Limosilactobacillus fermentum and use thereof

Abstract

The present invention relates to a gene for a molecular marker of Limosilactobacillus fermentum and a Limosilactobacillus fermentum containing the gene. The gene of the molecular marker thereof has a nucleotide sequence set forth in SEQ ID NO: 24. The Limosilactobacillus fermentum has good efficacy in preventing and / or treating vaginal infections and related diseases thereof, and the problems of increased drug resistance and high recurrence rate caused by using antibacterial drugs such as antibiotics in the treatment of vaginal infections and related diseases thereof can be avoided.

Description

A fermented mucus lactobacillus and its application Technical Field

[0001] The present invention belongs to the field of biomedicine and specifically relates to a fermented Lactobacillus mucilaginosus and its use in preparing a medicament for preventing and / or treating vaginal infection and related diseases. Background Art

[0002] With economic development and improvement of living standards, female reproductive tract diseases are becoming more and more common, and vaginitis is even more common, which has a serious impact on women's quality of life and physical and mental health.

[0003] 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 typical clinical features of BV are foul-smelling, watery, or gray vaginal discharge. Patients may also develop various complications due to ascending infection, such as chronic cervicitis, pelvic inflammatory disease, endometritis, and even infertility.

[0004] Vulvovaginal candidiasis (VVC) is the second most common vaginal infection after bacterial vaginosis. Studies have found that approximately 78% of women experience at least one episode of VVC in their lifetime, 65% experience three or fewer annual episodes, and 35% experience four or more annual episodes. VVC typically presents with vulvar itching, burning, painful urination, and a dreg-like vaginal discharge, often impairing patients' quality of life.

[0005] Currently, the treatment of BV is primarily based on Western medicine, often using antimicrobial agents such as antibiotics, including metronidazole, clindamycin, and tinidazole. However, the formation and persistence of pathogenic biofilms can lead to recurrence. Reports indicate that recurrence rates in BV patients treated with oral metronidazole were 23%, 49%, 59%, and 68% at 1, 3, 6, and 12 months, respectively. At 12 months after treatment, 84% of patients still had abnormal vaginal flora. Furthermore, long-term, high-dose antibiotic use not only increases pathogen resistance but also inhibits the growth of some vaginal flora, allowing the previously limited Candida albicans to proliferate, leading to vaginal flora disturbance. The role of live bacterial preparations in the treatment of BV remains controversial. Clinical studies have shown that the use of live bacterial preparations is primarily divided into two categories: conventional antibiotic treatment followed by probiotics or probiotics alone. The results of several clinical trials examining combination therapy have been inconsistent, making it uncertain whether the combined use of antibiotics and probiotics is effective for the treatment of bacterial vaginosis. Other studies have shown that some patients fail to achieve satisfactory results with probiotic preparations alone. Therefore, the effectiveness of probiotics alone in treating BV remains controversial, and extensive research is needed. Currently, the primary probiotic used clinically for the treatment of bacterial vaginosis is the Lactobacillus Live Vaginal Capsules (Dingjunsheng) developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd.

[0006] Currently, azoles and polyenes are the most commonly used drugs in the clinic for the treatment of VVC. For example, diazepam suppositories are often used to treat vulvovaginal candidiasis. However, with the long-term use of antifungal drugs in clinical practice, Candida species undergo phenotypic changes and virulence factor mutations, and infections with non-albicans Candida species are increasing, challenging the therapeutic efficacy of traditional antifungal drugs. Lactobacilli can be used as an adjunctive therapy in combination with antifungal drugs such as azoles in the treatment of various VVC patients, with some efficacy. However, compared with conventional antifungal drugs, there is currently insufficient evidence to demonstrate that the use of Lactobacillus preparations alone is effective in the treatment of VVC.

[0007] Summary of the Invention

[0008] In response to the above problems, the present invention aims to provide a fermented Lactobacillus muciniphila strain and its use in the preparation of a medicament for preventing and / or treating vaginal infections and related diseases. This Lactobacillus muciniphila strain is a new strain screened by the inventors and has excellent biological properties and is effective in preventing and / or treating vaginal infections and related diseases.

[0009] The above object of the present invention is achieved by providing the following technical solutions:

[0010] In a first aspect, the present invention provides a gene (or DNA molecule) for a molecular marker of fermenting Lactobacillus mucilaginosus, which has a nucleotide sequence selected from one of the following:

[0011] (1) the nucleotide sequence shown in SEQ ID NO: 24;

[0012] (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence shown in SEQ ID NO: 24;

[0013] (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;

[0014] Preferably, the gene of the molecular marker for fermenting Lactobacillus mucilaginosus has a nucleotide sequence as shown in SEQ ID NO: 24, or its nucleotide sequence is as shown in SEQ ID NO: 24.

[0015] In a second aspect, the present invention provides a fermentative Lactobacillus muciniphila comprising the gene of the molecular marker for fermenting Lactobacillus muciniphila according to the present invention.

[0016] In a third aspect, the present invention provides a primer set for identifying the Lactobacillus mucilaginosus according to the present invention, comprising a forward primer and a reverse primer, wherein:

[0017] The nucleotide sequence of the forward primer is shown in SEQ ID NO: 1;

[0018] The nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2.

[0019] In a fourth aspect, the present invention provides a method for identifying the fermentative Lactobacillus mucilaginosus according to the present invention, comprising: amplifying the genomic DNA of the strain to be identified using the primer set according to the present invention, and then detecting the amplified product.

[0020] Preferably, the method comprises the following steps: extracting a genomic DNA sample from the strain to be identified, performing PCR amplification on the genomic DNA sample using the primer set according to the present invention, and then comparing the amplified product with the gene of the molecular marker for fermenting Lactobacillus mucosus according to the present invention; if the amplified product contains the gene of the molecular marker for fermenting Lactobacillus mucosus according to the present invention, then the strain to be identified is the fermenting Lactobacillus mucosus according to the present invention.

[0021] Preferably, if the amplified product comprises the nucleotide sequence shown in SEQ ID NO: 24, the strain to be identified is the fermentative Lactobacillus muciniphila according to the present invention.

[0022] In a fifth aspect, the present invention provides a use of the gene for molecular marker of fermentative Lactobacillus mucilaginosus according to the present invention in identifying the fermentative Lactobacillus mucilaginosus according to the present invention.

[0023] The term "molecular marker" in the present invention refers to a specific DNA fragment that can reflect certain differences in the genomes of different individuals of the same bacterial species.

[0024] In a sixth aspect, the present invention provides a fermentative Lactobacillus mucilaginosus comprising a nucleotide sequence selected from one of the following:

[0025] (1) the nucleotide sequence shown in SEQ ID NO: 23;

[0026] (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology to the nucleotide sequence shown in SEQ ID NO: 23;

[0027] (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: 23.

[0028] Preferably, the genome of the fermentative Lactobacillus muciniphila comprises the nucleotide sequence shown in SEQ ID NO: 23.

[0029] Preferably, the 16S rDNA gene sequence of the fermentative Lactobacillus mucilaginosus is shown as SEQ ID NO: 23.

[0030] Preferably, the fermented mucus lactobacillus is the fermented mucus lactobacillus (Limosilactobacillus fermentum) with a deposit number of CGMCC No. 26501. Specifically, the fermented mucus lactobacillus (HY757) provided by the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (abbreviated as CGMCC), the depository address of which is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 26501 and a deposit date of February 7, 2023.

[0031] This strain has the following properties:

[0032] 1. Colony morphological characteristics:

[0033] The strain forms round, smooth-edged, milky white colonies with a protrusion in the middle after being cultured on MRS plates.

[0034] 2. Morphological characteristics of strains:

[0035] After staining, the strain was observed under an optical microscope, and it was determined that the bacteria were Gram-positive and had a short rod shape.

[0036] 3. Physiological and biochemical characteristics:

[0037] The culture temperature of this strain is 35-38℃, and the optimal growth temperature is 37℃.

[0038] The carbon sources that can be utilized by this strain include maltose, sucrose, raffinose and lactose.

[0039] 4. Nutritional characteristics:

[0040] The strain does not require any special nutrients for cultivation and is cultured in a basic medium, which is MRS medium, in a facultative anaerobic manner.

[0041] In the present invention, the fermented mucus Lactobacillus can be isolated from naturally fermented food or human samples, wherein the naturally fermented food is a naturally fermented food with regional characteristics; the human sample is selected from one or more of vaginal secretions, feces and breast milk.

[0042] In a seventh aspect, the present invention provides a microbial preparation for preventing and / or treating vaginal infection and related diseases, comprising the fermented Lactobacillus mucinus according to the present invention.

[0043] Preferably, the fermented Lactobacillus muciniphila is the only probiotic active ingredient in the microbial preparation.

[0044] Preferably, the total viable count of Lactobacillus mucilaginosus fermentation in the microbial preparation is not less than 1×10 5 CFU / g, preferably 1×10 5 to 1×10 10 CFU / g.

[0045] Preferably, the microbial preparation further comprises a pharmaceutically acceptable excipient.

[0046] Further preferably, the pharmaceutically acceptable excipient is selected from one or more of a surfactant, a preservative, an antioxidant, a hardener, a thickener and an absorption enhancer.

[0047] Preferably, the microbial preparation is a suppository.

[0048] In an eighth aspect, the present invention provides a combination drug for preventing and / or treating vaginal infection and related diseases, wherein the combination drug comprises the fermented Lactobacillus mucosa according to the present invention or the microbial preparation according to the present invention, and other antibacterial drugs.

[0049] In a ninth aspect, the present invention provides use of the fermented Lactobacillus muciniphila according to the present invention or the microbial preparation according to the present invention in the preparation of a medicament for preventing and / or treating vaginal infection and related diseases.

[0050] Preferably, the vaginal infection and related diseases are bacterial vaginosis and / or vulvovaginal candidiasis.

[0051] Further preferably, the pathogenic bacteria of bacterial vaginosis are selected from one or more of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Fannyhessea vaginae and Prevotella bivia.

[0052] More preferably, the Gardnerella vaginalis is the Gardnerella vaginalis with a deposit number of ATCC 14018.

[0053] More preferably, the Escherichia coli is the Escherichia coli with a deposit number of ATCC 25922.

[0054] More preferably, the Staphylococcus aureus is Staphylococcus aureus with a deposit number of ATCC 25923.

[0055] More preferably, the Atopobium vaginalis is Atopobium vaginalis with a deposit number of CCUG 38953.

[0056] More preferably, the Prevotella diversa is the Prevotella diversa with a deposit number of NCTC 11156.

[0057] In a tenth aspect, the present invention provides a method for preventing and / or treating vaginal infection and related diseases, comprising administering the fermented Lactobacillus muciniphila according to the present invention or the microbial preparation according to the present invention to a subject in need thereof.

[0058] Preferably, the vaginal infection and related diseases are bacterial vaginosis and / or vulvovaginal candidiasis.

[0059] Further preferably, the pathogenic bacteria of bacterial vaginosis are selected from one or more of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginalis and Prevotella diversa.

[0060] The present invention has at least the following beneficial effects:

[0061] The specific primers for fermentative Lactobacillus mucilaginosus provided by the present invention can highly match the DNA sequence of the fermentative Lactobacillus mucilaginosus of the present invention and can produce specific amplification only for the DNA sequence of the fermentative Lactobacillus mucilaginosus of the present invention. The method of the present invention can efficiently and conveniently identify and detect the fermentative Lactobacillus mucilaginosus of the present invention.

[0062] The present invention provides a novel fermented Lactobacillus muciniphila strain that is non-toxic, has excellent biological properties, and exhibits excellent therapeutic effects for preventing and / or treating vaginal infections and related diseases. Specifically, the fermented Lactobacillus muciniphila strain of the present invention can produce organic acids with antibacterial effects. The fermented Lactobacillus muciniphila strain of the present invention can inhibit biofilm formation of pathogenic bacteria that cause vaginal infections and related diseases, thereby exhibiting a strong inhibitory effect against pathogenic bacteria that cause vaginal infections and related diseases, such as Gardnerella vaginalis (GV) and Candida albicans (CA).

[0063] The present invention demonstrates the therapeutic potential of the strain for bacterial vaginosis caused by GV and vulvovaginal candidiasis caused by CA through conventional antibacterial experiments, co-culture antibacterial experiments and in vivo animal efficacy experiments.

[0064] The fermented mucus lactobacillus screened out by the present invention avoids the problems of increased drug resistance and high recurrence rate caused by the use of antibacterial drugs such as antibiotics in the treatment of vaginal infection and related diseases.

[0065] BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0067] FIG1 is a colony morphology diagram of the fermented Lactobacillus mucilaginosus of the present invention, wherein the left diagram is a colony diagram of the three-zone line method; the right diagram is a colony diagram of the plate coating method.

[0068] FIG2 is a diagram showing the bacterial morphology of the fermented Lactobacillus mucilaginosus of the present invention.

[0069] Figure 3 is an electrophoretic pattern of PCR amplification of specific nucleotide sequences of four fermentative Lactobacillus mucilaginosus using primer 2-p1 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY757 as a template, 2 uses the supernatant after lysis of HY00810 as a template, 3 uses the supernatant after lysis of HY10008 as a template, and 4 uses the supernatant after lysis of HY00406 as a template.

[0070] FIG4 is an electrophoretic pattern of PCR amplification of specific nucleotide sequences of four Lactobacillus mucilaginosus fermentants using primers 1-p1 according to an embodiment of the present invention, wherein M is a marker, 1 uses the supernatant after lysis of HY757 as a template, 2 uses the supernatant after lysis of HY00810 as a template, 3 uses the supernatant after lysis of HY10008 as a template, and 4 uses the supernatant after lysis of HY00406 as a template.

[0071] FIG5 shows the results of a hemolysis experiment using the fermented Lactobacillus mucilaginosus of the present invention.

[0072] Figure 6 shows the results of the VVC animal model efficacy test.

[0073] Best Mode for Carrying Out the Invention

[0074] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. Example

[0075] 1.1 Experimental methods

[0076] 1.1.1 Isolation of Lactobacillus mucilaginosus

[0077] (1) Collect samples

[0078] The samples were collected from vaginal secretion samples of volunteers who met the inclusion criteria (healthy, disease-free, no use of antibiotics or other drugs in the past month, and no oral probiotic products) by gynecologists at the Yuhang First People's Hospital in Hangzhou.

[0079] (2) Strain isolation

[0080] The sample was serially diluted tenfold with physiological saline. Appropriate dilutions were plated onto MPYG plates and anaerobic blood plates (purchased from Huankai Microbiology) and incubated in an anaerobic workstation at 37°C for 48-72 hours. Individual colonies of varying morphology were streaked onto the blood plates for purification and further incubation. Pure cultures were then selected for 16S rDNA sequencing and identification. Once the strain species was confirmed, the pure culture was inoculated into MPYG liquid medium and expanded. Once the strain reached an appropriate concentration, the mixture was mixed evenly with an equal volume of the liquid culture using sterile 50% (v / v) glycerol solution and stored in a -80°C strain bank.

[0081] 1.1.2 Identification of Lactobacillus muciferus

[0082] (1) Colony characteristics

[0083] Dip the bacterial solution in the culture tube with an inoculating loop, streak inoculate on an MRS plate, and incubate anaerobically at 37°C for 48 hours to observe the colony morphology (as shown in Figure 1, left); dilute the bacterial solution and spread it, incubate anaerobically at 37°C for 48 hours, and observe the colony morphology on the plate (as shown in Figure 1, right).

[0084] (2) Staining microscopy

[0085] Use an inoculating loop to transfer one loop of sterile distilled water onto a clean glass slide. Pick a single colony from the MRS plate, mix it with distilled water, and evenly spread it onto the glass slide. Stain according to the instructions of the Gram stain kit (purchased from Qingdao Haibo Biotechnology Co., Ltd.). Then observe the bacterial morphology.

[0086] (3) Biochemical identification and analysis

[0087] ① Lactobacillus culture: The target strain was inoculated into MRS broth medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), placed in an anaerobic incubator, and cultured at 37°C for 24 hours.

[0088] ② Preparation of bacterial suspension: centrifuge the fermentation liquid of the strain at 4000 rpm for 5 min, 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 mix evenly with the bacterial mud for later use.

[0089] 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.

[0090] ④ Other identification experiments: aspirate 50 μL of bacterial suspension and add it to the biochemical identification tube (Note: after adding the bacterial solution to the aesculin identification tube, the liquid surface 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.

[0091] (4) 16S rDNA identification

[0092] The fermentative Lactobacillus mucilaginosus was amplified and sequenced by 16S rDNA, and the obtained sequence was compared with BLAST in the NCBI database.

[0093] (5) Specific fragment molecular markers

[0094] ①Specific primer design

[0095] a. Specific nucleotide sequence screening: The HY757 strain was subjected to whole genome sequencing and genome analysis, and compared with the genome sequences of Lactobacillus fermentans strains included in the NCBI database to screen for specific nucleotide sequences of Lactobacillus fermentans HY757.

[0096] 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 these specific nucleotide sequences, as shown in Table 1. Also design 10 primer pairs for other specific nucleotide sequences across the entire genome as controls. See Table 1 for primer sequences.

[0097] Table 1 Primer list

[0098] c. Primer Screening: Template preparation was performed using Lactobacillus mucilaginosus HY757 and three strains of the same species as a control group (see Table 2 for specific strain information). Single colonies were picked from each strain in 50 μL of lysis buffer (TaKaRa), lysed at 80°C for 15 minutes, and centrifuged at 4000 rpm for 5 minutes. The supernatant was used as the template. After primer screening, 2-p1 was selected for PCR amplification. The PCR amplification system consisted of 12.5 μL of Taq enzyme, 1 μL of F, 1 μL of R, 1.5 μL of template, and ddHO to 25 μL. PCR reaction conditions are shown in Table 3. After the PCR experiment, 1.5 g of agarose was added to 100 mL of 1× TAE buffer and thoroughly heated to melt. 5 μL of Gel Red dye was added and the sample was poured onto a gel plate to solidify. 4 μL of the PCR product was subjected to agarose gel electrophoresis under the following conditions: voltage 130 V, current 400 mA, and time 35 minutes.

[0099] Table 2 Strain information Note: The strains numbered HY00810, HY10008, and HY00406 in the above table are Lactobacillus muciniphila screened by the inventors from the samples collected in Example 1. To verify the specificity of the above-mentioned specific nucleotide sequence to Lactobacillus muciniphila HY757, PCR amplification was performed on HY757 and three strains of the same species using the above-mentioned primers.

[0100] Table 3 PCR reaction conditions

[0101] 1.1.3 Characteristics of the strain

[0102] (1) Antibiotic sensitivity test

[0103] Lactobacillus muciniphilus HY757 was cultured and fermented in MRS broth. The culture solution was then evenly spread on an MRS plate. After the solution was absorbed and dried, antibiotic susceptibility paper was applied. The plate was incubated anaerobically at 37°C for 48 hours. The diameter of the inhibition zone was measured with a vernier caliper. The inhibition zone diameter was used to determine the strain's antibiotic sensitivity.

[0104] (2) Toxicity test

[0105] ①Hemolysis test

[0106] Dip the bacterial solution in the frozen tube of the culture and streak inoculate it on the anaerobic blood plate. Incubate it anaerobically at 37℃ for 48 hours and observe the color change of the blood plate around the colony.

[0107] ②Toxicity test in mice

[0108] Five mice weighing 18-22 g were used, and each mouse was orally gavaged with 0.5 mL of fresh bacterial solution (no less than 1.0 × 10 9 CFU / 0.5ml), once a day for 3 consecutive days, and the mice were observed continuously from the first day of gavage to the seventh day, and the survival of the mice was observed and weighed.

[0109] (3) Determination of metabolite content

[0110] ①D-lactic acid detection

[0111] The D-lactic acid production of the lactobacillus supernatant was detected using a D-lactic acid detection kit (purchased from Sigma-Aldrich). The principle used in this kit is that 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.

[0112] ②L-lactic acid detection

[0113] The supernatant was filtered through a 0.22 μm sterile filter membrane and the L-lactic acid concentration was measured using a biosensor.

[0114] 1.1.4 Application Function Analysis

[0115] (1) Antibacterial experiment

[0116] ① Preparation of working bacterial solution: Lactobacillus was inoculated into 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) at a 0.5% inoculum size and cultured in an anaesthetic workstation. The following six pathogenic bacteria (Gardnerella vaginalis ATCC14018, abbreviated as GV; Escherichia coli.ATCC 25922, abbreviated as EC; Staphylococcus aureus ATCC 25923, abbreviated as SA; Fannyhessea vaginae CCUG 38953, abbreviated as FV; Prevotella bivia NCTC 11156, abbreviated as PB; Canidia albicans ATCC 10231, abbreviated as CA) were cultured using adapted culture medium. After culturing the target strain, the supernatant was centrifuged and filtered through 0.22 μm to obtain a cell-free supernatant. The supernatant was used immediately or stored in a -80°C freezer. OD values ​​were measured after culturing the pathogenic bacteria. 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 to 5.0×10 6 CFU / mL) (WS / T 650-2019 Evaluation method for antibacterial and antibacterial effects).

[0117] ② Interaction: Take the same volume of supernatant and pathogenic bacteria solution and mix them evenly. Immediately take 100 μL of the mixed 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 Set up a blank control group, with 2 replicates for each sample, and calculate the inhibition rate of Lactobacillus against pathogenic bacteria according to the following formula: Inhibition rate = (AB) / A*100%

[0118] A: OD increase in the positive control group (blank culture medium) within 48 hours 600 value;

[0119] B: OD increase in the experimental group within 48 hours 600 value.

[0120] (2) Co-culture antibacterial experiment

[0121] ① Experiment on the interaction and co-culture of Lactobacillus and CA

[0122] Lactobacillus culture: Take a glycerol tube of fermented Lactobacillus mucilaginosus and inoculate it into 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 concentrate or dilute the cultured bacterial solution to about 1 × 10 9 CFU / mL, used as Lactobacillus working culture solution.

[0123] CA culture: Inoculate the CA glycerol tube into Sabouraud medium and culture aerobically at 37°C. Centrifuge the culture solution to remove the supernatant, and then adjust the concentration of the culture solution to about 1.0 × 10 7 CFU / mL, used as CA working bacterial solution.

[0124] 400 μL of each Lactobacillus and CA working culture solution was inoculated into 40 mL of MM liquid medium. CA alone was inoculated as a positive control. Two replicates were used for each group. The culture solution was gently shaken and incubated at 37°C in an anaerobic workbench. Samples were taken 20 hours after incubation, and viable CA cells were counted using Candida chromogenic medium (purchased from CHROMagar, France).

[0125] ② Experiment on the interaction and co-culture of Lactobacillus and GV

[0126] Lactobacillus culture: Take a glycerol tube of fermented Lactobacillus mucilaginosus and inoculate it into 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 concentrate or dilute the cultured bacterial solution to about 1 × 10 9 CFU / mL concentration is used as the Lactobacillus working culture solution.

[0127] 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.

[0128] 400 μL of Lactobacillus and GV working bacterial solutions were respectively inoculated into 40 mL of BHI liquid culture medium containing 10% fetal bovine serum. GV was inoculated alone as a positive control group. Two replicates were placed in an anaerobic workstation for culture. Samples were taken 27 hours after culture, and the number of viable GV bacteria was detected by fluorescent quantitative qPCR using GV-specific probe primers.

[0129] (3) Biofilm removal experiment

[0130] ① Experiment on the removal of GV biofilm by Lactobacillus

[0131] 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 the fermented Lactobacillus muciniphilus. The plates were incubated at 37°C in an anaerobic workstation for 24 hours.

[0132] After the culture, the inhibitory effect of fermented Lactobacillus mucilaginosus on GV biofilm formation was determined by microplate crystal violet staining.

[0133] ② Experiment on the removal of CA biofilm by Lactobacillus

[0134] 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. 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.

[0135] Biofilm removal experiment: adjust the bacterial solution after CA culture to 1.0×10 7CFU / mL, 100 μL was inoculated into 96-well plates, with two groups of four replicates each. 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. 100 μL 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 the plates were 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 microplates.

[0136] (4) Cell adhesion assay

[0137] ① Preparation of working bacterial solution: culture Lactobacillus and test the OD of bacterial solution 600 The cultured Lactobacillus culture was centrifuged at 4000 rpm for 5 min at 4°C, the supernatant was discarded, and the mixture 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.

[0138] 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.

[0139] 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. After trypsinization, each well was added to digest the cells, and then MEM complete medium was added to terminate the reaction. 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 method (L2).

[0140] ④ Calculate the number of adhesions and adhesion rate: average number of Lactobacillus cells adhered = L2 / C2, adhesion rate (%) = 100*L2 / L1.

[0141] (5) VVC animal model efficacy test

[0142] Healthy SPF-grade Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups: a model group (M), an experimental group (fermented Lactobacillus muciniphila HY757), and a positive control group (dimethoprim-3 suppositories). Each group received a subcutaneous injection of estradiol benzoate for pretreatment. CA (15 μL / mouse) was administered continuously from D0 to D2, and the experimental group received vaginal administration of Lactobacillus muciniphila HY757 (5×10 β-lactamase) daily from D3 to D7. 9 CFU / mL, 20 μL). A positive control group was given 30 mg of bisoprolol suppositories, and a model group was given an equal volume of normal saline (20 μL). Vaginal lavage was performed on day 8. The lavage fluid was diluted and spread on a Candida albicans identification medium (chromogenic medium). After incubation at 37°C for 48 hours, the number of green colonies on the culture plate was observed and counted. One-way analysis of variance was performed using GraphPad Prism 5 software, and P < 0.05 was considered statistically significant.

[0143] 1.2 Experimental Results

[0144] 1.2.1 Screening and Isolation of Fermentative Lactobacillus mucilaginosus

[0145] Through identification and screening, a strain of fermentative Lactobacillus muciniphila was obtained and named as fermentative Lactobacillus muciniphila HY757.

[0146] 1.2.2 Identification of Lactobacillus muciflorus

[0147] (1) Colony characteristics

[0148] As shown in Figure 1 , the strain formed round, smooth-edged, milky white colonies with a protrusion in the middle after being cultured on MRS plates.

[0149] (2) Staining microscopy

[0150] As shown in FIG2 , after the strain was stained and observed under an optical microscope, it was determined that the bacteria were Gram-positive and had a short rod shape.

[0151] (3) Biochemical identification and analysis

[0152] The results of the target lactobacillus biochemical identification tube were interpreted according to the instructions for use of the lactic acid bacteria biochemical identification tube. The interpretation results are detailed in Table 4.

[0153] Table 4 Interpretation results of the fermentative mucus Lactobacillus identification tube Note: + represents positive; - represents negative; +w represents weak positive.

[0154] The results of biochemical identification showed that Lactobacillus mucis fermentans could utilize maltose, sucrose, raffinose and lactose as carbon sources.

[0155] (4) 16S rDNA identification

[0156] The 16S rDNA comparison results showed that the strain belonged to Lactobacillus fermentans. The 16S rDNA gene sequence (SEQ ID NO: 23) is shown below:

[0157] (5) Specific fragment molecular labeling results

[0158] Among the primers designed for specific nucleotide sequences, PCR amplification experiments using 2-p1 as the primer revealed only a single, clear, and high-concentration amplified band for HY757, with relatively few primer dimers. No amplified bands were observed for HY00810, HY10008, or HY00406, as detected by agarose gel electrophoresis (Figure 3). However, PCR amplification experiments using primers designed for other nucleotide sequences revealed either no amplified band in any group or amplified bands in the control group (using 1-p1 as an example, as detected by agarose gel electrophoresis (Figure 4)). This indicates that the primer corresponding to 2-p1 is a molecular marker primer for HY757, and the amplification product generated by this primer is a molecular marker for Lactobacillus fermentans HY757. The amplification product of the 2-p1 primer is 261 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 2-p1 can be used as a specific primer for fermentative Lactobacillus mucronulatus HY757, and the nucleotide shown in SEQ ID NO:24 is a specific nucleotide sequence marker for fermentative Lactobacillus mucronulatus HY757. This provides a reliable tool and basis for identifying and detecting fermentative Lactobacillus mucronulatus HY757.

[0159] In addition, during the experiment, the inventors found that when sequencing multiple samples of fermentative Lactobacillus mucilaginosus HY757 using the 2-p1 primer, the amplified product sequences differed from the nucleotide sequence shown in SEQ ID NO: 24 at individual bases. However, compared with the control bacteria, all had specific amplification bands, and the bands were clear and high in concentration, and the primer dimers were relatively few.

[0160] Therefore, after multiple sequencing and alignment, the homology range between the molecular marker of fermentative Lactobacillus mucronulatus HY757 and the nucleotide sequence shown in SEQ ID NO: 24 was determined, that is, the nucleotide sequence of the molecular marker of fermentative Lactobacillus mucronulatus HY757 has at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 24.

[0161] 1.2.3 Characteristics of the strain

[0162] (1) Antibiotic sensitivity test

[0163] The antibiotic sensitivity of the strain was determined by the diameter of the bacterial ring on the antibiotic sensitivity paper. The results are shown in the table below. The strain was resistant to metronidazole, norfloxacin and kanamycin, moderately sensitive to ofloxacin and ciprofloxacin, and sensitive to clindamycin and cefuroxime.

[0164] Table 5 Antibiotic sensitivity test results Note: S: sensitive (15-20mm highly sensitive; >20mm extremely sensitive); I: intermediate (10-14mm moderately sensitive); R: resistant (<10mm insensitive)

[0165] (2) Toxicity test

[0166] ① Hemolytic

[0167] The results of the hemolysis test are shown in FIG5 . Small gray-white colonies appeared in the culture medium around the colonies, and no hemolysis ring appeared around the colonies, indicating that the fermentative Lactobacillus mucosa HY757 was γ-hemolytic, i.e., non-hemolytic.

[0168] ② After oral gavage with fresh fermented Lactobacillus muciniphila HY757, all mice survived healthily and gained weight.

[0169] (3) Determination of metabolite content

[0170] Table 6 Metabolite content determination results

[0171] Fermented Lactobacillus mucosa HY757 was cultured in MRS broth for 24 hours. The D-lactic acid and L-lactic acid levels were 4.52 g / L and 4.13 g / L, respectively, with a total acid production of 8.65 g / L, which was higher than the 5.48 g / L of DJS-Lactobacillus delbrueckii. These organic acids are antibacterial substances that can compete with pathogenic bacteria for nutrients and adhesion sites, thereby enhancing the host's mucosal immunity and anti-infection capabilities.

[0172] 1.2.4 Application Function Analysis

[0173] (1) Antibacterial experiment

[0174] Two strains of the same species, the positive drug DJS and the screened fermentative Lactobacillus muciflorus HY757 were selected to test their antibacterial properties.

[0175] Table 7 Antibacterial test results

[0176] The results are shown in the table above. Except for the inhibition rates of EC and SA, which were basically the same as those of the other control groups, the inhibition of Lactobacillus muciflorus HY757 against the other four pathogens was better than that of the same strain control group and the positive control group, especially for GV, PB and CA, which had better effects than the control groups.

[0177] (2) Results of co-culture antibacterial experiment

[0178] ① Results of antibacterial experiment of co-culture of Lactobacillus and CA

[0179] The number of viable CA bacteria was detected after Lactobacillus mucilaginosus was co-cultured with CA for 20 h.

[0180] Table 8 Antibacterial test results of co-culture of Lactobacillus and CA

[0181] The experimental results are shown in the table above. After Lactobacillus mucilaginosus was co-cultured with CA for 20 hours, the inhibition rate reached 89.2%, indicating that the strain had a good inhibitory effect on the growth of CA.

[0182] ② Antibacterial experiment of co-culture of Lactobacillus and GV

[0183] The number of viable GV bacteria was detected after Lactobacillus mucilaginosus was co-cultured with GV for 27 h.

[0184] Table 9 Antibacterial experiment of co-culture of Lactobacillus and GV

[0185] The experimental results are shown in the table above. After Lactobacillus mucilaginosus was co-cultured with GV for 27 hours, the inhibition rate reached 97%, indicating that the strain had a strong inhibitory effect on the growth of GV.

[0186] (3) Biofilm removal test results

[0187] ① Experimental results of Lactobacillus removal of GV biofilm

[0188] The effect of fermentative Lactobacillus mucilaginosus on GV biofilm formation was investigated.

[0189] Table 10 Lactobacillus removal test results of GV biofilm

[0190] The experimental results are shown in the table above. The supernatant of fermented Lactobacillus mucilaginosus has a strong inhibitory effect on the formation of GV biofilm, which can reach 91.5%.

[0191] ② Experimental results on the removal of CA biofilm by Lactobacillus

[0192] The effects of fermentative Lactobacillus mucilaginosus on the formation of CA biofilm and the removal of formed biofilm were investigated.

[0193] Table 11 Results of the experiment on the removal of CA biofilm by lactobacilli

[0194] The experimental results are shown in the table above. Compared with the inhibition of biofilm formation ability, the fermented mucus lactobacillus has a stronger clearing effect on the formed CA biofilm.

[0195] (4) Cell adhesion assay

[0196] The number of single-cell adhesion of fermentative Lactobacillus mucinous HY757 to Hela cells was 12.31 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epithelial cells.

[0197] (5) VVC animal model efficacy test

[0198] The model group (M), experimental group (HY757) and positive control group (Dapoxetine suppository) were used to establish VVC model. After successful model establishment, the experimental group was given Lactobacillus mucinus HY757 (5×10 9 CFU / mL, 20 μL), the positive control group was given bisoprolol suppository (30 mg), and the model group was given an equal volume of normal saline (20 μL). Finally, the content of Candida albicans in the vaginal lavage fluid was detected. The experimental results are shown in Figure 6.

[0199] The experimental results showed that compared with the model group, the content of pathogenic bacteria in the vaginal lavage fluid of the HY757 group was significantly reduced after treatment. Although there was no significant difference compared with the positive control group, it was slightly lower than the number of pathogenic bacteria in the diazopoxetine suppository group, indicating that this strain has a good therapeutic effect on VVC in mice.

Claims

1. A gene for a molecular marker of Lactobacillus mucosae for fermentation, 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; Preferably, the gene sequence of the molecular marker of Lactobacillus mucosae for fermentation has the nucleotide sequence shown in SEQ ID NO:24, or its nucleotide sequence is as shown in SEQ ID NO:

24.

2. A Lactobacillus mucosae for fermentation, comprising the gene of the molecular marker of Lactobacillus mucosae for fermentation according to claim 1.

3. A primer set for identifying the Lactobacillus mucosae for fermentation according to claim 2, comprising a forward primer and a reverse primer, wherein: The nucleotide sequence of the forward primer is as shown in SEQ ID NO:1; The nucleotide sequence of the reverse primer is as shown in SEQ ID NO:

2.

4. A method for identifying Lactobacillus mucosae according to claim 2, comprising: The genomic DNA of the strain to be identified is amplified using the primer set according to claim 3, and then the amplification product is detected.

5. The method according to claim 4, the method comprising the following steps: Extract a genomic DNA sample from the strain to be identified, perform PCR amplification on the genomic DNA sample using the primer set according to claim 3, and then compare the amplification product with the gene of the molecular marker of Lactobacillus mucosae for fermentation according to claim 1. If the amplification product contains the gene of the molecular marker of Lactobacillus mucosae for fermentation according to claim 1, the strain to be identified is the Lactobacillus mucosae for fermentation according to claim 2; Preferably, if the amplification product contains the nucleotide sequence shown in SEQ ID NO:24, the strain to be identified is the Lactobacillus mucosae for fermentation according to claim 2.

6. Use of the gene of the molecular marker of Lactobacillus mucosae for fermentation according to claim 1 for identifying the Lactobacillus mucosae for fermentation according to claim 2.

7. A Lactobacillus mucosae for fermentation, comprising a nucleotide sequence selected from one of the following: (1) The nucleotide sequence shown in SEQ ID NO:23; (2) A nucleotide sequence having at least 99.8%, 99.9% or higher homology with the nucleotide sequence shown in SEQ ID NO:23; (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:23; Preferably, the gene sequence of the Lactobacillus mucosae for fermentation contains the nucleotide sequence shown in SEQ ID NO:23; Preferably, the 16S rDNA gene sequence of the Lactobacillus mucosae for fermentation is as shown in SEQ ID NO:

23.

8. The fermented Lactobacillus mucosae according to claim 2 or 7, wherein, The fermented Lactobacillus mucosae is Lactobacillus mucosae with the preservation number of CGMCC No. 26501.

9. A microbial preparation for preventing and / or treating vaginal infections and related diseases, which comprises the fermented Lactobacillus mucosae according to claim 2 or 7.

10. The microbial agent according to claim 9, wherein, The fermented Lactobacillus mucosae is the only probiotic active ingredient in the microbial preparation.

11. The microbial preparation according to claim 9 or 10, wherein The total number of viable bacteria of Lactobacillus mucilaginosus fermentation in the microbial preparation is not less than 1×10 5 CFU / g, preferably 1×10 5 to 1×10 10 CFU / g.

12. The microbial preparation according to any one of claims 9 to 11, wherein The microbial preparation further comprises pharmaceutically acceptable excipients.

13. A combined drug for preventing and / or treating vaginal infections and related diseases, the combined drug comprising the fermented Lactobacillus mucosae according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12, and other antibacterial drugs.

14. Use of the fermented Lactobacillus mucosae according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12 in the preparation of a drug for preventing and / or treating vaginal infections and related diseases; Preferably, the vaginal infections and related diseases are bacterial vaginitis and / or vulvovaginal candidiasis.

15. The use according to claim 14, wherein, The pathogenic bacteria of the bacterial vaginitis are selected from one or more of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginae and Prevotella bivia.

16. A method for preventing and / or treating vaginal infections and related diseases, the method comprising administering to a subject in need the fermented Lactobacillus mucosae according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12; Preferably, the vaginal infections and related diseases are bacterial vaginitis and / or vulvovaginal candidiasis.

17. The method according to claim 16, wherein, The pathogenic bacteria of the bacterial vaginitis are selected from one or more of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginae and Prevotella bivia.

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