Lactobacillus jensenii and application thereof

Abstract

The invention provides a microorganism, which is Lactobacillus jensenii, is preserved in the China General Microbiological Culture Collection Center on February 7, 2023, and has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.26505. The invention further provides a preparation method of the microorganism. The lactobacillus jensenii has a relatively strong inhibition effect on pathogenic bacteria such as gardnerella vaginalis, escherichia coli, staphylococcus aureus and two-way proteurella, so that the lactobacillus jensenii has the capabilities of maintaining the weak acidity of the environment in the vagina and further resisting invasion of other pathogenic bacteria, and can be used for preventing and treating vaginal infection and inflammation; the steady state of the environment in the vagina is maintained, improved or recovered.

Description

Technical Field

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

[0002] With the economic development and the improvement of living standards, the disease spectrum of humans has changed greatly, and the high incidence of female reproductive tract diseases has become increasingly prominent. Among them, the incidence of vaginitis is particularly common, which has seriously affected the quality of life, physical and mental health of women. Among the diseases related to vaginal infections, bacterial vaginosis (BV) is the most common. Epidemiological investigations show that the incidence of BV in China ranges from 4.96% to 36.00%. The main reason for the occurrence of this disease is the reduction of dominant lactobacilli that maintain the normal acidic vaginal environment, while the increase of various other anaerobic microorganisms. The dominant lactobacilli are replaced by Gardnerella vaginalis and mixed anaerobic bacteria. Gardnerella vaginalis is one of the most common pathogenic bacteria of BV in women. The typical clinical features of bacterial vaginosis are that the patient's vagina shows fetid, watery, grayish secretions. The patient may also have many complications due to the infection of pathogens, such as chronic cervicitis, pelvic inflammatory disease, endometritis, and even infertility.

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

[0004] Currently, the treatment of BV mainly relies on Western medicine, with the frequent use of antibacterial drugs such as antibiotics, including metronidazole, clindamycin, tinidazole, etc. However, the generation and persistent existence of pathogenic bacterial biofilms can lead to disease recurrence. According to the "Diagnosis and Treatment Guidelines for Bacterial Vaginosis (2021 Revised Edition)", the recurrence rate of BV is 20% one month after treatment, 40% three months after treatment, and as high as 60% twelve months after treatment. By the 12th month after treatment, 84% of patients have abnormal vaginal flora. In addition, long-term and excessive use of antibiotics not only increases the drug resistance of pathogenic bacteria but also inhibits the reproduction of some vaginal flora, while enabling the relatively small amount of Candida albicans to multiply significantly, resulting in vaginal flora disorder. Currently, the role of live bacteria preparations in the treatment of BV is still controversial. Relevant clinical studies show that the application of current live bacteria preparations is mainly divided into two types: the application of probiotics after conventional antibiotic treatment or the application of probiotics alone. The results of multiple clinical trials of combined treatment show inconsistent results, and it is impossible to determine whether antibiotics combined with probiotics can be used for the treatment of bacterial vaginosis. In addition, some studies have shown that some patients did not obtain satisfactory efficacy when using probiotic preparations alone. Therefore, there is still a great deal of controversy about the effect of probiotics in the treatment of BV, and a large number of experiments are still needed for exploration and research. The currently marketed probiotic drug for the treatment of bacterial vaginosis is mainly Ding Junsheng (Vaginal Lactobacillus Live Capsules) developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd.

[0005] Therefore, there is an urgent need for a lactobacillus that has both good cell adhesion and antibacterial ability, so as to inhibit the growth of a variety of pathogenic bacteria. Summary of the Invention

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

[0007] Therefore, the inventor isolated a strain from the vaginal secretions of healthy childbearing-aged women in Zhejiang. After identification, the strain was Lactobacillus jensenii (Lactobacillus jensenii HY1335). Through experiments, it was found that this strain can produce a large amount of hydrogen peroxide H2O2. H2O2 can directly kill pathogenic bacteria or form halides with stronger bactericidal effects through enzymatic catalytic oxidation. Therefore, this Lactobacillus jensenii has the potential to maintain the vaginal environment at a weakly acidic state, thereby resisting the invasion of other pathogenic bacteria and maintaining and improving the homeostasis of the vaginal environment, thus preventing vaginal infections and treating inflammation.

[0008] In view of this, in the first 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 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 7, 2023, with the deposit number 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 inventor isolated this strain from the vaginal secretions of healthy reproductive-aged women in Zhejiang. After further identifying the biochemical characteristics of this strain, it was found that this bacterium can produce a large amount of hydrogen peroxide H2O2, and H2O2 can directly kill pathogenic bacteria and can also be catalytically oxidized by biological enzymes to form halides with stronger bactericidal effects; in addition, this bacterium has strong inhibitory effects on pathogenic bacteria Gardnerella vaginalis (GV), Escherichia coli (EC), Staphylococcus aureus (SA) and Prevotella bivia (PB). Therefore, this Lactobacillus jensenii has the potential to maintain a weakly acidic environment in the vagina, thereby resisting the invasion of other pathogenic bacteria and maintaining and improving the homeostasis of the vaginal environment, thus preventing vaginal infections and treating inflammation.

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

[0010]

[0011] In a second 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), and the 16S rDNA of the microorganism has one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO:1; (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO:1; (3) a nucleotide sequence having one or more, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO:1. The microorganism of the present invention has strong inhibitory effects on 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 pathogenic bacteria and maintaining and improving the homeostasis of the vaginal environment, thus preventing vaginal infections and treating inflammation.

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

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

[0014] According to an embodiment of the present invention, the gene sequence of the microorganism further contains a nucleotide sequence selected from 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. Therefore, the microorganism of the present invention can be specifically identified by the above nucleotide sequence.

[0015] According to an embodiment of the present invention, the gene sequence of the microorganism further contains the nucleotide sequence shown in SEQ ID NO:24.

[0016] According to specific embodiments of the present invention, the microorganism is capable of producing at least 5.0 - 5.5 g / L of lactic acid under effective cultivation.

[0017] According to specific embodiments 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, 5.5 g / L of lactic acid under effective cultivation.

[0018] According to specific embodiments of the present invention, the microorganism is capable of producing at least 3000 - 3200 μM of hydrogen peroxide under effective cultivation.

[0019] According to specific embodiments 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, 3200 μM of hydrogen peroxide under effective cultivation.

[0020] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 80 - 85% against GV.

[0021] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 80%, 81%, 82%, 83%, 84%, 85% against GV.

[0022] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 94 - 98% against PB.

[0023] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 94%, 95%, 96%, 97%, 98% against PB.

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

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

[0026] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 98 - 100% against SA.

[0027] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 98%, 99%, 100% against SA.

[0028] According to specific embodiments of the present invention, the microorganism has an antibacterial rate of at least 45 - 50% against CA.

[0029] According to specific embodiments of the present invention, the microorganism has an antibacterial rate against CA of at least 45%, 46%, 47%, 48%, 49%, 50%.

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

[0031] In the fourth aspect of the present invention, the present invention provides a microbial preparation. According to embodiments of the present invention, the microbial preparation contains the microorganism described in the first aspect or the second aspect. As previously mentioned, the inventors of the present invention isolated and obtained a new Lactobacillus jensenii, which has strong inhibitory effects on pathogenic bacteria GV, EC, SA, and PB. Therefore, this Lactobacillus jensenii can be made into a microbial preparation to facilitate patients' taking.

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

[0033] In the fifth aspect of the present invention, the present invention provides a single-dose preparation. According to embodiments of the present invention, it includes 1×10 6 ~1×10 11 CFU of the microorganism described in the first aspect or the second aspect as an active ingredient.

[0034] According to embodiments of the present invention, it includes 10 6 CFU, 10 7 CFU, 10 8 CFU, 10 9 CFU, 10 10 CFU, 10 11 CFU of the microorganism described in the first aspect or the second aspect as an active ingredient.

[0035] According to embodiments of the present invention, it includes 1×10 8 ~1×10 10 CFU of the microorganism described in the first aspect or the second aspect as an active ingredient.

[0036] In a sixth aspect of the present invention, there is provided the use of the microorganism according to the first or second aspect, the microbial preparation according to the fourth aspect, or the single-dose preparation according to the fifth aspect in the preparation of a medicament for treating and / or preventing vaginal pathogenic bacterial infection or related diseases caused by vaginal pathogenic bacterial infection.

[0037] In a seventh aspect of the present invention, there is provided the use of the microorganism according to the first or second aspect, the microbial preparation according to the fourth aspect, or the single-dose preparation according to the fifth aspect in the preparation of a medicament for inhibiting vaginal pathogenic bacteria.

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

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

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

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

[0042] In an eighth aspect of the present invention, there is provided the application of the microorganism according to the first or second aspect, the microbial preparation according to the fourth aspect, or the single-dose preparation according to the fifth aspect in the preparation of a product for regulating the balance of vaginal flora.

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

[0044] In a ninth aspect of the present invention, the present invention provides a molecular marker of the microorganism described in the first aspect or the second aspect. According to an embodiment of the present invention, the molecular marker comprises a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 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.

[0045] According to an embodiment of the present invention, the molecular marker has the nucleotide sequence shown in SEQ ID NO: 24.

[0046] GGATCGGGTAATGGCACCAATATAATAAATGATAAAGTTAAAATTCCAACCGGTGCTTTTCAGTCTAAACAAATGCAAACTGAAAATTGCAAAGTAAATAATCAGATGTTGGATAAAACTGAAGTAACAAATTCTACTAAAAATTCTTCTACACTAAATGTGGAAAATTCCAAGACTGCTGATTTGTCGAGATTTGATTACTCTTTATATACAAAAAAAGTAAAGAGTTTTGAATTTAGAAATTCAGATAATAACGATGTAATTAGAACAGTAATATTAAACAAACCTACCGGTGTTGAAACTGTAACTATGACTTTGAATGTTAGCTGCTTAAAAGGGAGAAAGCATCAGCATCAATCGCAC(SEQ ID NO: 24)

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

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

[0049] According to an embodiment of the present invention, the product of the amplification treatment has the nucleotide sequence shown in SEQ ID NO: 24, which is an indication that the strain to be detected is the microorganism described in the first aspect or the second aspect. Using the primer set described in the third aspect can amplify the nucleotide sequence shown in SEQ ID NO: 24. The nucleotide sequence shown in SEQ ID NO: 24 is a specific fragment marker of the microorganism of the present invention, providing a reliable tool and basis for identifying and detecting the microorganism of the present invention.

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

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

[0052] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0053] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where:

[0054] Figure 1 is the colony morphology diagram of Lactobacillus jensenii HY1335 according to an embodiment of the present invention;

[0055] Figure 2 is the cell morphology diagram of Lactobacillus jensenii HY1335 according to an embodiment of the present invention;

[0056] Figure 3 is the PCR amplification electrophoresis pattern of primer 1-p2 for 4 Lactobacillus jensenii strains according to an embodiment of the present invention, where M is 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;

[0057] Figure 4 is the hemolysis test result diagram of Lactobacillus jensenii HY1335 according to an embodiment of the present invention;

[0058] Figure 5 It is a graph showing the results of BV pharmacodynamic animal experiments according to an embodiment of the present invention. Detailed implementation manners

[0059] The following specific embodiments illustrate the implementation manners of the invention of the present application. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0060] Term definitions

[0061] In the present application, 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.

[0062] In the present application, the term "effective culture conditions" generally refers to the environment in which Lactobacillus jensenii is placed or exposed to promote the growth of the bacterium. Therefore, this term generally refers to the culture medium, temperature, atmospheric conditions, substrate, stirring conditions, etc. that can affect the growth of the bacterium.

[0063] In the present application, the term "antibiotic-sensitive" means that the bacterium has weak resistance to antibiotics, and under the condition of micro-dose administration, it can affect the normal growth of the bacterium. According to an embodiment of the present invention, the Lactobacillus jensenii HY1335 is sensitive to antibiotics such as clindamycin and cefuroxime.

[0064] In the present application, the term "vagina" generally refers to the vaginal region or division or the surrounding area, including the labia, vulva, cervix, uterus, fallopian tubes, ovaries, urethra, bladder, anus and rectum, including their mucosal tissues.

[0065] In the present application, the terms "disease" or "disorder" can be used interchangeably and generally refer to any change in the state of the body or certain organs that hinders or disrupts the performance of functions, and / or causes symptoms such as discomfort, dysfunction, pain or even death in the diseased person or those in contact with it.

[0066] In the present application, the term "pathogenic" (e.g., "pathogenic bacterium") generally refers to a substance, microorganism or condition that can cause a disease. In certain situations, pathogens also include microorganisms (e.g., bacteria) that are related to a disease or disorder 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 a disease or dysbiosis, depending on various factors (e.g., the immune status of the site, the abundance of the microbial taxon, etc.). Such microorganisms are referred to as "pathogenic organisms".

[0067] In the present application, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms colonizing the vagina.

[0068] In the present application, the term "inhibit" generally refers to the process of inhibiting or impeding the growth, reproduction, and activity of bacteria.

[0069] In the present application, the term "isolated", when 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 associated in its natural state. For example, it can be in a homogeneous state and can be in a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0070] The term "isolated", when applied to bacteria, generally means bacteria that have been (1) separated from at least some of the components with which they were associated at the time of their original production (whether in nature or in an experimental setting), and / or (2) produced, prepared, purified, and / or manufactured artificially, such as by using artificial culture conditions, such as (but not limited to) culturing on plates and / or in fermenters. Isolated bacteria include those bacteria that are cultured, even if such cultures are not pure cultures. Isolated bacteria can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were 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 embodiments of the present invention, the bacterial populations provided in the present application include isolated bacteria. According to embodiments of the present invention, the compositions provided in the present application include isolated bacteria. According to embodiments of the present invention, the bacteria administered are isolated bacteria.

[0071] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a substance that aids in the administration of an active agent to a subject and is absorbed by the subject and that can be included in the compositions of the present application without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline solution, lactated Ringer's solution, common sucrose, common glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and dyes, among others. Such products can be sterilized, and those skilled in the art will recognize that other pharmaceutical carriers can also be used in the present application.

[0072] In the present application, the term "CFU (Colony-Forming Units)" generally refers to the total number of colonies of microorganisms such as bacteria, fungi, and yeast in a product, and is usually used for calculating the number of viable bacteria.

[0073] The term "CFU / dose" means the amount of bacteria present in the composition / food or dietary supplement / drug provided to a subject per day or per administration. For example, in certain embodiments, Lactobacillus gasseri in the food or dietary supplement is present in an amount of 10 6 to 10 11 CFU / dose (e.g., 10 8 to 10 10 CFU / dose). In such an embodiment, if Lactobacillus gasseri is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject per day or per administration may contain approximately 10 6 to 10 11 CFU of Lactobacillus gasseri. Of course, alternatively, the amount of such bacteria can be administered in multiple doses, as long as the total amount of Lactobacillus gasseri received by the subject within any specific time period (e.g., every 24-hour period) is from approximately 10 6 to approximately 10 11 CFU of bacteria, that is, the Lactobacillus gasseri in the above-mentioned food product or dietary supplement is present in an amount of 10 6 to 10 11 CFU / dose (e.g., 10 8 to 10 10 CFU / dose).

[0074] In the present application, the term "treatment and / or prevention" not only includes treating and / or preventing a disease, but generally includes preventing the onset of the disease, slowing or reversing the disease process, 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 a further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial for treating the patient. The compositions of the present application form viable therapeutic agents without the need to achieve complete cure or eradication of any symptoms or manifestations of the disease. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, prophylactic administration of a treatment to form a viable prophylactic agent does not need to be completely effective in preventing the onset of the condition. Simply reducing the impact of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by enhancing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of the disease occurring or worsening is sufficient.

[0075] In the present application, the term "about" generally refers to a variation in the range of about 0.5% - 10% above or below the specified value, for example, in 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.

[0076] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention. For those not specified in the embodiments in terms of specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0077] Example 1 Screening and Isolation of Lactobacillus jensenii

[0078] (1) Collection of samples

[0079] The sample sources are naturally fermented foods with regional characteristics, vaginal secretion samples of volunteers collected by hospital gynecologists who meet the inclusion criteria, and human samples (feces and breast milk).

[0080] (2) Isolation of strains

[0081] The vaginal secretions of healthy women of childbearing age were serially diluted tenfold with normal saline, and the diluent of an appropriate dilution gradient was spread on an anaerobic blood agar plate (purchased from Huankai Microorganism), and cultured at 37 °C for 48 - 72 h in an anaerobic workstation. Single colonies with different morphologies were picked and streaked and purified on the blood agar plate, and continued to be cultured. The pure culture was picked for strain identification (16S rDNA sequencing). Through identification and screening, a strain of Lactobacillus jensenii was obtained and named Lactobacillus jensenii HY1335.

[0082] After the strain species was determined, the pure culture (i.e., pure Lactobacillus jensenii HY1335) was inoculated into MRS broth liquid medium for expansion culture. When the strain grew to an appropriate concentration, a sterile 50% glycerol solution was mixed evenly with the same volume of liquid culture, and stored in a -80 °C strain bank.

[0083] Example 2 Identification of Lactobacillus jensenii

[0084] (1) Colony characteristics

[0085] Dip the inoculation loop into the bacterial liquid in the bacterial tube and streak-inoculate it on the MRS plate, and culture it anaerobically at 37 °C for 48 h, and observe the colony morphology on the plate. As Figure 1 shown, after the strain was cultured on the MRS plate, round, rough-edged, milky white colonies were formed, and the middle of the colony protruded.

[0086] (2) Staining and microscopic examination

[0087] Pick 1 loop of sterilized distilled water with an inoculation loop onto a clean glass slide, pick a single colony from the MRS plate, mix it evenly with the distilled water and spread it on the glass slide, stain it according to the instructions of the Gram staining solution kit (purchased from Qingdao Haibo), and then observe the cell morphology under an electron microscope. As Figure 2 shown, after the strain was stained and observed through an optical microscope, it could be judged that the cells were stained Gram-positive and the cells were short rod-shaped. Through the observation of colony characteristics and Gram staining identification, the isolated strain was initially determined to be Lactobacillus.

[0088] (3) Biochemical identification analysis

[0089] ① Lactobacillus culture: Inoculate the target strain into MRS broth medium (purchased from Qingdao Haibo), and culture it in an anaerobic incubator at 37 °C for 24 h.

[0090] ② Bacterial suspension preparation: Centrifuge the strain fermentation broth at 4000 rpm for 5 min in a centrifuge, discard the supernatant, wash the bacterial sludge with normal saline, centrifuge at 4000 rpm for 5 min, discard the supernatant, wash the bacterial sludge with normal saline again, and centrifuge at 4000 rpm for 5 min to discard the supernatant, and add normal saline and mix evenly with the bacterial sludge for standby.

[0091] ③1% Sodium Hippurate Identification Experiment: Pipette 50 μL of the bacterial suspension and add it to the 1% sodium hippurate identification tube. Seal the tube with a sealing film and incubate it in a water bath at 37°C for 2 h. Then, slowly add 200 μL of ninhydrin solution (3.5% ninhydrin solution: 0.175 g of ninhydrin hydrate, 2.5 mL of acetone, 2.5 mL of butanol) along the tube wall without shaking. After placing it in a water bath at 37°C for 10 min, read the result.

[0092] ④Other Identification Experiments: Pipette 50 μL of the bacterial suspension and add it to the biochemical identification tube (Note: For the aesculin identification tube, after adding the bacterial suspension, the liquid surface needs to be covered with sterile liquid paraffin). Seal the tube with a sealing film and place it in an anaerobic incubator. Incubate it at 37°C for 48 h and then read the result. The read results are shown in Table 1:

[0093] Table 1 Reading Results of Lactobacillus jensenii Identification Tubes

[0094]

[0095] Note: + represents positive; - represents negative; +w represents weakly positive.

[0096] The biochemical identification results show that Lactobacillus jensenii can utilize aesculin, cellobiose, maltose, salicin, sucrose, and inulin as carbon sources.

[0097] (4) 16S rDNA Identification

[0098] Amplify and sequence the above Lactobacillus jensenii through 16S rDNA, and perform a BLAST comparison in the NCBI database after obtaining the sequence. The 16S rDNA comparison results show that the above strain is Lactobacillus jensenii, named Lactobacillus jensenii HY1335. The determined gene sequence of 16S rDNA is as follows:

[0099] AGTCGAGCGAGCTTGCCTATAGAAATTCTTCGGAATGGACATAGATACAAGCTAGCGGCGGAT

[0100] GGGTGAGTAACGCGTGGGTAACCTGCCCTTAAGTCTGGGATACCATTTGGAAACAGATGCTAATAC

[0101] CGGATAAAAGCTACTTTCGCATGAAAGAAGTTTAAAAGGCGGCGTAAGCTGTCGCTAAAGGATGG

[0102] ACCTGCGATGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTG

[0103] AGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGG

[0104] AATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATC

[0105] GTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAAC

[0106] CAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGG

[0107] ATTTATTGGGCGTAAAGCGAGCGCAGGCGGATTGATAAGTCTGATGTGAAAGCCTTCGGCTCAACC

[0108] GAAGAACTGCATCAGAAACTGTCAATCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGC

[0109] GGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACG

[0110] CTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATG

[0111] AGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGG

[0112] GGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCAT

[0113] GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGA

[0114] TTAGGTTTTCCCTTCGGGGACAAAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA

[0115] TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTAATAGTTGCCAGCATTAAGTTGGGCACTCT

[0116] ATTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGA

[0117] CCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAACCTGTGAAGGCAAGCGGAT

[0118] CTCTTAAAGCTGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGCTGGAATCGCTAG

[0119] TAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA

[0120] TGAGAGNTTGTAACACCCAAAGTCGGTGAGGTAACCNTTGGAGCCAGCCGCCTAA(SEQ ID NO:1)

[0121] (5) Specific nucleotide sequence molecular marker

[0122] ① Specific primer design

[0123] a. Screening of specific nucleotide sequence: The whole genome of strain HY1335 was sequenced and genome analysis was carried out. It was compared and analyzed with the genome sequences of Lactobacillus jensenii strains deposited in the NCBI database to screen out the specific nucleotide sequence of Lactobacillus jensenii HY1335.

[0124] b. Primer design: Design primers for the specific nucleotide sequences screened in step a. Design primer fragments with a predicted product length of approximately 200 to 800 bp for the screened specific nucleotide sequences, and design 10 pairs of primers for other nucleotide sequences on the whole genome as controls. The primer sequences are shown in Table 2:

[0125] Table 2 Primer List

[0126]

[0127]

[0128] c. Primer screening: Use Lactobacillus jensenii HY1335 and 3 strains of the same species as the control group (specific strain information is shown in Table 3) to prepare templates. Pick single colonies into 50 μL of lysis buffer (purchased from TaKaRa) respectively, after simple centrifugation, lyse at 80 °C for 15 min, centrifuge at 4000 rpm for 5 min, and the supernatant is the template. Through primer screening, select 1-p2 for PCR amplification experiment. The PCR amplification system is 12.5 μL of Taq enzyme, 1 μL of F primer, 1 μL of R primer, 1.5 μL of template, and make up to 25 μL with ddH2O; the PCR reaction conditions are as shown in Table 4 below, and perform the PCR experiment. After the PCR experiment, add 1.5 g of agarose to 100 mL of 1×TAE buffer, heat and melt it thoroughly, add 5 μL of Gel Red dye, pour it into the gel plate and let it solidify for use. Take 4 μL of the PCR product for agarose gel electrophoresis. The electrophoresis conditions are as follows: voltage 130 V, current 400 mA, time 35 min.

[0129] Table 3 Strain Information

[0130] Strain number Identified name HY1335 Lactobacillus jensenii HY00888 Lactobacillus jensenii HY01360 Lactobacillus jensenii HY02445 Lactobacillus jensenii

[0131] Note: The strains with strain numbers HY00888, HY01360, and HY02445 in Table 3 are Lactobacillus jensenii strains screened from the samples collected by the inventors according to Example 1. In order to verify the specificity of the above specific nucleotide sequences for Lactobacillus jensenii HY1335, the above primers were used to perform PCR amplification on HY1335 and 3 strains of the same species respectively.

[0132] Table 4 PCR Reaction Conditions

[0133]

[0134] Among the primers designed for specific nucleotide sequences, when using 1-p2 as the primer for PCR amplification experiments, only HY1335 had a specific amplification band, the band was clear and had a high concentration, and there were relatively few primer dimers. For HY00888, HY01360, and HY02445, no amplification bands were observed. The agarose gel electrophoresis detection pattern is shown in Figure 3 . However, when using primers designed for other nucleotide sequences for PCR amplification experiments, there were cases where bands were also amplified in the control bacteria group. Therefore, it shows that primer 1-p2 is a molecular marker primer for HY1335. The amplification product generated by this primer is the molecular marker of Lactobacillus jensenii HY1335. The size of the amplification product (molecular marker) of primer 1-p2 is 363 bp, and the nucleotide sequence is as shown in SEQ ID NO:24. Thus, it is determined that the nucleotide sequence shown in SEQ ID NO:24 is a 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 the specific nucleotide sequence marker of Lactobacillus jensenii HY1335. This provides a reliable tool and basis for the identification and detection of Lactobacillus jensenii HY1335.

[0135] GGATCGGGTAATGGCACCAATATAATAAATGATAAAGTTAAAATTCCAACCGGTGCTTTTCAGTCTAAACAAATGCAAACTGAAAATTGCAAAGTAAATAATCAGATGTTGGATAAAACTGAAGTAACAAATTCTACTAAAAATTCTTCTACACTAAATGTGGAAAATTCCAAGACTGCTGATTTGTCGAGATTTGATTACTCTTTATATACAAAAAAAGTAAAGAGTTTTGAATTTAGAAATTCAGATAATAACGATGTAATTAGAACAGTAATATTAAACAAACCTACCGGTGTTGAAACTGTAACTATGACTTTGAATGTTAGCTGCTTAAAAGGGAGAAAGCATCAGCATCAATCGCAC(SEQ IDNO:24).

[0136] d. The inventors found during the experiment that when using primer 1-p2 to sequence multiple Lactobacillus jensenii HY1335 samples, it was found that there were differences in individual bases between the amplified product sequences and the nucleotide sequence shown in SEQ ID NO:24. However, compared with the control bacteria, they all had specific amplification bands, the bands were clear and had a high concentration, and there were relatively few primer dimers.

[0137] In an experiment, it was found that the nucleotide sequence of the product amplified from the Lactobacillus jensenii HY1335 sample using the 1-p2 primer pair is shown in SEQ ID NO:25, and the result of alignment with the nucleotide sequence shown in SEQ ID NO:24 is 99.17%.

[0138] GATCGGGTAATGGCACCAATATAATAAATGATAAAGTTAAAATTCCAACCGGTGCTTTTCAGTCTAAACAAATGCAAACTGAAAATTGCAAAGTAAATAATCAGATGTTGGATAAAACTGAAGTAACAAATTCTACTAAAAATTCTTCTACACTAAATGTGGAAAATTCCAAGACTGCTGATTTGTCGAGATTTGATTACTCTTTATATACAAAAAAAGTAAAGAGTTTTGAATTTAGAAATTCAGATAATAACGATGTAATTAGAACAGTAATATTAAACAAACCTACCGGTGTTGAAACTGTAACTATGACTTTGAATGTTAGCTGCTTAAAAGGGAGAAAGCATCAGACCAAATCGCACA(SEQ IDNO:25)

[0139] Therefore, after multiple sequencing and alignment, the homology range of the molecular marker of Lactobacillus jensenii HY1335 with the nucleotide sequence shown in SEQ ID NO:24 was determined, that is, the molecular marker of Lactobacillus jensenii HY1335 has a nucleotide sequence with at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:24.

[0140] Example 3 Strain Characteristics

[0141] (1) Antibiotic Sensitivity Test

[0142] Lactobacillus jensenii HY1335 was cultured in MRS broth, and then the bacterial solution was evenly spread on an MRS plate. After the bacterial solution was absorbed and dried, antibiotic susceptibility test strips were applied, and anaerobic culture was carried out at 37°C for 48 h. The diameter of the inhibition zone was measured using a vernier caliper. The antibiotic sensitivity of the strain was judged by the diameter of the inhibition zone of the strain to the antibiotic susceptibility test strips. The results are shown in Table 5. The strain is resistant to metronidazole, norfloxacin, ofloxacin, ciprofloxacin, and kanamycin, and sensitive to clindamycin and cefuroxime.

[0143] Table 5 Results of Antibiotic Sensitivity Test

[0144]

[0145] Note: S: Sensitive (15 - 20 mm highly sensitive; > 20 mm extremely sensitive) I: Intermediate (10 - 14 mm moderately sensitive) R: Resistant (< 10 mm insensitive)

[0146] (2) Toxicity test

[0147] ① Hemolysis experiment

[0148] Dip the bacterial liquid in the bacterial strain cryopreservation tube and streak inoculate it on an anaerobic blood agar plate, and culture it anaerobically at 37 °C for 48 h, and observe the change in the color of the blood agar plate around the colony. The results of the hemolysis experiment are as Figure 4 shown. Grayish-white fine colonies appear in the medium around the colony, and there is no hemolysis ring around the colony, indicating that Lactobacillus jensenii HY1335 is γ-hemolytic, that is, non-hemolytic.

[0149] ② Mouse toxicity experiment

[0150] Use 5 mice weighing 18 - 22 g, and orally gavage 0.5 ml of fresh bacterial liquid (not less than 1.0×10 CFU / 0.5 ml) for each mouse once a day for 3 consecutive days. Continuously observe from the first day of gavage until the 7th day. The mice should all survive healthily and gain weight. The experimental results show that after the mice were gavaged with the fresh bacterial liquid of Lactobacillus jensenii HY1335, they all survived healthily and gained weight.

[0151] (3) Determination of metabolite content

[0152] ① D-lactic acid detection

[0153] Use a D-lactic acid detection kit (purchased from Sigma-Aldrich) to detect the D-lactic acid production in the supernatant of Lactobacillus. The method used by this kit is: D-lactic acid is oxidized by a specific D-lactic acid dehydrogenase to produce a color reaction, and it has a proportional relationship with the D-lactic acid concentration. Measure the absorbance at 450 nm.

[0154] ② L-lactic acid detection

[0155] Filter the supernatant with a 0.22 μm sterile filter membrane. Use a biosensor to determine the L-lactic acid concentration.

[0156] ③ Hydrogen peroxide detection

[0157] Prepare 100 μl of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mM hydrogen peroxide standard solutions with PBS solution, add 200 μl of enzyme reagent (0.5 mM / L 4 - aminophenazone (4 - AA), 0.2 mM / L peroxidase), add 200 μl of phenol solution (19.66 mM / L), mix well, incubate at 37 °C for 20 min, evenly take 200 μL and place it in a 96 - well plate, and measure the absorbance of each well at a wavelength of 505 nm. Establish a standard curve of hydrogen peroxide solution based on the detection results.

[0158] Inoculate Lactobacillus at an inoculation amount of 0.5% into MRS medium and culture it in an anaerobic chamber at 37 °C for 24 h. When the Lactobacillus is cultured for 24 h, centrifuge at 4000 rpm at 4 °C for 20 min, discard the supernatant, wash it with PBS buffer, and resuspend the cells with 0.5% glucose solution until OD 600 = 1.0. Incubate at 37 °C and 170 rpm for 5 hours, centrifuge at 10,000 rpm for 5 min, collect the supernatant, filter and sterilize it with a 0.22 μm filter for standby. Take 100 μl of the supernatant, add 200 μL of enzyme reagent and phenol reagent respectively, mix well, incubate at 37 °C for 20 min. Evenly take 200 μL and place it in a 96 - well plate, and measure the absorbance of each well at a wavelength of 505 nm.

[0159] Lactobacillus jensenii HY1335 was cultured in MRS broth for 24 h. The detection 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 is 5.23 g / L, the content of L - lactic acid is 0 g / L, and the total acid production of Lactobacillus jensenii HY1335 is 5.23 g / L, which is basically the same as that of Lactobacillus delbrueckii subsp. bulgaricus DJS - 5.48 g / L; H2O2 can directly kill pathogenic bacteria, and can also be catalytically oxidized by biological enzymes to form halides with stronger bactericidal effects. As can be seen from Table 6, Lactobacillus jensenii HY1335 can produce 3170 μM hydrogen peroxide, while the amount of hydrogen peroxide produced by Lactobacillus delbrueckii subsp. bulgaricus DJS is only 80.67 μM. The ability of Lactobacillus jensenii HY1335 to produce hydrogen peroxide is much higher than that of Lactobacillus delbrueckii subsp. bulgaricus DJS. Therefore, Lactobacillus jensenii HY1335 has the potential to kill pathogenic bacteria, inhibit the growth and reproduction of harmful bacteria, prevent vaginal infections and treat inflammation.

[0160] Table 6 Determination results of metabolite contents

[0161] Metabolite Lactobacillus jensenii HY1335 Lactobacillus delbrueckii subsp. bulgaricus DJS D-lactic acid 5.23 g / L 5.24 g / L L-lactic acid 0 g / L 0.24 g / L Hydrogen peroxide 3170 μM 80.67 μM

[0162] Example 4 Application function analysis

[0163] (1) Bacteriostatic experiment

[0164] ① Preparation of working bacterial solution: Lactobacillus was inoculated into MM medium (modified MRS broth, with the following 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 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) at an inoculation amount of 0.5%, and cultured in an anaerobic workstation. Six pathogenic bacteria were cultured using a suitable medium (Gardnerella vaginalis ATCC14018, abbreviated as GV; Escherichia coli ATCC 25922, abbreviated as EC; Staphylococcus aureus ATCC 25923, abbreviated as SA; Prevotella bivia NCTC 11156, abbreviated as PB; Canidia albicans ATCC 10231, abbreviated as CA). After culturing the target strains, the supernatant was obtained by centrifugation, and the cell-free supernatant was obtained by filtration through a 0.22 μm filter, and used immediately or stored in a -80°C refrigerator. After culturing the pathogenic bacteria, the OD 600 value was measured and diluted to an OD 600 value of about 0.005 (the viable cell count was maintained at 5.0×10 5 CFU / mL - 5.0×10 6 CFU / mL) (WS / T 650—2019 Antibacterial and bacteriostatic effect evaluation method).

[0165] ② Interaction: The same volume of supernatant and pathogenic bacteria solution were mixed evenly. Immediately, 100 μL of the evenly mixed bacterial solution was taken and placed in a blank 96-well plate to measure the OD 600 ; the remaining culture solution was placed at 37°C and cultured anaerobically or aerobically according to the culture conditions of the pathogenic bacteria. After 48 h of culture, 100 μL of the evenly mixed bacterial solution was taken and placed in a blank 96-well plate to measure the OD 600 . A blank control group was set up, with 2 replicates for each sample. According to the following formula, the bacteriostatic rate of Lactobacillus against pathogenic bacteria was calculated.

[0166] Bacteriostatic rate = (A - B) / A * 100%

[0167] A: The OD 600 value increased within 48 h in the positive control group (i.e., blank medium);

[0168] B: The OD 600 value increased within 48 h in the experimental group.

[0169] Two different strains of the same bacterial species and the positive drug Ding Junsheng were selected, and the antibacterial performance of Lactobacillus jensenii HY1335 screened out was detected. The results are shown in Table 7. This strain has a strong antibacterial rate against GV, PB, EC, SA, and CA, and its antibacterial performance against GV, PB, EC, and SA is better than that of the control group of the same bacterial species and the positive control group. This strain can simultaneously have a strong inhibitory effect on these four pathogenic bacteria, while neither the positive control nor the control group of different strains of the same bacterial species can achieve this effect.

[0170] Table 7 Antibacterial experiment results

[0171]

[0172] Note: The strains with strain numbers HY02405 and HY01361 in Table 7 are Lactobacillus jensenii screened from the samples collected by the inventors according to Example 1 to verify the inhibition rates of Lactobacillus jensenii HY1335 and two strains of the same bacterial species against different pathogenic bacteria.

[0173] (2) Co-culture experiment of Lactobacillus and GV

[0174] Lactobacillus culture: Take the glycerol tube of Lactobacillus jensenii and inoculate it into MM medium (modified MRS broth, the composition of the components is: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) respectively. Concentrate or dilute the cultured bacterial solution to about 1×10 9 CFU / mL concentration as the working bacterial solution of Lactobacillus.

[0175] GV culture: Take the GV glycerol tube and inoculate it into BHI liquid medium containing 10% fetal bovine serum, and culture it anaerobically at 37°C. Centrifuge the cultured bacterial solution to remove the supernatant, and then adjust the bacterial solution concentration to about 1.0×10 7 CFU / mL as the working bacterial solution of GV.

[0176] Take 400 μL of the working bacterial solutions of Lactobacillus and GV and inoculate them into 40 ml of BHI liquid medium containing 10% fetal bovine serum respectively. GV inoculated alone is used as the blank control group, with 2 replicates in each group. Place them in an anaerobic workstation for culture, and sample at 27 h after culture respectively. The viable count of GV is detected by fluorescence quantitative qPCR method using GV-specific probe primers. The viable count of GV was detected after co-culturing Lactobacillus jensenii and GV for 27 h. The experimental results are shown in Table 8. After co-culturing Lactobacillus jensenii HY1335 and GV for 27 h, the antibacterial rate reached 89%, indicating that this strain has a strong inhibitory effect on the growth of GV.

[0177] Table 8 Antibacterial experiment of co - culture of Lactobacillus and GV

[0178]

[0179] (3) Biofilm clearance experiment

[0180] 1) Biofilm clearance experiment of Lactobacillus on GV

[0181] Adjust the bacterial liquid of GV after cultivation to 1.0×10 7 CFU / mL, inoculate it into a 96 - well plate, and set up 2 groups in total, with 4 parallels in each group. The two groups were respectively added with the same volume of blank MM liquid medium (modified MRS broth, the composition of the components is: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and the supernatant of Lactobacillus jensenii HY1335 after cultivation. Incubate in a 37℃ anaerobic workstation for 24 h.

[0182] After the cultivation, the microplate crystal violet staining method was used to determine the inhibitory effect of Lactobacillus jensenii HY1335 on the formation of GV biofilm. The experimental results are shown in Table 9. The supernatant of Lactobacillus jensenii HY1335 has a strong inhibitory effect on the formation of GV biofilm, reaching 91.4%. Therefore, it can effectively inhibit the growth of GV.

[0183] Table 9 Results of biofilm clearance experiment of Lactobacillus on GV

[0184] Strain Inhibitory rate of Lactobacillus supernatant on GV biofilm formation Lactobacillus jensenii HY1335 91.4％±2.4％

[0185] 2) Biofilm clearance experiment of Lactobacillus on CA

[0186] Inhibitory biofilm clearance experiment: Adjust the bacterial liquid of CA after cultivation to 1.0×10 7 CFU / mL, inoculate it into a 96 - well plate, and set up 2 groups in total, with 4 parallels in each group. The two groups were respectively added with the same volume of Sabouraud dextrose liquid medium and the supernatant of Lactobacillus. Incubate in a 37℃ anaerobic workstation for 24 h. After the cultivation, the microplate crystal violet staining method was used to determine the inhibitory effect of Lactobacillus on the formation of CA biofilm.

[0187] Biofilm clearance experiment: Adjust the bacterial liquid of CA after cultivation to 1.0×10 7CFU / mL, inoculated into a 96-well plate, with a total of 2 groups set up, 4 parallels in each group. Incubated in an anaerobic workstation at 37 °C for 24 h. After 24 h of incubation, the mixture in the wells was discarded and washed with sterile PBS. The same volume of MM medium (modified MRS broth, with the following 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 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and the supernatant of Lactobacillus were added to each group in turn, and incubated at 37 °C for 24 h. After the incubation was completed, the microplate crystal violet staining method was used to determine the clearance effect of the Lactobacillus supernatant on mature CA biofilm.

[0188] 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 formed CA biofilm.

[0189] Table 10 Experimental results of the clearance of CA biofilm by Lactobacillus

[0190]

[0191] (4) Cell adhesion experiment

[0192] ① Preparation of working bacterial solution: Culture Lactobacillus, detect the OD 600 value of the bacterial solution. Centrifuge the cultured Lactobacillus culture solution at 4 °C and 4000 rpm for 5 min, discard the supernatant, wash it three times repeatedly with PBS, and finally resuspend Lactobacillus with MEM complete medium (purchased from Zhongqiao Xinzhou). Take an appropriate amount of the resuspended solution and detect the viable cell count by the plate spreading method, which is L1.

[0193] ② Cell culture: Hela cells were inoculated into a 24-well plate, with 1.5×10 5 cells / mL in each well, and cultured for 16 h until the confluence rate reached 90%. The HeLa cells cultured in the 24-well plate were washed with serum-free MEM medium (purchased from Zhongqiao Xinzhou), and then counted, recorded as C1.

[0194] ③ Interaction: Bacteria and cells were mixed at a ratio of 100:1. Lactobacillus was inoculated into the cells and incubated in an environment of 37 °C and 5% CO2 for 1.5 h. The centrifuged supernatant was added to the cells as a blank control group. After 1.5 h, the culture solution in the wells was collected, the cells in the wells were washed with MEM medium, then trypsin was added to each well for digestion, and then MEM complete medium was added to terminate the reaction. The suspension was collected, a part of it was taken to count the cell number C2, and the plate spreading method was used to detect the viable cell count L2 of Lactobacillus adhering to the cells.

[0195] ④ Calculate the number of adhesions and the adhesion rate: Average number of Lactobacillus cell adhesions = L2 / C2.

[0196] The number of single-cell adhesions of Lactobacillus jensenii HY1335 to Hela cells is 3.60 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epidermal cells.

[0197] (5) Efficacy test on the BV (bacterial vaginitis) animal model

[0198] Healthy SPF-grade Balb / c mice, female, 6 - 8 weeks old were used for modeling. After adaptive cultivation of the animals, they were randomly divided into groups of 8 each, including a model group (M), an experimental group (Lactobacillus jensenii HY1335), and a positive control group (DJS). Before inoculating the pathogenic bacteria in each group of animals, estradiol benzoate injection was subcutaneously injected, and then the same concentration of GV (20 μl) was vaginally administered to establish a BV pathogenic bacteria model. After successful modeling, the experimental group was continuously given Lactobacillus jensenii HY1335 solution (1×10 10 CFU / mL, 20 μL) vaginally for 5 consecutive days, and the positive control group was continuously given an equal amount of Lactobacillus delbrueckii solution (the strain in Ding Junsheng, 1×10 10 CFU / mL, 20 μL) vaginally for 5 consecutive days. The model group was treated with an equal volume of normal saline. After the treatment was completed, each animal was lavaged vaginally with 50 μL / time of PBS, 4 times per animal. The liquid was collected and placed in a 1.5 mL Ep tube and stored at -80 °C. The qPCR method was used to detect the load of GV in the lavage fluid, and then the differences in the load of GV in the lavage fluid among the groups were compared.

[0199] The experimental results are as Figure 5 shown. The experimental results show that using Lactobacillus jensenii HY1335 to treat the BV-infected animal model significantly reduced the content of GV in its vagina, and the treatment effect was significantly better than that of the positive drug (DJS), indicating that this strain has a good treatment effect on BV in mice.

[0200] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0201] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A microorganism, which is Lactobacillus jensenii, was deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on February 7, 2023, and the deposit number is CGMCC No. 26505.

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

1.

3. A primer set for detecting the microorganism according to claim 1 or 2, 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.

4. A microbial preparation, characterized in that, Comprising the microorganism according to claim 1 or 2.

5. The microbial agent according to claim 4, characterized in that, Further comprising a pharmaceutically acceptable carrier or excipient.

6. A single-dose preparation, characterized in that, Includes 1×10 6 ~1×10 11 The microorganism according to claim 1 is used as an active ingredient.

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

8. Use of the microorganism according to claim 1 or 2, the microbial preparation according to claim 4 or 5, or the single-dose preparation according to claim 6 in the preparation of a drug for inhibiting vaginal pathogenic bacteria.

9. The use according to claim 7 or 8, 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.

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

11. Application of the microorganism according to claim 1 or 2, the microbial preparation according to claim 4 or 5, or the single-dose preparation according to claim 6 in the preparation of a product for regulating vaginal flora balance.

12. A molecular marker of the microorganism according to claim 1 or 2, characterized in that, The molecular marker comprises a nucleotide sequence selected from one of the following: (1) The nucleotide sequence shown in SEQ ID NO: 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.

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

24.

14. The molecular marker according to claim 12, characterized in that, The primer of the molecular marker comprises the primer group according to claim 3.

15. A method for detecting the microorganism according to claim 1 or 2, characterized in that, Comprising: Using the primer group according to claim 3 to perform amplification treatment on the DNA of the strain to be detected, so as to obtain the microorganism according to claim 1 or 2.

16. The method according to claim 15, wherein The product of the amplification treatment has the nucleotide sequence shown in SEQ ID NO: 24, which is an indication that the strain to be detected is the microorganism described in claim 1 or 2.

17. The method according to claim 15, wherein It includes the following steps: extracting a DNA sample from the strain to be detected, performing PCR amplification treatment on the DNA sample using the primer set described in claim 3, and then comparing the amplification result with the molecular marker in claim 12, so as to identify whether the strain to be detected is the microorganism described in claim 1 or 2.

18. Use of the molecular marker according to claim 12 for identifying and detecting the microorganism according to claim 1 or 2.

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