpm for 5 min, remove the supernatant, wash the bacterial mud with physiological saline again, centrifuge at 4000 rpm for 5 min, remove the supernatant, add physiological saline and the bacterial mud and mix evenly for later use.

[0105] ③1% sodium hippurate identification test: aspirate 50 μL of bacterial suspension, add it to 1% sodium hippurate identification tube, seal it with sealing film and incubate it in a 37°C water bath for 2 hours, then slowly add 200 μL of ninhydrin solution (3.5% ninhydrin solution: 0.175 g of hydrated ninhydrin, 2.5 mL of acetone, and 2.5 mL of butanol) along the wall of the tube. Do not shake it, place it in a 37°C water bath for 10 minutes and then read the results.

[0106] ④ Other identification experiments: Pipette 50 μL of bacterial suspension and add it to the biochemical identification tube (Note: After adding the bacterial solution, the liquid surface of the aesculin identification tube needs to be covered with sterile liquid paraffin), seal it with sealing film, place it in an anaerobic incubator, and incubate it at 37°C for 48 hours before interpreting the results. The interpretation results are shown in Table 1:

[0107] Table 1 Interpretation results of Lactobacillus gasseri identification tube Note: + represents positive; - represents negative; +w represents weak positive.

[0108] The results of biochemical identification showed that Lactobacillus gasseri could utilize all nine carbon sources (esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin, and lactose) except raffinose and 1% sodium hippurate.

[0109] (4) 16S rDNA identification

[0110] The above-mentioned Lactobacillus gasseri was amplified and sequenced by 16S rDNA. After obtaining the sequence, a BLAST comparison was performed in the NCBI database. The 16S rDNA comparison results showed that the above-mentioned strain was Lactobacillus gasseri (Lactobacillus gasseri), and was named Lactobacillus gasseri HY1124. The 16S rDNA gene sequence was determined as follows:

[0111] (5) Specific nucleotide sequence molecular markers

[0112] ①Specific primer design

[0113] a. Specific nucleotide sequence screening: The HY1124 strain was subjected to whole genome sequencing and genome analysis, and compared with the genome sequence of Lactobacillus gasseri strains included in the NCBI database to screen for the specific nucleotide sequence of Lactobacillus gasseri HY1124.

[0114] b. Primer Design: Design primers for the specific nucleotide sequences screened in step a. Design primer fragments with predicted product lengths of approximately 200 to 800 bp for the screened specific nucleotide sequences. Also design two pairs of primers for other specific nucleotide sequences across the genome as controls. See Table 2 for primer sequences:

[0115] Table 2 Primer list

[0116] c. Primer Screening: Template preparation was performed using Lactobacillus gasseri HY1124 and three strains of the same species as controls (see Table 3 for specific strain information). Single colonies were picked from each strain in 50 μL of lysis buffer (TaKaRa), lysed at 80°C for 15 minutes, and centrifuged at 4000 rpm for 5 minutes. The supernatant was used as the template. Primer screening was performed, and 1-p1 was selected for PCR amplification. The PCR amplification system consisted of 12.5 μL of Taq enzyme, 1 μL of F, 1 μL of R, 1.5 μL of template, and ddHO to 25 μL. PCR reaction conditions are shown in Table 4. After the PCR experiment, 1.5 g of agarose was added to 100 mL of 1× TAE buffer and heated thoroughly to melt. 5 μL of Gel Red dye was added and the sample was poured onto a gel plate to solidify. 4 μL of the PCR product was subjected to agarose gel electrophoresis under the following conditions: voltage 130 V, current 400 mA, and time 35 minutes.

[0117] Table 3 Strain information

[0118] Note: The strains numbered HY02772, HY09235, and HY11276 in Table 3 are Lactobacillus gasseri 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 to Lactobacillus gasseri HY1124, PCR amplification was performed on HY1124 and three strains of the same species using the above-mentioned primers.

[0119] Table 4 PCR reaction conditions

[0120] Among the primers designed for specific nucleotide sequences, PCR amplification experiments were performed using 1-p1 as a primer. Only HY1124 had a single amplified band, and the band was clear and high in concentration, with relatively few primer dimers, while HY02772, HY09235, and HY11276 all had no amplified bands, and the agarose gel electrophoresis detection pattern was shown in Figure 3. When PCR amplification experiments were performed using primers designed with other nucleotide sequences, the control bacteria group also amplified bands (taking 2-p1 as an example, the agarose gel electrophoresis detection pattern was shown in Figure 4). Therefore, it was shown that primer 1-p1 was a molecular marker primer for HY1124, and the amplified product produced by this primer was a molecular marker for Lactobacillus gasseri HY1124. The amplified product of the 1-p1 primer was 800bp in size, and the nucleotide sequence was as shown in SEQ ID NO:8. Thus, it was determined that the nucleotide sequence shown in SEQ ID NO:8 was part of the specific nucleotide sequence screened in step a, further verifying that primer 1-p1 can be used as a specific primer for Lactobacillus gasseri HY1124, and the nucleotide shown in SEQ ID NO:8 is a specific nucleotide sequence marker for Lactobacillus gasseri HY1124, which provides a reliable tool and basis for the identification and detection of Lactobacillus gasseri HY1124.

[0121] d. During the experiment, the inventors discovered that when sequencing multiple Lactobacillus gasseri HY1124 samples using the 1-p1 primer, the amplified product sequences differed from the nucleotide sequence shown in SEQ ID NO: 8 at individual bases. However, compared with the control bacteria, all of the amplified products had specific, clear, and high-concentration amplified bands, and the primer dimers were relatively rare.

[0122] In one 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, which aligned 99.87% with the nucleotide sequence shown in SEQ ID NO: 8.

[0123] 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 that is at least 97%, 98%, 99% or higher homologous to the nucleotide sequence shown in SEQ ID NO: 8.

[0124] Example 3 Bacterial characteristics

[0125] (1) Antibiotic sensitivity test

[0126] Lactobacillus gasseri HY1124 was cultured in MRS broth, and the culture solution was evenly spread on MRS plates. After the solution was absorbed and dried, antibiotic susceptibility paper was applied and incubated anaerobically at 37°C for 48 hours. The diameter of the inhibition zone was measured using a vernier caliper. The antibiotic susceptibility of the strain was determined by the ring diameter on the antibiotic susceptibility paper. 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.

[0127] Table 5 Antibiotic sensitivity test results Note: S: sensitive (15-20mm high sensitivity; >20mm extremely sensitive) I: intermediate (10-14mm moderately sensitive) R: resistant (<10mm insensitive)

[0128] (2) Toxicity test

[0129] ①Hemolysis test

[0130] The bacterial solution from the frozen tube was streaked onto an anaerobic blood plate. The plate was then incubated anaerobically at 37°C for 48 hours, and the color change of the blood plate surrounding the colonies was observed. The results of the hemolysis test are shown in Figure 5. Small, off-white colonies appeared in the culture medium surrounding the colonies, and no hemolytic rings appeared around the colonies, indicating that Lactobacillus gasseri HY1124 is gamma-hemolytic, meaning it is non-hemolytic.

[0131] ②Toxicity test in mice

[0132] Five mice weighing 18-22 g were used, and each mouse was orally gavaged with 0.5 ml of fresh bacterial solution (no less than 1.0 × 10 9 CFU / 0.5 ml) once daily for 3 consecutive days. The mice were observed for survival and weight from day 1 to day 7. The experimental results showed that all mice survived and gained weight after oral gavage with fresh Lactobacillus gasseri HY1124 solution.

[0133] (3) Determination of metabolite content

[0134] ①D-lactic acid detection

[0135] The D-lactic acid production of the lactobacillus supernatant was detected using a D-lactic acid detection kit (purchased from Sigma-Aldrich). The method used in this kit is as follows: D-lactic acid is oxidized by a specific D-lactate hydrogenase, producing a color reaction that is proportional to the D-lactic acid concentration, and the absorbance at 450 nm is measured.

[0136] ②L-lactic acid detection

[0137] The supernatant was filtered through a 0.22 μm sterile filter membrane and the L-lactic acid concentration was measured using a biosensor.

[0138] ③ Hydrogen peroxide detection

[0139] Prepare 100 μl of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 mM hydrogen peroxide standard solutions in PBS. Add 200 μl of enzyme reagent (0.5 mM / L 4-aminoantipyrine (4-AA), 0.2 mM / L peroxidase) and 200 μl of phenol solution (19.66 mM / L). Mix thoroughly and incubate at 37°C for 20 minutes. Dispense 200 μl evenly into a 96-well plate and measure the absorbance of each well at 505 nm. Use the test results to construct a hydrogen peroxide solution standard curve.

[0140] Inoculate Lactobacillus into MRS medium at a 0.5% inoculum volume and culture at 37°C for 24 hours. When the Lactobacillus is cultured for 24 hours, centrifuge at 4000 rpm for 20 minutes at 4°C, discard the supernatant, wash 1-2 times with PBS buffer, and resuspend the cells in 0.5% glucose solution to an OD of 0. 600 = 1.0. Incubate at 37°C and 170 rpm for 5 hours. Centrifuge at 10,000 rpm for 5 minutes, collect the supernatant, and sterilize by 0.22 μm filtration. Add 200 μL each of enzyme reagent and phenol reagent to 100 μL of the supernatant, mix thoroughly, and incubate at 37°C for 20 minutes. Dispense 200 μL evenly into a 96-well plate and measure the absorbance of each well at 505 nm.

[0141] Lactobacillus gasseri HY1124 was cultured in MRS broth for 24 h, and the test results of D-lactic acid, L-lactic acid and hydrogen peroxide were shown in Table 6. Organic acids such as D-lactic acid and L-lactic acid are antibacterial substances that can compete with pathogenic bacteria for nutrients and adhesion sites, thereby improving the host mucosal immunity and anti-infection ability. The content of D-lactic acid and L-lactic acid in the metabolites of Lactobacillus gasseri HY1124 was 2.07 g / L and 3.9 g / L, respectively. The total acid production of Lactobacillus gasseri HY1124 was 5.97 g / L, while the total acid production of DJS-Lactobacillus delbrueckii was only 5.48 g / L. The acid production of Lactobacillus gasseri HY1124 was higher than that of DJS-Lactobacillus delbrueckii. H2O2 can directly kill pathogens and can also be 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 DJS-Lactobacillus delbrueckii is only 80.67 μM. The ability of Lactobacillus gasseri HY1124 to produce hydrogen peroxide is much higher than that of DJS-Lactobacillus delbrueckii. Therefore, Lactobacillus gasseri HY1124 has the potential to kill pathogens, enhance host mucosal immunity and anti-infection, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0142] Table 6 Metabolite content determination results

[0143] Example 4 Application Function Analysis

[0144] (1) Antibacterial experiment

[0145] 1. Preparation of working bacterial solution: Lactobacillus was inoculated into MM medium (MRS broth modified medium, composed 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 tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) at a 0.5% inoculum size and cultured in an anaerobic workstation. Five pathogenic bacteria (Gardnerella vaginalis ATCC14018, abbreviated as GV; Escherichia coli ATCC 25922, abbreviated as EC; Staphylococcus aureus ATCC 25923, abbreviated as SA; Prevotella bivia NCTC 11156, abbreviated as PB; Canidia albicans ATCC 10231, abbreviated as CA) were cultured in an adapted culture medium. After the target strains were cultured, the supernatant was centrifuged and filtered through 0.22 μm to obtain a cell-free supernatant, which was used immediately or stored in a -80°C refrigerator. OD values ​​were measured after the pathogens were cultured. 600 value, diluted to about OD 600 The value is 0.005 (the number of viable bacteria is maintained at 5.0×10 5 CFU / mL~5.0×10 6 CFU / mL) (WS / T 650-2019 Evaluation method for antibacterial and antibacterial effects).

[0146] ② Interaction: Take the same volume of supernatant and pathogenic bacteria solution and mix them evenly. Immediately take 100 μL of the mixed bacteria solution and place it into a blank 96-well plate to measure OD 600 The remaining culture medium was placed at 37°C and cultured anaerobically or aerobically according to the culture conditions of the pathogen. After 48 hours of culture, 100 μL of the mixed bacterial solution was taken and placed into a blank 96-well plate to measure the OD 600 A blank control group was set up, with two replicate wells for each sample, and the inhibition rate of Lactobacillus against pathogenic bacteria was calculated according to the following formula.

[0147] Antibacterial rate = (AB) / A*100%

[0148] A: OD increase in the positive control group (blank culture medium) within 48 hours 600 value;

[0149] B: OD increase in the experimental group within 48 hours 600 value.

[0150] Three different strains of the same bacterial species, along with a strain from the positive drug Dingjunsheng, were selected to test their antibacterial properties alongside the selected Lactobacillus gasseri HY1124. The results, as shown in Table 7, showed that this strain exhibited strong antibacterial activity against GV, PB, EC, SA, and CA bacteria, with the latter exhibiting significantly better antibacterial activity than the same-species control group and the positive control group.

[0151] Table 7 Antibacterial test results Note: The strains numbered HY02772, HY01880, and HY02172 in Table 7 are Lactobacillus gasseri screened from samples collected by the inventors according to Example 1, in order to verify the inhibition rates of Lactobacillus gasseri HY1124 and three strains of the same species against different pathogens.

[0152] (2) Co-culture antibacterial experiment

[0153] 1) Interaction and co-culture experiment of Lactobacillus and CA

[0154] Lactobacillus culture: Take a glycerol tube of Lactobacillus gasseri and inoculate it into MM medium (MRS broth modified medium, the composition is: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPO4 2.0g / L, ammonium citrate tribasic 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L), and concentrate or dilute the cultured bacterial liquid to about 1×10 9 CFU / mL, used as Lactobacillus working culture solution.

[0155] CA culture: Inoculate the CA glycerol tube into Sabouraud medium and culture aerobically at 37°C. Centrifuge the culture solution to remove the supernatant, and then adjust the concentration of the culture solution to about 1.0 × 10 7 CFU / mL, used as CA working bacterial solution.

[0156] 400 μL of each Lactobacillus and CA working culture solution were inoculated into 40 mL of MRS liquid medium. CA alone was inoculated as a blank control. Two replicates were performed in each group. The culture solution was gently shaken and incubated at 37°C under an anaerobic bench. Samples were taken 20 hours after incubation, and viable CA bacteria were counted using Candida albicans chromogenic medium (purchased from CHROMagar, France). The experimental results are shown in Table 8. After 20 hours of co-culture of Lactobacillus gasseri and CA, the inhibition rate reached 31.2%, indicating that this strain has a certain inhibitory effect on the growth of CA.

[0157] Table 8 Antibacterial test results of co-culture of Lactobacillus and CA

[0158] 2) Lactobacillus and GV co-culture experiment

[0159] Lactobacillus culture: Take a glycerol tube of Lactobacillus gasseri and inoculate it into MM medium (MRS broth modified medium, the composition is: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPO42.0g / L, ammonium citrate 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L), and concentrate or dilute the cultured bacterial solution to about 1×10 9 CFU / mL concentration is used as the Lactobacillus working culture solution.

[0160] GV culture: GV glycerol tubes were inoculated into BHI liquid medium containing 10% fetal bovine serum and cultured anaerobically at 37°C. The cultured bacterial solution was centrifuged to remove the supernatant, and then the bacterial solution concentration was adjusted to about 1.0 × 10 7 CFU / mL, used as GV working bacterial solution.

[0161] 400 μL of lactobacillus and GV working bacterial solution were respectively inoculated into 40 ml of BHI liquid medium containing 10% fetal bovine serum. GV was inoculated alone as a blank control group, with 2 replicates per group. As for the culture in the anaerobic workstation, samples were taken 27 hours after culture, and the viable count of GV was detected by fluorescence quantitative qPCR using GV-specific probe primers. After Lactobacillus gasseri and GV were co-cultured for 27 hours, the viable count of GV was detected. The experimental results are shown in Table 9. After Lactobacillus gasseri HY1124 was co-cultured with GV for 27 hours, the inhibition rate reached 94%, indicating that the strain had a strong inhibitory effect on the growth of GV.

[0162] Table 9 Antibacterial test results of co-culture of Lactobacillus and GV

[0163] (3) Biofilm removal experiment

[0164] 1) Lactobacillus removal of GV biofilm

[0165] After GV culture, the bacterial solution was adjusted to 1.0×10 7CFU / mL were inoculated into 96-well plates in two groups, each with four replicates. The two groups were supplemented with equal volumes of blank MM liquid medium (MRS broth modified medium, consisting of: 10 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 15 g / L glucose, 2.0 g / L K2HPO4, 1.0 g / L triammonium citrate, 2.5 g / L sodium acetate, 0.2 g / L magnesium sulfate, and 0.05 g / L manganese sulfate) and supernatant from Lactobacillus gasseri HY1124. The plates were incubated at 37°C in an anaerobic workstation for 24 hours.

[0166] After the incubation period, the inhibitory effect of Lactobacillus gasseri HY1124 on GV biofilm formation was determined using a microplate crystal violet staining method. The experimental results, shown in Table 10, show that the supernatant of Lactobacillus gasseri HY1124 had a strong inhibitory effect on GV biofilm formation, reaching 89.6%. Therefore, it can effectively inhibit the growth of GV.

[0167] Table 10 Lactobacillus removal test results of GV biofilm

[0168] 2) Lactobacillus removal of CA biofilm

[0169] Inhibition of biofilm removal experiment: The bacterial solution after CA culture was adjusted to 1.0×10 7 CFU / mL were inoculated into 96-well plates in two replicate groups, each containing four replicates. Equal volumes of Sabouraud dextrose broth and Lactobacillus supernatant were added to each group. The plates were incubated at 37°C in an anaerobic workstation for 24 hours. After incubation, the inhibitory effect of Lactobacillus on CA biofilm formation was determined using crystal violet staining in microplates.

[0170] Biofilm removal experiment: adjust the bacterial solution after CA culture to 1.0×10 7 CFU / mL, inoculated into 96-well plates, with four replicates in each group. The plates were incubated at 37°C in an anaerobic workstation for 24 hours. After 24 hours of incubation, the mixture was discarded and the wells were washed with sterile PBS. An equal volume of MM medium (MRS broth modified medium, composition: 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 tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and Lactobacillus supernatant were added to each group and incubated at 37°C for 24 hours. After incubation, the effect of the Lactobacillus supernatant on the removal of mature CA biofilms was determined using crystal violet staining in microtiter plates.

[0171] The experimental results are shown in Table 11. Compared with the ability to remove the formed biofilm, the Lactobacillus gasseri has a stronger inhibitory effect on the formation of CA biofilm, with an inhibition rate of 51.2%.

[0172] Table 11 Results of the experiment on the removal of CA biofilm by lactobacilli

[0173] (4) Cell adhesion assay

[0174] ① Preparation of working bacterial solution: culture Lactobacillus and test the OD of bacterial solution 600 The cultured Lactobacillus culture solution was centrifuged at 4°C and 4000 rpm for 5 min, the supernatant was discarded, and the solution was washed three times with PBS. Finally, the Lactobacillus was resuspended in MEM complete medium (purchased from Zhongqiao Xinzhou). An appropriate amount of the resuspended solution was spread on a plate to detect the number of viable bacteria, which was L1.

[0175] ②Cell culture: HeLa cells were seeded in 24-well plates, with 1.5×10 cells per well. 5 The HeLa cells cultured in 24-well plates were washed with serum-free MEM medium (purchased from Zhongqiao Xinzhou) and then counted, recording C1.

[0176] ③ Interaction: Lactobacillus was inoculated into the cells at a bacteria-to-cell ratio of 100:1 and incubated at 37°C in a 5% CO2 environment for 1.5 hours. The supernatant after centrifugation was added to the cells as a blank control. After 1.5 hours, the culture medium in the wells was collected, and the cells in the wells were washed with MEM medium. Trypsin was then added to each well for digestion, and the reaction was terminated by adding MEM complete medium. The suspension was collected, and a portion of the cell count (C2) was taken. The number of viable Lactobacillus bacteria adhering to the cells was determined by plate spreading (L2).

[0177] ④Calculate the adhesion number and adhesion rate: average Lactobacillus cell adhesion number = L2 / C2.

[0178] The single cell adhesion number of Lactobacillus gasseri HY1124 to Hela cells was 12.31 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epithelial cells.

[0179] (5) BV (bacterial vaginosis) animal model efficacy test

[0180] Healthy SPF Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups, each consisting of a model group (M), an experimental group (Lactobacillus gasseri HY1124), a control group with the same bacterial strain (Lactobacillus gasseri HY02172), and a positive control group (DJS). Before inoculation with pathogenic bacteria, each group of animals received a subcutaneous injection of estradiol benzoate injection, and then received the same concentration of GV (20 μl) vaginally to establish a BV pathogenic bacteria model. After successful modeling, the experimental group and the control group with the same bacterial strain were given the corresponding lactobacillus solution (1×10 10 CFU / mL, 20 μL), and the positive control group was given an equal amount of Lactobacillus delbrueckii solution (Dingjunshengzhong strain, 1×10 10 CFU / mL, 20 μL). The model group received an equal volume of normal saline. After treatment, vaginal lavage was performed four times per animal with 50 μL of PBS. The fluid was collected, placed in a 1.5 mL Eppendorf tube, and frozen at -80°C. The GV load in the lavage fluid was measured using qPCR, and the differences in GV load in the lavage fluid were compared between the groups.

[0181] The experimental results are shown in Figure 6. The experimental results show that the use of Lactobacillus gasseri HY1124 to treat BV-infected animal models significantly reduced the GV content in the vagina, and the therapeutic effect was significantly better than the positive drug (DJS) and slightly better than the same strain HY02172, indicating that this strain has a good therapeutic effect on BV in mice.

[0182] (6) VVC (vaginal candidiasis) animal model efficacy test

[0183] Healthy SPF-grade Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups: a model group (M), an experimental group (Lactobacillus gasseri HY1124), and a positive control group (Dapoxetine suppository). Each group received a subcutaneous injection of estradiol benzoate injection for pretreatment. CA (15 μL / mouse) was continuously administered from D0 to D2, and the experimental group received vaginal administration of Lactobacillus gasseri HY1124 (5×10 β-lactamase) daily from D3 to D7. 9 CFU / mL, 20 μL). A positive control group was given 30 mg of bisoprolol suppositories, and a model group was given an equal volume of normal saline (20 μL). Vaginal lavage was performed on day 8. The lavage fluid was diluted and spread on a Candida albicans identification medium (chromogenic medium). After incubation at 37°C for 48 hours, the number of green colonies on the culture plate was observed and counted. One-way analysis of variance was performed using GraphPad Prism 5 software, and P < 0.05 was considered statistically significant.

[0184] The experimental results are shown in Figure 7. Compared with the model group, the pathogenic bacteria content in the vaginal lavage fluid of the Lactobacillus gasseri HY1124 group was significantly reduced after treatment, which is equivalent to the therapeutic effect of the positive drug diazepam suppository, indicating that this strain has a good therapeutic effect on VVC in mice.

[0185] 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.

[0186] 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 molecular marker of Lactobacillus gasseri, characterized in that, Having 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.

2. The molecular marker according to claim 1, wherein The molecular marker has the nucleotide sequence shown in SEQ ID NO:

8.

3. A Lactobacillus gasseri, characterized in that, Including the molecular marker according to claim 1 or 2.

4. The Lactobacillus gasseri according to claim 3, characterized in that, Deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on February 7, 2023, with the deposit number CGMCC No. 26504.

5. The Lactobacillus gasseri according to claim 3, characterized in that, The 16S rDNA of the Lactobacillus gasseri has a nucleotide sequence selected from one of the following: (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.

6. A primer set for detecting Lactobacillus gasseri according to any one of claims 3-5, characterized in that, Including 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.

7. A microbial preparation, characterized in that, Including the Lactobacillus gasseri according to any one of claims 3-5.

8. The microbial agent according to claim 7, wherein Further comprising a pharmaceutically acceptable carrier or excipient.

9. A single-dose preparation, characterized in that, Including 1×10 6 ~1×10 10 CFU Lactobacillus gasseri according to any one of claims 3-5 as an active ingredient.

10. Use of the Lactobacillus gasseri according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 in the preparation of a drug for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection.

11. Use of the Lactobacillus gasseri according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 in the preparation of a drug for inhibiting vaginal pathogenic bacteria.

12. The use according to claim 10 or 11, characterized in that, The pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Prevotella bivia, and Candida albicans.

13. Use of the Lactobacillus gasseri according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 in the preparation of a product for regulating vaginal flora balance.

14. A method for inhibiting the growth of vaginal pathogenic bacteria, characterized in that, Including: Contact treatment of at least one of the Lactobacillus gasseri according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9 with the vaginal pathogenic bacteria.

15. A method for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection, characterized in that, Including: Administering to a patient at least one of Lactobacillus gasseri according to any one of claims 3-5, the microbial preparation according to claim 7 or 8, or the single-dose preparation according to claim 9, in a pharmaceutically acceptable dose.

16. The method according to claim 14 or 15, characterized in that, The vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Prevotella bivia, and Candida albicans.

17. A method for detecting Lactobacillus gasseri according to any one of claims 3-5, characterized in that, Comprising: Performing an amplification treatment on the DNA of the strain to be detected using the primer set according to claim 6 to obtain Lactobacillus gasseri according to any one of claims 3-5.

18. The method according to claim 17, wherein The product of the amplification treatment has the molecular marker according to claim 1 or 2, which is an indication that the strain to be detected is Lactobacillus gasseri according to any one of claims 3-5.

19. The method according to claim 18, characterized in that, Comprising the following steps: extracting a DNA sample from the strain to be detected, performing a PCR amplification treatment on the DNA sample using the primer set according to claim 6, and then comparing the amplification result with the molecular marker according to claim 1 or 2 to identify whether the strain to be detected is Lactobacillus gasseri according to any one of claims 3-5.

20. Use of the molecular marker according to claim 1 or 2 for identifying and detecting Lactobacillus gasseri according to any one of claims 3-5.

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