Lactiplantibacillus plantarum and use thereof

Abstract

The present invention provides Lactiplantibacillus plantarum and a use thereof. A molecular marker gene for Lactiplantibacillus plantarum has a nucleotide sequence selected from one of the following: (1) a nucleotide sequence as shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence as shown in SEQ ID NO: 24; and (3) a nucleotide sequence having one or more, e.g., 1, 2, 3, 4, 5, or more, nucleotide substitutions, deletions, or insertions in the nucleotide sequence as shown in SEQ ID NO: 24. The Lactiplantibacillus plantarum provided by the present invention has non-toxicity and good biological characteristics and has a good curative effect on the prevention and / or treatment of vaginal infection and related diseases thereof.

Description

(2) Toxicity test @ Hemolysis test The bacterial suspension in the strain cryopreservation tube was dipped and streaked onto an anaerobic blood agar plate, cultured anaerobically at 37C for 48 h, and the color change of the blood agar plate around the colonies was observed. @ Mouse toxicity test Five mice with a body weight of 18-22 g were used. Each mouse was orally gavaged with 0.5 mL of fresh bacterial suspension (not less than 1.0^109 CFU / 0.5 mL) once a day for 3 consecutive days, and continuously observed from the first day of gavage to the 7th day. The survival of the mice was observed and the mice were weighed. (3) Determination of metabolite content @ D-lactic acid detection A D-lactic acid detection kit (purchased from Sigma-Aldrich) was used to detect the D-lactic acid content of the Lactiplantibacillus plantarum supernatant. The kit is based on the principle that D-lactic acid is oxidized by a specific D-lactate dehydrogenase to produce a color reaction that is proportional to the D-lactic acid concentration, and the absorbance is measured at 450 nm. @ L-lactic acid detection The supernatant was filtered using a 0.22 um sterile filter membrane. The L-lactic acid concentration was determined using a biosensor. 1.1.4 Application function analysis (1) Antibacterial experiment @ Preparation of working bacterial suspension: The Lactiplantibacillus plantarum strain was inoculated into MM medium (modified MRS broth medium, having 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, triammonium citrate 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.05 g / L) at an inoculum size of 0.5%, and cultured in an anaerobic workstation. The following 6 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; Candida albicans ATCC 10231, abbreviated as CA, the above six pathogenic bacteria can all be obtained commercially) were cultured using appropriate media. After the target strain was cultured, the supernatant was obtained by centrifugation, and a cell-free supernatant was obtained by 0.22 um filtration, which was used immediately or stored in a -80C refrigerator. After the pathogenic bacteria were cultured, the OD600 value was measured and diluted to an OD600 value of about 0.005 (the viable count was maintained at 5.0^105 CFU / mL to 5.0^106 CFU / mL) (WS / T 650—2019 Evaluation method for antibacterial and bacteriostatic effects). @ Interaction: An equal volume of the supernatant and the pathogenic bacterial suspension were mixed evenly, and 100 uL of the mixture was immediately taken and placed in a blank 96-well plate to measure OD600; the remaining culture was placed at 37C and subjected to anaerobic or aerobic culture according to the culture conditions of the pathogenic bacteria. After 48 h of culture, 100 uL of the evenly mixed bacterial suspension was pipetted and placed in a blank 96-well plate to measure OD600. A blank control group was set, each sample had 2 replicate wells, and according to the following formula, the inhibition rate of the Lactiplantibacillus plantarum strain against pathogenic bacteria was calculated. Inhibition rate = (A-B) / A*100% A: the increase in OD600 value within 48 h in the positive control group (i.e., blank medium); B: the increase in OD600 value within 48 h in the experimental group. (2) Co-culture antibacterial experiment @ Interaction and co-culture experiment of Lactiplantibacillus plantarum and CA Culture of Lactiplantibacillus plantarum: A glycerol stock of Lactiplantibacillus plantarum was taken and inoculated into MM medium (modified MRS broth medium, having 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, triammonium 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 cultured bacterial suspension was concentrated or diluted to about 1^109 CFU / mL to serve as the Lactiplantibacillus plantarum working bacterial suspension. Culture of CA: A glycerol stock of CA was taken and inoculated into Sabouraud medium, and cultured aerobically at 37°C. The resulting culture was centrifuged to remove the supernatant, and then the cell pellet was resuspended in fresh Sabouraud medium and adjusted to about 1.0^107 CFU / mL to serve as the CA working suspension. 400 liL of Lactiplantibacillus plantarum working bacterial suspension and 400 liL of CA working suspension were taken and co-inoculated into 40 mL of MM liquid medium. CA alone was inoculated as a positive control group, each group had 2 replicates. The suspension was gently shaken and cultured on a 37°C anaerobic workbench. Samples were taken at 20 h of culture, and the viable count of CA was counted using Candida chromogenic medium (purchased from CHROMagar, France). @ Interaction and co-culture experiment of Lactiplantibacillus plantarum and GV Culture of Lactiplantibacillus plantarum: Glycerol stocks of Lactiplantibacillus plantarum were taken and respectively inoculated into MM medium (modified MRS broth medium, having 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, triammonium 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 cultured bacterial suspension was concentrated or diluted to a concentration of about 1*109 CFU / mL to serve as the Lactiplantibacillus plantarum working bacterial suspension. Culture of GV: A glycerol stock of GV was taken and inoculated into BHI liquid medium containing 10% fetal bovine serum, and cultured anaerobically at 37°C. The resulting culture was centrifuged to remove the supernatant, and then the cell pellet was resuspended in fresh Sabouraud medium and adjusted to about 1.0x107 CFU / mL to serve as the GV working suspension. 400 uL of Lactiplantibacillus plantarum working bacterial suspension and 400 uL of GV working bacterial suspension were taken and co-inoculated into 40 mL of BHI liquid medium containing 10% fetal bovine serum. GV alone was inoculated as a positive control group, each group had 2 replicates, placed in an anaerobic workstation for culture, samples were taken at 27 h of culture, respectively, and the viable count of GV was detected by fluorescence quantitative qPCR method using GV-specific probe primers. (3) Biofilm removal experiment @ Biofilm removal experiment of Lactiplantibacillus plantarum against GV The cultured GV bacterial suspension was adjusted to 1.0x107 CFU / mL and inoculated into a 96-well plate. 2 groups were set up in total, with 4 replicates per group. The 2 groups were respectively added with an equal volume of blank MM liquid medium (modified MRS broth medium, having 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, triammonium 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 Lactiplantibacillus plantarum HY2946. The plate was placed in a 37°C anaerobic workstation and cultured for 24 h. After the culture was completed, the inhibitory effect of Lactiplantibacillus plantarum HY2946 on GV biofilm formation was determined by microplate crystal violet staining method. @ Biofilm removal experiment of Lactiplantibacillus plantarum against CA Inhibition of biofilm formation experiment: The cultured CA bacterial suspension was adjusted to 1.0^107 CFU / mL and inoculated into a 96-well plate. 2 groups were set up in total, with 4 replicates per group. The 2 groups were respectively added with an equal volume of Sabouraud dextrose liquid medium and the supernatant of the Lactiplantibacillus plantarum strain. The plate was placed in a 37°C anaerobic workstation and cultured for 24 h. After the culture was completed, the inhibitory effect of the Lactiplantibacillus plantarum strain on CA biofilm formation was determined by microplate crystal violet staining method. Biofilm removal experiment: The cultured CA bacterial suspension was adjusted to 1.0x107 CFU / mL. 100 liL of the adjusted CA bacterial suspension was taken and inoculated into a 96-well plate. 2 groups were set up in total, with 4 replicates per group. The plate was placed in a 37°C anaerobic workstation and cultured for 24 h. After 24 h of culture, the mixture in the wells was discarded, and the wells were washed with sterile PBS. An equal volume of MM medium (modified MRS broth medium, having 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, triammonium 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 the Lactiplantibacillus plantarum strain were respectively added to each group, and each group was cultured at 37°C for 24 h. After the culture was completed, the removal effect of the Lactiplantibacillus plantarum supernatant on mature CA biofilm was determined by microplate crystal violet staining method. (4) Cell adhesion experiment @ Preparation of working bacterial suspension: The Lactiplantibacillus plantarum strain was cultured, and the OD600 value of the bacterial suspension was measured. The cultured Lactiplantibacillus plantarum culture solution was centrifuged at 4000 rpm for 5 min at 4°C, and the supernatant was discarded. The bacterial pellet was washed repeatedly three times with PBS, and finally the Lactiplantibacillus plantarum pellet was resuspended in MEM complete medium (purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.). An appropriate amount of the resuspension was taken, and the viable count was determined by plate spreading method, which was L1. @ Cell culture: HeLa cells were inoculated into a 24-well plate at a density of 1.5^105 cells per well, and cultured for 16 h until the confluence reached 90%. The HeLa cells cultured in the 24-well plate were washed with serum-free MEM medium (purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.), and the cells were then counted and recorded as C1. @ Interaction:The Lactiplantibacillus plantarum strain was inoculated into the cells at a bacteria-to-cell ratio of 100: 1 and incubated at 37°C under 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 medium 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 portion of the suspension was taken to count the cell number C2, and the viable count L2 of the Lactiplantibacillus plantarum strain adhered to the cells was detected by plate spreading method. @ Calculation of adhesion number and adhesion rate: Average cell adhesion number of Lactiplantibacillus plantarum = L2 / C2, adhesion rate (%) = 100*L2 / L1. (5) Efficacy test in BV animal model Healthy SPF-grade Balb / c mice (female, 6-8 weeks old) were used for modeling. After adaptive culture, the animals were randomly divided into groups, with 8 mice per group: a model group (M), an experimental group (Lactiplantibacillus plantarum HY2946), and a positive control group (DJS). Before inoculation with pathogenic bacteria, each group of animals was subcutaneously injected with estradiol benzoate injection, and then GV (20 uL) at the same concentration was administered vaginally for BV pathogenic bacteria modeling. After successful modeling, treatments were carried out as follows: the experimental group was vaginally administered the corresponding Lactiplantibacillus plantarum suspension (1x1010 CFU / mL, 20 gL) for 5 consecutive days, the positive control group was vaginally administered an equal amount of Lactobacillus delbrueckii solution (strain in Ding'unsheng, 1^1010 CFU / mL, 20 gL) for 5 consecutive days, and the model group was administered an equal volume of normal saline. After the treatment, each animal was lavaged vaginally with 50 uL PBS / time, 4 times / animal, the lavage fluid was collected, placed in a 1.5 mL Ep tube, and frozen at -80C. The load of GV in the lavage fluid was detected by qPCR method, and then the difference in GV load in the lavage fluid among the groups was compared. (6) Efficacy test in VVC animal model Healthy SPF-grade Balb / c mice (female, 6-8 weeks old) were used for modeling. The animals were adaptively cultured, then randomly grouped, 8 mice per group, divided into a model group (M), an experimental group (Lactiplantibacillus plantarum HY2946), and a positive control group (Shuangzuotai suppositories). Each group was pretreated by subcutaneous injection of estradiol benzoate injection; from D0 to D2, CA (15 uL / mouse) was continuously administered, from D3 to D7, the experimental group was vaginally administered Lactiplantibacillus plantarum HY2946 (5^109 CFU / mL, 20 uL) every day, the positive control group was administered Shuangzuotai suppositories (30 mg), and the model group was administered an equal volume of normal saline (20 uL). On D8, the vagina was lavaged, the lavage fluid was diluted and spread on Candida albicans identification medium (chromogenic medium), and after culturing at 37C for 48 h, the number of green colonies on the culture dish was observed and counted. GraphPad Prism 5 software was used for one-way analysis of variance, and P<0.05 was considered statistically significant. 1.2 Experimental results 1.2.1 Screening and isolation of Lactiplantibacillus plantarum Through identification and screening, a strain of Lactiplantibacillus plantarum was obtained and named Lactiplantibacillus plantarum HY2946. 1.2.2 Identification of Lactiplantibacillus plantarum (1) Colony characteristics As shown in Fig. 1, after the strain was cultured on an MRS plate, it formed round, smooth-edged, milky white colonies with a raised center. (2) Staining and microscopic examination / electron microscopy As shown in Fig. 2, the strain was observed under an optical microscope after stained, and it could be determined that the cells were Gram-positive and short rod-shaped. (3) Biochemical identification analysis The results of the biochemical identification tubes of the target Lactiplantibacillus plantarum strain were read according to the instructions for use of the lactic acid bacteria biochemical identification tubes. The reading results are shown in detail in Table 4. Table 4 Reading results of identification tubes of the Lactiplantibacillus plantarum strain Identification substance Esculin Cellobiose Maltose Mannitol Salicin Sorbitol Reading result + + + + + + Identification substance Sucrose Raffinose Inulin Lactose 1% Sodium hippurate / Reading result + + + + - / Note: + represents positive; - represents negative; +w represents weakly positive. The biochemical identification results show that, except for 1% sodium hippurate, the other 10 carbon sources (esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose) can all be utilized by the Lactiplantibacillus plantarum strain. (4) 16S rDNA identification The 16S rDNA alignment results show that the strain belongs to Lactiplantibacillus plantarum. The gene sequence determined by 16S rDNA (SEQ ID NO: 23) is as follows: AGGCGGCTGGTTCCTAAANGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGG TGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC GATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAA TGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTA GCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCT CCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATA - 18 - AGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAA CCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATA GTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACC GCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGG CGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGC ATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCG AGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATA TCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCC CAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAAC CGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACC GCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAAC AGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTT CACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTG CCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGT CGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGG GACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGCGGTCCAAGT TGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCC ACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATC AATACCAGAGTTCGTTCGACNTGC (5) Results of specific fragment molecular marker Among the primers designed for the specific nucleotide sequence, when primer 3-p3 was used for PCR amplification experiments, only HY2946 produced a specific amplification band. The band was clear and high in concentration, and primer dimers were relatively few, whereas no amplification bands were observed for HY02938, HY00854, and HY12772. The agarose gel electrophoresis detection pattern is shown in Fig. 3. However, when primers designed for other nucleotide sequences were used for PCR amplification experiments, there was a situation where the control bacterial group also amplified a band (taking 2-p3 as an example, the agarose gel electrophoresis detection pattern is shown in Fig. 4). Therefore, this indicates that primer 3-p3 is the molecular marker primer of HY2946, and the amplification product produced by this primer is the molecular marker of Lactiplantibacillus plantarum HY2946. The amplification product size of primer 3-p3 is 578 bp, and the nucleotide sequence is as shown in SEQ ID NO: 24. Thus, it was determined that the nucleotide sequence as shown in SEQ ID NO: 24 is a part of the specific nucleotide sequence screened out in step a, further verifying that primer 3-p3 can serve as a specific primer for Lactiplantibacillus plantarum HY2946, and that the nucleotide as shown in SEQ ID NO: 24 is a specific nucleotide sequence marker of Lactiplantibacillus plantarum HY2946, which provides a reliable tool and basis for identifying and detecting Lactiplantibacillus plantarum HY2946. TGTCCATGTCCTTGTCCTGCCCCAAATCTATCATTAATCTGATTTTCTCCTAAATCATATG TAACTGAAGTATTTACGCTCGATAATTGTACACTTTCTCCAATCTTACCGTACTTAGATAT CTCGCTAGCTTGTTTCGTGGCTCTATCTAAGTCTCTATTAATTTGATCCTTAGTTGTCTCA GCCTCAGTCCACTCACTACTATTTTTAGTCCATCCAGTATTAATTGCAATTTCAAGTATTC TTTTAATTTCATCTAATTTACTAGCATCGAGATTTATAGTCTTAGTTGAATGATTAGCATA ATATACAACAATATGGTTTTCATCTGCATCGATTCGATATATATCTTGAAAAGGTACTTGC GTAACTTCGATATCGTATTCATTTCCACTGCCCTTGCTTTTAACTGGTTCTTTAATAATTA TTCCAAACGTAGTTATTAAATATCCAAACTCTCTAAACAACATTTCATCATTTAGTGGCT TCGCATCATAAACCAATGGAAAATCATTTTCTGTATCTATATTCTCAATTCCTGAACTAA AATATTTCGCACGGTTATATGGCATTGCCGC (SEQ ID NO: 24). In addition, the inventors found during the experiments that when primer 3-p3 was used to sequence multiple samples of Lactiplantibacillus plantarum HY2946, it was found that the sequences of the amplified products had differences in individual bases from the nucleotide sequence as shown in SEQ ID NO: 24. However, compared with the control bacteria, all exhibited a specific amplification band, which was clear and high in concentration, and primer dimers were relatively few. In one experiment, it was found that the nucleotide sequence of the product amplified from a sample of Lactiplantibacillus plantarum HY2946 using primer 3-p3 was as shown in SEQ ID NO: 25, and the result of alignment with the nucleotide sequence as shown in SEQ ID NO: 24 was 99.65% identity. TGTCCATGGTCTTGTCCTGCCCCAAATCTATCATTAATCTGATTTTCTCCTAAATCATATG TAACTGAAGTATTTACGCTCGATAATTGTACACTTTCTCCAATCTTACCGTACTTAGATAT CTCGCTAGCTTGTTTCGTGGCTCTATCTAAGTCTCTATTAATTTGATCCTTAGTTGTCTCA GCCTCAGTCCACTCACTACTATTTTTAGTCCATCCAGTATTAATTGCAATTTCAAGTATTC TTTTAATTTCATCTAATTTACTAGCATCGAGATTTATAGTCTTAGTTGAATGATTAGCATA ATATACAACAATATGGTTTTCATCTGCATCGATTCGATATATATCTTGAAAAGGTACTTGC GTAACTTCGATATCGTATTCATTTCCACTGCCCTTGCTTTTAACTGGTTCTTTAATAATTA TTCCAAACGTAGTTATTAAATATCCAAACTCTCTAAACAACATTTCATCATTTAGTGGCT TCGCATCATAAACCAATGGAAAATCATTTTCTGTATCTATATTCTCAATTCCTGAACTAA AATATTTCGCACGGTTATA (SEQ ID NO: 25) Therefore, after multiple sequencing and alignment, the range of sequence identity between the molecular marker of Lactiplantibacillus plantarum HY2946 and the nucleotide sequence as shown in SEQ ID NO: 24 was determined. That is, the nucleotide sequence of the molecular marker of Lactiplantibacillus plantarum HY2946 has at least 97%, 98%, 99% or more identity relative to the nucleotide sequence as shown in SEQ ID NO: 24. 1.2.3 Strain characteristics (1) Antibiotic sensitivity test The antibiotic sensitivity of the strain was judged by the diameter of the inhibition zone of the strain against antibiotic susceptibility disks. The results are shown in the following table. The strain is resistant to metronidazole, norfloxacin, ciprofloxacin and kanamycin, moderately sensitive to ofloxacin, and sensitive to clindamycin and cefuroxime. Table 5 Antibiotic sensitivity test results Antimicrobial susceptibility disk Metronidazole 5 ug / disk Clindamycin 2 ug / disk Cefuroxime 30 Lig / disk Norfloxacin 10 ^g / disk Inhibition zone diameter / mm 0.0 15.0 38.0 0.0 Determination R S S R Antimicrobial susceptibility disk Ofloxacin 5 ug / disk Ciprofloxacin 5 ug / disk Kanamycin 30 Lig / disk / Inhibition zone diameter / mm 11.0 0.0 0.0 / Determination I R R / Note: S: sensitive (15-20 mm: highly sensitive; >20 mm: extremely sensitive); I: intermediate (1014 mm: moderately sensitive); R: resistant (<10 mm: non-sensitive). (2) Toxicity test @ Hemolysis The hemolysis test results are shown in Fig. 5. Gray-white fine colonies appeared on the medium around the colonies, and no hemolytic ring appeared around the colonies, indicating that Lactiplantibacillus plantarum HY2946 is Y—hemolytic, that is, non-hemolytic. @ After oral gavage of fresh bacterial suspension of Lactiplantibacillus plantarum HY2946 to mice, all mice survived and gained weight. (3) Determination of metabolite content Table 6 Results of metabolite content determination Metabolite Lactiplantibacillus plantarum HY2946 DJS-Lactobacillus delbrueckii D-lactic acid 13.23 g / L 5.24 g / L L-lactic acid 7.01 g / L 0.24 g / L Lactiplantibacillus plantarum HY2946 was cultured in MRS broth for 24 h. After detection, D-lactic acid was 13.23 g / L and L-lactic acid was 7.01 g / L, and the total acid production was 20.24 g / L, which is higher than 5.48 g / L of DJS-Lactobacillus delbrueckii. These organic acids are antibacterial substances, can compete with pathogenic bacteria for nutrients and adhesion sites, and improve host mucosal immunity and anti-infection ability. 1.2.4 Application function analysis (1) Antibacterial experiment Two strains of the same species, the positive drug DJS, and the screened Lactiplantibacillus plantarum HY2946 were selected for antibacterial performance testing. Table 7 Antibacterial experiment results Strain Inhibition rate IR GV PB FV EC SA CA HY2946 70.67% 74.81% 35.56% 91.80% 87.32% 93.28% HY00854 73.62% 67.84% 35.89% 91.49% 88.14% 88.37% HY02938 69.94% 80.00% 31.09% 91.49% 87.22% 90.29% DJS 55.45% 78.81% 28.36% 98.91% 97.84% 48.74% The experimental results are shown in the above table. HY2946 has a good inhibitory effect on GV, PB, EC, and CA. For CA, compared with the positive drug DJS, which showed an inhibition rate of only 48.74% and was essentially non-inhibitory, the strain exhibited a strong inhibitory effect on CA. (2) Co-culture antibacterial experiment results @ Co-culture antibacterial experiment results of Lactiplantibacillus plantarum and CA After the Lactiplantibacillus plantarum strain and CA were co-cultured for 20 h, the viable count of CA was detected. Table 8 Co-culture antibacterial experiment results of Lactiplantibacillus plantarum and CA Strain Viable count of CA in the culture after 20 h (CFU / mL) Inhibition rate of Lactiplantibacillus plantarum against CA after 20 h CA positive control 4.17E+06 0% Lactiplantibacillus plantarum HY2946 8.20E+05 80.3% The experimental results are shown in the above table. After the Lactiplantibacillus plantarum strain and CA were co-cultured for 20 h, the inhibition rate reached 80.3%, indicating that the strain has a good inhibitory effect on the growth of CA. @ Co-culture antibacterial experiment of Lactiplantibacillus plantarum and GV After the Lactiplantibacillus plantarum strain and GV were co-cultured for 27 h, the viable count of GV was detected. Table 9 Co-culture antibacterial experiment of Lactiplantibacillus plantarum and GV Strain Viable count of GV in the culture after 27 h (CFU / mL) Inhibition rate of Lactiplantibacillus plantarum against GV after 27 h GV positive control 4.06E+08 0% Lactiplantibacillus plantarum HY2946 3.02E+07 86% The experimental results are shown in the above table. After the Lactiplantibacillus plantarum strain and GV were co-cultured for 27 h, the inhibition rate reached 86%, indicating that the strain has a strong inhibitory effect on the growth of GV. (3) Biofilm removal experiment results @ Biofilm removal experiment results of Lactiplantibacillus plantarum against GV The effect of the Lactiplantibacillus plantarum strain on GV biofilm formation was explored. Table 10 Biofilm removal experiment results of Lactiplantibacillus plantarum against GV Strain Inhibition rate of Lactiplantibacillus plantarum supernatant on GV biofilm formation Lactiplantibacillus plantarum HY2946 29.4%±20.7% The experimental results are shown in the above table. The supernatant of the Lactiplantibacillus plantarum strain has a certain inhibitory effect on the formation of GV biofilm. @ Biofilm removal experiment results of Lactiplantibacillus plantarum against CA The effects of the Lactiplantibacillus plantarum strain on the formation of CA biofilm and the removal of formed biofilm were explored. Table 11 Biofilm removal experiment results of Lactiplantibacillus plantarum against CA Strain Inhibition rate of the supernatant of the Lactiplantibacillus plantarum strain on CA biofilm formation Removal rate of the supernatant of the Lactiplantibacillus plantarum strain on CA biofilm Lactiplantibacillus plantarum HY2946 84.3%±1.7% 73.3%±25.7% The experimental results are shown in the above table. The strain not only has a strong inhibitory effect on the formation of CA biofilm, but also has a strong destructive ability on the formed CA biofilm. (4) Cell adhesion experiment The single-cell adhesion number of Lactiplantibacillus plantarum HY2946 to HeLa cells was 5.05 CFU, indicating that the strain has good adhesion or colonization characteristics to vaginal epidermal cells. (5) Efficacy test in BV animal model The model group (M), experimental group (HY2946) and positive control group (DJS) were respectively subjected to BV pathogenic bacteria modeling. After successful modeling, the experimental group was vaginally administered the corresponding Lactiplantibacillus plantarum suspension (1x1010 CFU / mL, 20 uL) for 5 consecutive days, the positive control group was vaginally administered an equal amount of Lactobacillus delbrueckii solution (strain in Dingjunsheng, 1x1010 CFU / mL, 20 uL) for 5 consecutive days, and the model group was administered an equal volume of normal saline for treatment. Finally, the difference in GV load in the lavage fluid among the groups was detected and compared. The experimental results are shown in Fig. 6. The experimental results show that treatment of the BV-infected animal model with HY2946 significantly reduced the GV content in the vagina, achieving a certain therapeutic effect, whereas the positive drug did not show a therapeutic effect. (6) Efficacy test in VVC animal model The model group (M), experimental group (HY2946) and positive control group (Shuangzuotai suppositories) were subjected to VVC modeling. After successful modeling, from D3 to D7, the experimental group was vaginally administered Lactiplantibacillus plantarum HY2946 (5x109 CFU / mL, 20 uL) every day, the positive control group was administered Shuangzuotai suppositories (30 mg), and the model group was administered an equal volume of normal saline (20 u-L). Finally, the content of Candida albicans in the vaginal lavage fluid was detected. The experimental results are shown in Fig. 7. The experimental results show that compared with the model group, the pathogenic bacteria content in the vaginal lavage fluid of the HY2946 group was extremely significantly reduced after treatment, indicating that the strain has a good therapeutic effect on VVC in mice.

Claims

1. A molecular marker gene for Lactiplantibacillus plantarum, comprising a nucleotide sequence selected from the group consisting of:(1) the nucleotide sequence as shown in SEQ ID NO: 24;(2) a nucleotide sequence having at least 97%, 98%, 99% or more identity relative to the nucleotide sequence as shown in SEQ ID NO: 24;(3) a nucleotide sequence having one or more, for example 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 24;preferably, the molecular marker gene for Lactiplantibacillus plantarum has the nucleotide sequence as shown in SEQ ID NO: 24, or its nucleotide sequence is as shown in SEQ ID NO: 24.

2. A Lactiplantibacillus plantarum strain comprising the molecular marker gene for Lactiplantibacillus plantarum according to claim 1.

3. A primer set for identifying the Lactiplantibacillus plantarum strain 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 the Lactiplantibacillus plantarum strain according to claim 2, which comprises: amplifying the genomic DNA of a strain to be identified using the primer set according to claim 3, and then detecting the amplification product.

5. The method according to claim 4, wherein 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 claim 3, and then aligning the amplification product with the molecular marker gene for Lactiplantibacillus plantarum according to claim 1; if the amplification product comprises the molecular marker gene for Lactiplantibacillus plantarum according to claim 1, the strain to be identified is the Lactiplantibacillus plantarum strain according to claim 2;preferably, if the amplification product comprises the nucleotide sequence as shown in SEQ ID NO: 24, the strain to be identified is the Lactiplantibacillus plantarum strain according to claim 2.

6. Use of the molecular marker gene for Lactiplantibacillus plantarum according to claim 1 inidentifying the Lactiplantibacillus plantarum strain according to claim 2.

7. A Lactiplantibacillus plantarum strain comprising a nucleotide sequence selected from the group consisting of:(1) the nucleotide sequence as shown in SEQ ID NO: 23;(2) a nucleotide sequence having at least 99.8%, 99.9% or more identity relative to the nucleotide sequence as shown in SEQ ID NO: 23;(3) a nucleotide sequence having one or more, for example 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence as shown in SEQ ID NO: 23;preferably, the genome of the Lactiplantibacillus plantarum strain comprises the nucleotide sequence as shown in SEQ ID NO: 23;preferably, the 16S rDNA gene sequence of the Lactiplantibacillus plantarum strain is as shown in SEQ ID NO: 23.

8. The Lactiplantibacillus plantarum strain according to claim 2 or 7, wherein the strain is deposited under CGMCC No. 26502.

9. A microbial preparation for preventing and / or treating vaginal infection and related diseases thereof, comprising the Lactiplantibacillus plantarum strain according to claim 2 or 7.

10. The microbial preparation according to claim 9, wherein the Lactiplantibacillus plantarum strain is the sole probiotic active ingredient in the microbial preparation.

11. The microbial preparation according to claim 9 or 10, wherein the total viable count of the Lactiplantibacillus plantarum strain in the microbial preparation is not less than 1x105 CFU / g, preferably 1x105 to 1x1010 CFU / g.

12. The microbial preparation according to any one of claims 9 to 11, wherein the microbial preparation further comprises a pharmaceutically acceptable excipient;preferably, the pharmaceutically acceptable excipient is one or more selected from the group consisting of a surfactant, a preservative, an antioxidant, a hardening agent, a thickening agent and an absorption enhancer.

13. A combined drug for preventing and / or treating vaginal infection and related diseases thereof, the combined drug comprising the Lactiplantibacillus plantarum strain according to claim 2 or 7 orthe microbial preparation according to any one of claims 9 to 12, and other antibacterial drugs.

14. Use of the Lactiplantibacillus plantarum strain according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12 in the preparation of a medicament for preventing and / or treating vaginal infection and related diseases thereof;preferably, the vaginal infection and related diseases thereof are bacterial vaginosis and / or vulvovaginal candidiasis.

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

16. A method for preventing and / or treating vaginal infection and related diseases thereof, the method comprising administering to a subject in need thereof the Lactiplantibacillus plantarum strain according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12; preferably, the vaginal infection and related diseases thereof are bacterial vaginosis and / or vulvovaginal candidiasis.

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

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