Lactobacillus jensenii and molecular marker and use thereof

Abstract

The present invention provides lactobacillus jensenii and a molecular marker and use thereof. The molecular marker of lactobacillus jensenii has a nucleotide sequence selected from one of the following: (1) a nucleotide sequence as shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or more homology to the nucleotide sequence as shown in SEQ ID NO: 24; and (3) a nucleotide sequence having one or more, such as one, two, three, four, five or more nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 24.

Description

Lactobacillus jensenii and its molecular markers and uses Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a Lactobacillus jensenii and a molecular marker and application thereof. 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.

[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 results with probiotic preparations alone. Therefore, the effectiveness of probiotics in treating BV remains controversial, and extensive research is still needed. Currently, the only marketed probiotic for the treatment of bacterial vaginosis is the Lactobacillus Live Vaginal Capsules (Dingjunsheng) developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd.

[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 healthy women of childbearing age in Zhejiang Province. The strain was identified as Lactobacillus jensenii (Lactobacillus jensenii HY1335). Experiments found that the strain can produce a large amount of hydrogen peroxide (H2O2). H2O2 can directly kill pathogens and can also be oxidized by biological enzymes to form halides with stronger bactericidal effects. Therefore, the Lactobacillus jensenii has the potential to maintain a weakly acidic environment in the vagina, thereby resisting the invasion of other pathogens and maintaining and improving the homeostasis of the vaginal environment, thereby preventing vaginal infections and treating inflammation.

[0008] In view of this, in the first aspect of the present invention, the present invention proposes a molecular marker for Lactobacillus jensenii. 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: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 24; (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: 24. Therefore, the molecular marker of the present invention can not only quickly and easily screen for the target Lactobacillus jensenii, but also can highly specifically identify the target Lactobacillus jensenii, 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:24.

[0010] In a second aspect, the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is Lactobacillus jensenii (Lactobacillus jensenii HY1335), and the Lactobacillus jensenii includes the molecular marker described in the first aspect.

[0011] According to an embodiment of the present invention, the microorganism was deposited in the General Microbiology Center of the China Culture Collection Administration on February 7, 2023, with the deposit number being CGMCC No. 26505.

[0012] According to an embodiment of the present invention, the 16S rDNA of the microorganism has one of the following nucleotide sequences: (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 or 5 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 microorganism has the nucleotide sequence shown in SEQ ID NO:1.

[0014] In a third aspect of the present invention, the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is Lactobacillus jensenii (Lactobacillus jensenii HY1335), which was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on February 7, 2023, with a deposit number of CGMCC No. 26505, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. As mentioned above, the inventors isolated the strain from the vaginal secretions of healthy women of childbearing age in Zhejiang Province. After further identification of the biochemical characteristics of the strain, it was found that the bacteria can produce a large amount of hydrogen peroxide H2O2, which can 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). Therefore, this Lactobacillus jensenii has the potential to maintain a weakly acidic environment in the vagina, thereby resisting the invasion of other pathogens and maintaining and improving the homeostasis of the vaginal environment, thereby preventing vaginal infections and treating inflammation.

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

[0016] In a fourth aspect, the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism is Lactobacillus jensenii (Lactobacillus jensenii HY1335), and the 16S rDNA of the microorganism has a nucleotide sequence of 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 microorganisms described in the present invention have a strong inhibitory effect on the pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA) and Prevotella bivia (PB). Therefore, the Lactobacillus jensenii has the potential to maintain a weakly acidic environment in the vagina, thereby resisting the invasion of other pathogens and maintaining and improving the homeostasis of the vaginal environment, thereby preventing vaginal infection and treating inflammation.

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

[0018] According to an embodiment of the present invention, the microorganism of 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 microorganism further comprises a nucleotide sequence selected from the following: (1) a nucleotide sequence as shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence as shown in SEQ ID NO: 24; (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: 24. Therefore, the microorganism 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 the microorganism further comprises a nucleotide sequence as shown in SEQ ID NO:24.

[0021] According to a specific embodiment of the present invention, the microorganism is capable of producing at least 5.0-5.5 g / L of lactic acid under effective culture.

[0022] According to a specific embodiment of the present invention, the microorganism is capable of producing at least 5.0 g / L, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, or 5.5 g / L of lactic acid under effective culture.

[0023] According to a specific embodiment of the present invention, the microorganism is capable of producing at least 3000-3200 μM hydrogen peroxide under effective culture.

[0024] According to a specific embodiment of the present invention, the microorganism is capable of producing at least 3000 μM, 3020 μM, 3050 μM, 3080 μM, 3100 μM, 3120 μM, 3150 μM, 3170 μM, or 3200 μM of hydrogen peroxide under effective culture.

[0025] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 80-85% against GV.

[0026] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 80%, 81%, 82%, 83%, 84%, or 85% against GV.

[0027] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 94-98% against PB.

[0028] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 94%, 95%, 96%, 97%, or 98% against PB.

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

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

[0031] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 98-100% against SA.

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

[0033] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 45-50% against CA.

[0034] According to a specific embodiment of the present invention, the microorganism has an inhibition rate of at least 45%, 46%, 47%, 48%, 49%, or 50% against CA.

[0035] In a fifth aspect, the present invention provides a primer set for detecting the microorganisms described in the second, third, or fourth aspects. According to an embodiment of the present invention, the primer set includes a forward primer and a reverse primer, wherein the forward primer has the nucleotide sequence shown in SEQ ID NO: 2; and the reverse primer has the nucleotide sequence shown in SEQ ID NO: 3. The primer set described in the present invention can achieve a high degree of match with the DNA sequence of the microorganism described in the second, third, or fourth aspects, and can produce specific amplification only in the microorganism. This enables the primers to accurately and reliably identify the microorganism, 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 microorganisms described in the second, third, or fourth aspects. As previously mentioned, the inventors of the present invention have isolated a new strain of Lactobacillus jensenii that exhibits strong inhibitory effects against pathogenic bacteria GV, EC, SA, and PB. Therefore, this Lactobacillus jensenii can be formulated into a microbial preparation for convenient patient administration.

[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 11 The microorganisms described in the second, third or fourth aspects of CFU are used as active ingredients.

[0039] According to an embodiment of the present invention, comprising 10 6 CFU, 10 7 CFU, 10 8 CFU, 10 9 CFU, 10 10 CFU, 10 11 The microorganisms described in the second, third or fourth aspects of CFU are used as active ingredients.

[0040] According to an embodiment of the present invention, including 1×10 8 ~1×10 10 The microorganisms described in the second, third or fourth aspects of CFU are used as active ingredients.

[0041] In the eighth aspect of the present invention, the present invention proposes the use of the microorganism 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 microorganism 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 drug 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 pathogenic bacteria 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 microorganism described in the second aspect, the third aspect or the 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 the eleventh aspect of the present invention, the present invention provides a molecular marker for the microorganism according to 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: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence as shown in SEQ ID NO: 24; (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: 24.

[0050] According to an embodiment of the present invention, the molecular marker has a nucleotide sequence as shown in SEQ ID NO:24.

[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 the twelfth aspect of the present invention, a method for detecting the microorganisms 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 to obtain the microorganisms described in the second, third, or fourth aspects. As described above, the primer set described in the fifth aspect is specific for the microorganisms described in the present invention, is highly compatible with the DNA sequence of the microorganisms described in the second, third, or fourth aspects, and produces specific amplification only in the microorganisms. Using the method of the present invention, the microorganisms of the present invention can be efficiently and conveniently identified and detected.

[0053] According to embodiments 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 microorganism 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 fragment marker for the microorganism described in the present invention, providing a reliable tool and basis for identifying and detecting the microorganism described in the present invention.

[0054] According to an embodiment of the present invention, the method for detecting the microorganism 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 microorganism described in the second, third, or fourth aspects. The method of the present invention can efficiently and conveniently identify and detect the microorganism 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 microorganism described in the first aspect or the second aspect.

[0056] In the fourteenth aspect of the present invention, the present invention proposes the use of the microorganism described in the second aspect, the third aspect or the 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 microorganism described in the second aspect, the third aspect or the fourth aspect, the microbial preparation described in the sixth aspect or the single-dose preparation described in the seventh aspect in the treatment and / or prevention of 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 microorganisms 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 microorganisms 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 Lactobacillus jensenii HY1335 according to an embodiment of the present invention;

[0067] FIG2 is a bacterial morphology diagram of Lactobacillus jensenii HY1335 according to an embodiment of the present invention;

[0068] FIG3 is an electrophoretic pattern of PCR amplification of four Lactobacillus jensenii by primers 1-p2 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY1335 as a template, 2 uses the supernatant after lysis of HY00888 as a template, 3 uses the supernatant after lysis of HY01360 as a template, and 4 uses the supernatant after lysis of HY02445 as a template;

[0069] FIG4 is a graph showing the results of a hemolysis experiment of Lactobacillus jensenii HY1335 according to an embodiment of the present invention;

[0070] FIG5 is a diagram showing the results of a BV efficacy animal experiment according to an embodiment of the present invention. DETAILED DESCRIPTION

[0071] 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.

[0072] Definition of terms

[0073] In the present invention, the term "Lactobacillus jensenii" or "L. jensenii" 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.

[0074] In the present invention, the term "effective culture conditions" generally refers to the environment in which Lactobacillus jensenii 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.

[0075] 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 jensenii HY1335 is sensitive to clindamycin and cefuroxime antibiotics.

[0076] 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.

[0077] 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.

[0078] 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."

[0079] In the present invention, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms that colonize the vagina.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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 jensenii 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, adhesives, fillers, disintegrants, lubricants, coatings, sweeteners, spices, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinyl pyrrolidine, and dyes. Such preparations can be sterilized, and those skilled in the art will recognize that other pharmaceutical carriers can also be used in this application.

[0084] 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.

[0085] 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 jensenii present in the food or dietary supplement is 10 6 to 10 11The amount of CFU / dose present (e.g. 10 8 to 10 10 In this embodiment, if Lactobacillus jensenii 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 11 Alternatively, of course, this amount of bacteria can be divided into multiple administrations, so long as the total amount of Lactobacillus jensenii received by the subject in any particular time (e.g., per 24-hour period) is from about 10 6 to about 10 11 CFU of bacteria, that is, Lactobacillus jensenii in food products or dietary supplements that meet the above requirements, is 10 6 to 10 11 The amount of CFU / dose present (e.g. 10 8 to 10 10 CFU / dose).

[0086] 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, and 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 jensenii of the present invention to achieve a complete cure or eradication of any symptoms or manifestations of the disease in order for it 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. Simply reducing 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 reducing the likelihood of the disease occurring or worsening is sufficient.

[0087] 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.

[0088] 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.

[0089] Example 1 Screening and Isolation of Lactobacillus jensenii

[0090] (1) Collect samples

[0091] 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).

[0092] (2) Strain isolation

[0093] 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 jensenii was isolated from the vaginal secretion sample and named Lactobacillus jensenii HY1335.

[0094] After the strain species is determined, the pure culture (i.e., pure Lactobacillus jensenii HY1335) is inoculated into MRS broth liquid culture medium for expansion. When the strain grows to an appropriate concentration, sterile 50% glycerol solution is used to mix the liquid culture with the same volume and the culture is stored in a -80°C strain bank.

[0095] Example 2 Identification of Lactobacillus jensenii

[0096] (1) Colony characteristics

[0097] 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, milky white colonies with rough edges and a central protrusion when cultured on the MRS plate.

[0098] (2) Staining microscopy

[0099] Pick 1 loop of sterilized distilled water onto a clean glass slide with an inoculating loop, pick a single bacterium colony from the MRS flat plate, mix it with distilled water and evenly coat it on a glass slide, dye according to the Gram staining solution kit (purchased from Qingdao Haibo) instructions, and then observe the bacterial morphology under an electron microscope. As shown in Figure 2, after the bacterial strain is dyed, it is observed by light microscopy that the bacterial staining is Gram-positive, and the bacterial body is short rod-shaped. The observation of colony characteristics and Gram staining identification preliminarily determine that the isolated bacterial strain is a lactobacillus.

[0100] (3) Biochemical identification and analysis

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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:

[0105] Table 1 Interpretation results of Lactobacillus jensenii identification tube Note: + represents positive; - represents negative; +w represents weak positive.

[0106] The results of biochemical identification showed that Lactobacillus jensenii could utilize esculin, cellobiose, maltose, salicin, sucrose and inulin as carbon sources.

[0107] (4) 16S rDNA identification

[0108] The above-mentioned Lactobacillus jensenii 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 jensenii (Lactobacillus jensenii), and was named Lactobacillus jensenii HY1335. The 16S rDNA gene sequence was determined as follows:

[0109] (5) Specific nucleotide sequence molecular markers

[0110] ①Specific primer design

[0111] a. Specific nucleotide sequence screening: The HY1335 strain was subjected to whole genome sequencing and genome analysis, and compared with the genome sequence of Lactobacillus jensenii strains included in the NCBI database to screen for specific nucleotide sequences of Lactobacillus jensenii HY1335.

[0112] 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 10 pairs of primers for other specific nucleotide sequences across the genome as controls. See Table 2 for primer sequences:

[0113] Table 2 Primer list

[0114] c. Primer screening: Lactobacillus jensenii HY1335 and three strains of the same species were used as controls (see Table 3 for specific strain information) for template preparation. Single colonies were picked from 50 μL of lysis buffer (purchased from TaKaRa), lysed at 80°C for 15 minutes, and centrifuged at 4000 rpm for 5 minutes. The supernatant was used as the template. After primer screening, 1-p2 was selected for PCR amplification. The PCR amplification system consisted of 12.5 μL of Taq enzyme, 1 μL of F primer, 1 μL of R primer, 1.5 μL of template, and ddH2O to 25 μL. The PCR reaction conditions are shown in Table 4 below, and the PCR experiment was performed. After the PCR experiment was completed, 1.5 g of agarose was added to 100 mL of 1× TAE buffer, heated thoroughly to melt, and 5 μL of Gel Red dye was added. The product was poured into a gel plate and solidified for use. 4 μL of the PCR product was subjected to agarose gel electrophoresis. The electrophoresis conditions were as follows: voltage 130 V, current 400 mA, and time 35 min.

[0115] Table 3 Strain information Note: The strains numbered HY00888, HY01360, and HY02445 in Table 3 are Lactobacillus jensenii screened from samples collected by the inventors according to Example 1. In order to verify the specificity of the above-mentioned specific nucleotide sequence to Lactobacillus jensenii HY1335, PCR amplification was performed on HY1335 and three strains of the same species using the above-mentioned primers.

[0116] Table 4 PCR reaction conditions

[0117] Among the primers designed for specific nucleotide sequences, PCR amplification experiments using primer 1-p2 revealed that only HY1335 produced a single, clear, high-concentration amplified band with relatively few primer dimers, while HY00888, HY01360, and HY02445 all showed no amplified bands. The agarose gel electrophoresis pattern is shown in Figure 3. However, when PCR amplification experiments were performed using primers designed for other nucleotide sequences, bands were also amplified in the control bacterial group. Therefore, primer 1-p2 is a molecular marker primer for HY1335, and the amplification product generated by this primer is a molecular marker for Lactobacillus jensenii HY1335. The amplification product (molecular marker) produced by the 1-p2 primer is 363 bp in size, and the nucleotide sequence is shown in SEQ ID NO: 24. Thus, it was determined that the nucleotide sequence shown in SEQ ID NO:24 was part of the specific nucleotide sequence screened in step a, further verifying that primer 1-p2 can be used as a specific primer for Lactobacillus jensenii HY1335, and the nucleotide shown in SEQ ID NO:24 is a specific nucleotide sequence marker for Lactobacillus jensenii HY1335, which provides a reliable tool and basis for the identification and detection of Lactobacillus jensenii HY1335.

[0118] d. During the experiment, the inventors discovered that when sequencing multiple Lactobacillus jensenii HY1335 samples using the 1-p2 primer, the amplified product sequences differed from the nucleotide sequence set forth in SEQ ID NO: 24 at individual bases. However, compared to the control bacteria, all samples had specific amplified bands that were clear and high in concentration, and relatively few primer dimers.

[0119] In one experiment, it was found that the nucleotide sequence of the product amplified from the Lactobacillus jensenii HY1335 sample using primer 1-p2 was shown in SEQ ID NO: 25, which aligned 99.17% with the nucleotide sequence shown in SEQ ID NO: 24.

[0120] Therefore, after multiple sequencing and alignment, the homology range between the molecular marker of Lactobacillus jensenii HY1335 and the nucleotide sequence shown in SEQ ID NO: 24 was determined, that is, the molecular marker of Lactobacillus jensenii HY1335 has a nucleotide sequence that is at least 97%, 98%, 99% or higher homologous to the nucleotide sequence shown in SEQ ID NO: 24.

[0121] Example 3 Bacterial characteristics

[0122] (1) Antibiotic sensitivity test

[0123] Lactobacillus jensenii HY1335 was cultured in MRS broth, and the culture solution was evenly spread on an MRS plate. 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, ciprofloxacin, and kanamycin, but sensitive to clindamycin and cefuroxime.

[0124] 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)

[0125] (2) Toxicity test

[0126] ①Hemolysis test

[0127] 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 4. Small, off-white colonies appeared in the culture medium surrounding the colonies, and no hemolytic rings appeared around the colonies, indicating that Lactobacillus jensenii HY1335 is gamma-hemolytic, meaning it is non-hemolytic.

[0128] ②Toxicity test in mice

[0129] Five mice weighing 18-22g were orally gavaged with 0.5ml of fresh bacterial solution (minimum 1.0×10 CFU / 0.5ml) once daily for three consecutive days. All mice were observed from day 1 to day 7 to ensure they remained healthy and gained weight. The results showed that mice maintained healthy survival and gained weight after gavage with fresh Lactobacillus jensenii HY1335.

[0130] (3) Determination of metabolite content

[0131] ①D-lactic acid detection

[0132] 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.

[0133] ②L-lactic acid detection

[0134] The supernatant was filtered through a 0.22 μm sterile filter membrane and the L-lactic acid concentration was measured using a biosensor.

[0135] ③ Hydrogen peroxide detection

[0136] 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.

[0137] Lactobacillus was inoculated into MRS medium at a 0.5% inoculum volume and cultured at 37°C for 24 hours. When the lactobacillus was cultured for 24 hours, it was centrifuged at 4000 rpm for 20 minutes at 4°C, the supernatant was discarded, the cells were washed with PBS buffer, and the cells were resuspended 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.

[0138] Lactobacillus jensenii HY1335 was cultured in MRS broth for 24 h. The results of D-lactic acid detection, L-lactic acid detection and hydrogen peroxide detection are shown in Table 6. The content of D-lactic acid in the metabolites of Lactobacillus jensenii HY1335 was 5.23 g / L, and the content of L-lactic acid was 0 g / L. The total acid production of Lactobacillus jensenii HY1335 was 5.23 g / L, which was basically the same as that of DJS-Lactobacillus delbrueckii 5.48 g / L. H2O2 has the ability to directly kill pathogens and can also be used as a bioreactor. The enzyme catalyzes the oxidation of halides to form halides with stronger bactericidal effects. As can be seen from Table 6, Lactobacillus jensenii HY1335 can produce 3170 μM hydrogen peroxide, while the amount of hydrogen peroxide produced by DJS-Lactobacillus delbrueckii is only 80.67 μM. The ability of Lactobacillus jensenii HY1335 to produce hydrogen peroxide is much higher than that of DJS-Lactobacillus delbrueckii. Therefore, Lactobacillus jensenii HY1335 has the potential to kill pathogens, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0139] Table 6 Metabolite content determination results

[0140] Example 4 Application Function Analysis

[0141] (1) Antibacterial experiment

[0142] 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 6CFU / mL) (WS / T 650-2019 Evaluation method for antibacterial and antibacterial effects).

[0143] 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.

[0144] Antibacterial rate = (AB) / A*100%

[0145] A: OD increase in the positive control group (blank culture medium) within 48 hours 600 value;

[0146] B: OD increase in the experimental group within 48 hours 600 value.

[0147] Two different strains of the same bacterial species, along with the positive drug Dingjunsheng, were selected to test their antibacterial properties with the selected Lactobacillus jensenii HY1335. The results, as shown in Table 7, showed that the strain had a strong inhibition rate against GV, PB, EC, SA, and CA, and its inhibition against GV, PB, EC, and SA was superior to that of the same bacterial species control group and the positive control group. This strain had a strong inhibitory effect on these four pathogens simultaneously, an effect that could not be achieved by either the positive control or the same bacterial species different strain control group.

[0148] Table 7 Antibacterial test results

[0149] Note: The strains numbered HY02405 and HY01361 in Table 7 are Lactobacillus jensenii screened from the samples collected by the inventors according to Example 1, in order to verify the inhibition rates of Lactobacillus jensenii HY1335 and two strains of the same species against different pathogens.

[0150] (2) Experiment on the interaction and co-culture of Lactobacillus and GV

[0151] Lactobacillus culture: Take a glycerol tube of Lactobacillus jensenii 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 solution to about 1×10 9 CFU / mL concentration is used as the Lactobacillus working culture solution.

[0152] 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.

[0153] 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. Two replicates were placed in an anaerobic workstation for cultivation. Samples were taken 27 hours after cultivation, and the viable count of GV was detected by fluorescence quantitative qPCR using GV-specific probe primers. After Lactobacillus jensenii and GV were co-cultured for 27 hours, the viable count of GV was detected. The experimental results are shown in Table 8. After Lactobacillus jensenii HY1335 was co-cultured with GV for 27 hours, the inhibition rate reached 89%, indicating that the strain had a strong inhibitory effect on the growth of GV.

[0154] Table 8 Antibacterial experiment of co-culture of Lactobacillus and GV

[0155] (3) Biofilm removal experiment

[0156] 1) Lactobacillus removal of GV biofilm

[0157] After GV culture, the bacterial solution was adjusted to 1.0×10 7 CFU / 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 the supernatant of Lactobacillus jensenii HY1335. The plates were incubated at 37°C in an anaerobic workstation for 24 hours.

[0158] After the incubation period, the inhibitory effect of Lactobacillus jensenii HY1335 on GV biofilm formation was determined using a microplate crystal violet staining method. The experimental results, shown in Table 9, show that the supernatant of Lactobacillus jensenii HY1335 had a strong inhibitory effect on GV biofilm formation, reaching 91.4%. Therefore, it can effectively inhibit the growth of GV.

[0159] Table 9 Lactobacillus removal test results of GV biofilm

[0160] 2) Lactobacillus removal of CA biofilm

[0161] 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.

[0162] 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.

[0163] The experimental results are shown in Table 10. Lactobacillus jensenii HY1335 has a certain inhibitory or destructive effect on the formation of CA biofilm and the existing CA biofilm.

[0164] Table 10 Results of the experiment on the removal of CA biofilm by lactobacilli

[0165] (4) Cell adhesion assay

[0166] ① Preparation of working bacterial solution: culture Lactobacillus and test the OD of bacterial solution 600The 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.

[0167] 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.

[0168] 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).

[0169] ④Calculate the adhesion number and adhesion rate: average Lactobacillus cell adhesion number = L2 / C2.

[0170] The single cell adhesion number of Lactobacillus jensenii HY1335 to Hela cells was 3.60 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epithelial cells.

[0171] (5) BV (bacterial vaginosis) animal model efficacy test

[0172] 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, each consisting of a model group (M), an experimental group (Lactobacillus jensenii HY1335), 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 vaginal administration of the same concentration of GV (20 μl) to establish a BV pathogen model. After successful modeling, the experimental group received vaginal administration of Lactobacillus jensenii HY1335 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 10CFU / 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.

[0173] The experimental results are shown in Figure 5. The experimental results show that the use of Lactobacillus jensenii HY1335 to treat BV-infected animal models significantly reduced the GV content in the vagina, and the therapeutic effect was significantly better than that of the positive drug (DJS), indicating that this strain has a good therapeutic effect on BV in mice.

[0174] 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.

[0175] 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 jensenii, characterized in that, having a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO:24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:24; (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:

24.

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

24.

3. A microorganism, which is Lactobacillus jensenii, characterized in that, Comprising the molecular marker according to claim 1 or 2.

4. The microorganism 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. 26505.

5. The microorganism according to claim 3, characterized in that, The 16S rDNA of the microorganism 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 or 5 nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:1; Preferably, the 16S rDNA gene sequence of the microorganism comprises the nucleotide sequence shown in SEQ ID NO:

1.

6. A primer set for detecting the microorganism according to any one of claims 3-5, characterized in that, Comprising 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, Comprising the microorganism 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, Comprising 1×10 6 ~1×10 11 CFU of the microorganism according to any one of claims 3-5 as an active ingredient.

10. Use of the microorganism 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 microorganism 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 vaginal pathogenic bacteria are selected from at least one of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Prevotella bivia, and Candida albicans.

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

14. Use of the microorganism 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.

15. A method for inhibiting the growth of vaginal pathogenic bacteria, characterized in that, Comprising: Contact treating at least one of the microorganism 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.

16. A method for treating and / or preventing vaginal pathogenic bacteria infection or related diseases caused by vaginal pathogenic bacteria infection, characterized in that, Comprising: The right to administer to a patient a pharmaceutically acceptable dose of at least one of the microorganisms 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.

17. The method according to claim 15 or 16, 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.

18. A method for detecting the microorganism according to any one of claims 3-5, characterized in that, Comprising: Using the primer set according to claim 6 to amplify the DNA of the strain to be detected, so as to obtain the microorganism according to any one of claims 3-5.

19. The method according to claim 18, 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 the microorganism according to any one of claims 3-5.

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

21. Use of the molecular marker according to any one of claims 1-2 for identifying and detecting the microorganism according to any one of claims 3-5.

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