Probiotics with effect of improving vaginitis and composite microecological preparation and application thereof

Abstract

The invention discloses probiotics with an effect of improving vaginitis and a composite microecological preparation and application of the probiotics. The probiotics are characterized by comprising at least one of a plant lactobacillus 22-9 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.33867, a plant lactobacillus XA-9 strain with the preservation number of CGMCC No.33868 and a plant lactobacillus pentosus B-1-35 strain with the preservation number of CGMCC No.33870, the preparation method comprises the following steps: inoculating a lactic acid bacteria starter, which is prepared by mixing lactic acid bacteria cultures of plant lactobacillus 22-9, plant lactobacillus XA-9 and plant lactobacillus pentosus B-1-35 according to a volume ratio of 2: 1: 3, into a fermentation culture medium according to a volume ratio of 1-5%, so as to prepare the probiotic compound microecological preparation. The number of inflammatory cells in the vagina is reduced, and the levels of inflammatory factors IL-1beta, IL-6 and TNF-alpha in serum are reduced.

Description

Technical Field

[0001] The present invention relates to a probiotic complex microecological preparation, and in particular to a probiotic and its complex microecological preparation and application with the efficacy of improving vaginitis. Background Art

[0002] Vaginitis presents symptoms such as vulvovaginal pruritus, burning sensation, irritation, pain, "fishy" vaginal odor, and abnormal vaginal discharge. Vaginitis can affect the health of the reproductive system, increase the risk of sexually transmitted disease infection, and at the same time induce other gynecological inflammations, affect conception, increase the risks of miscarriage, premature birth, infertility, and ectopic pregnancy. The infected population accounts for about 26%-33% of all women, the recurrence rate of vaginitis is about 72%, and the recurrence rate of vulvovaginal candidiasis (VVC) is as high as 89%. Among them, bacterial vaginosis (BV), which accounts for the largest proportion of vaginitis, and VVC are mainly caused by Gardnerella vaginalis ( Gardnerella vaginalis ), and Candida albicans ( Candida albicans ); after adhering and colonizing in the vaginal environment, they aggregate other pathogenic bacteria to form a biofilm, resisting the host's immune defense environment and exacerbating the symptoms of vaginitis.

[0003] When the vagina is invaded by a large number of pathogens, the body can be regulated through the self-regulatory function of the vagina. It mainly includes: the vagina secretes acidic secretions to expel some pathogens; the innate immune cells in the vagina can recognize and phagocytose pathogens to initiate an immune response to prevent the spread of infection; the lactic acid bacteria in the vagina create and maintain an acidic environment in the vagina by producing substances such as lactic acid and hydrogen peroxide, thereby inhibiting the growth of other harmful bacteria. However, the self-regulatory function of the body is limited and cannot completely eliminate the pathogens in the vagina, and treatment intervention is required. At present, the treatment methods for vaginal infections are mainly divided into drug treatment and biological treatment. While drug treatment inhibits pathogenic bacteria, it has an adverse effect on the vaginal lactic acid bacteria flora and is prone to recurrence and chronic infection. And through biological treatment means such as lactic acid bacteria treatment, it inhibits the invasion and overgrowth of exogenous bacteria and conditional pathogenic bacteria, and has obvious advantages such as low recurrence rate, high safety, small side effects, and no drug resistance problems. For the health of the vaginal microecology, it is urgent to develop a probiotic complex microecological preparation with the efficacy of improving vaginitis. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a probiotic and its complex microecological preparation and application with the efficacy of improving vaginitis, which can increase the content of lactic acid bacteria in the vagina, reduce the content of pathogenic bacteria, reduce the number of inflammatory cells in the vagina, and reduce the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum.

[0005] The technical solution adopted by the present invention to solve the above technical problems is as follows: The present invention provides a probiotic with the efficacy of improving vaginitis, and the probiotic includes at least one of Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) strain 22-9 with the preservation number of CGMCC No. 33867, Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) strain XA-9 with the preservation number of CGMCC No. 33868, and Lactiplantibacillus pentosus ( Lactiplantibacillus pentosus ) strain B-1-35 with the preservation number of CGMCC No. 33870.

[0006] The present invention also provides a probiotic complex microecological preparation with the efficacy of improving vaginitis. The probiotic complex microecological preparation includes at least one strain of Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) strain 22-9 with the preservation number of CGMCC No. 33867, Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) strain XA-9 with the preservation number of CGMCC No. 33868, and Lactiplantibacillus pentosus ( Lactiplantibacillus pentosus ) strain B-1-35 with the preservation number of CGMCC No. 33870. The active ingredient of the probiotic complex microecological preparation includes the probiotic cells.

[0007] Further, the number of effective cells in the probiotic complex microecological preparation is not less than 1×10 7 CFU / mL.

[0008] Further, the concentrations of the Lactiplantibacillus plantarum 22-9 bacterial liquid, the Lactiplantibacillus plantarum XA-9 bacterial liquid, and the Lactiplantibacillus pentosus B-1-35 bacterial liquid are the same, and the mixing volume ratio is 2:1:3.

[0009] The present invention also provides a preparation method of the above probiotic complex microecological preparation with the efficacy of improving vaginitis, including the following steps: (1) Inoculate Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 into a fermentation medium at a ratio of 1-5% by volume respectively, and culture at 35-40 °C for 15-20 h to obtain lactic acid bacteria cultures. Mix the lactic acid bacteria cultures of Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 at a ratio of (1-3):(1-3):(1-3) by volume to obtain a mixed starter. Inoculate the mixed starter into the fermentation medium at a ratio of 2% by volume and culture at 37 °C for 18 h to obtain a lactic acid bacteria mixed culture; (2) Centrifuge the lactic acid bacteria culture obtained in step (1) to obtain lactic acid bacteria precipitate, and adjust the cell concentration to 1×10 7 -1×10 10 CFU / mL using sterile PBS buffer. Add a freeze-drying protectant with the same volume as the sterile PBS buffer, mix well, place it in a -80°C refrigerator for overnight pre-freezing, and then freeze-dry the pre-frozen sample to obtain the probiotic complex microecological preparation.

[0010] Further, in step (1), the lactic acid bacteria cultures of Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 are mixed in a volume ratio of 2:1:3 to obtain a mixed starter, and the cell concentrations of each lactic acid bacteria culture are the same.

[0011] Further, the fermentation medium in step (1) is prepared by dissolving MRS solid medium in 1 L of distilled water and sterilizing it at 121°C for 15 min. The formula of the MRS solid medium is 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 80 mL of Tween 80, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate, and 15 g of agar powder.

[0012] Further, the formula of the freeze-drying protectant in step (2) is 118.2 g / L trehalose, 17.1 g / L L-cysteine, 10.3 g / L sorbitol, 1.7 g / L sodium acetate, and 120 g / L skim milk powder.

[0013] The present invention also provides the application of the above probiotics in the preparation of inhibitors of Gardnerella vaginalis and / or Candida albicans.

[0014] The present invention also provides the application of the above probiotic complex microecological preparation in the preparation of drugs for improving or treating vaginitis symptoms.

[0015] Compared with the prior art, the advantages of the present invention are as follows: A probiotic and its complex microecological preparation and application with the efficacy of improving vaginitis. Three strains of lactic acid bacteria with significant inhibitory effects on Gardnerella vaginalis and Candida albicans are screened out. The microecological preparation obtained by using 3 kinds of lactic acid bacteria has a scientific and reasonable compatibility, can greatly inhibit the growth of vaginal pathogenic bacteria, can effectively increase the proportion of lactic acid bacteria in the vagina, reduce the number of inflammatory cells in the vagina, lower the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum, relieve the excessive inflammatory response, and has an obvious improvement effect on mice with vaginitis models, filling the gap in the market for probiotic preparation products for relieving vaginitis symptoms.

[0016] The above Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum)The strain 22-9, with the preservation number of CGMCC No. 33867, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 19, 2025, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] The above Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum )The strain XA-9, with the preservation number of CGMCC No. 33868, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 19, 2025, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] The above Lactiplantibacillus pentosus ( Lactiplantibacillus pentosus )The strain B-1-35, with the preservation number of CGMCC No. 33870, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 19, 2025, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the Drawings

[0019] Figure 1 is the inhibition rate of lactic acid bacteria against Gardnerella vaginalis; Figure 2 is the inhibition rate of lactic acid bacteria against Candida albicans; Figure 3 is the growth and acid production curve of lactic acid bacteria; Figure 4 is the survival rate and OD value of lactic acid bacteria at different pH values, where (A) is the OD value of lactic acid bacteria at different pH values, (B) is the survival rate of lactic acid bacteria at pH 3.0; (C) is the survival rate of lactic acid bacteria at pH 3.5; Figure 5 is the survival rate and OD value of lactic acid bacteria under different bile salts, where (A) is the OD value of lactic acid bacteria under different bile salts, (B) is the survival rate of lactic acid bacteria at 0.3% bile salt; (C) is the survival rate of lactic acid bacteria at 0.6% bile salt; (D) is the survival rate of lactic acid bacteria at 0.9% bile salt; Figure 6 is the evaluation of the adhesion ability of lactic acid bacteria (A) the self-aggregation ability of lactic acid bacteria; (B) the hydrophobic ability of lactic acid bacteria; Figure 7 is the hemolysis experiment of 3 strains of lactic acid bacteria; Figure 8 is the transmission electron micrograph of the cell morphology of 3 strains of lactic acid bacteria at 30000×; Figure 9 is the electrophoresis of the PCR products of the 16S rDNA of 3 strains of lactic acid bacteria; Figure 10 is the phylogenetic tree of 3 strains of lactic acid bacteria; Figure 11The inhibitory ability of the probiotic preparation with different compound ratios of lactic acid bacteria against Gardnerella vaginalis; Figure 12 The inhibitory ability of the probiotic preparation with different compound ratios of lactic acid bacteria against Candida albicans; Figure 13 The total number of colonies before and after freeze-drying of the undiluted probiotic preparation; Figure 14 The total number of colonies of the undiluted probiotic preparation at different storage temperatures; Figure 15 Gram staining of the vaginal lavage fluid on Day 4 during the administration period of the mouse vaginitis model; Figure 16 Vaginal and uterine tissues after the end of the administration period of the mouse vaginitis model; Figure 17 HE×400 staining result diagram of mouse vaginal tissue (inflammatory cells are indicated by black arrows); Figure 18 PAS×200 staining result diagram of mouse vaginal tissue (fungal positivity is indicated by green arrows); Figure 19 The number of inflammatory cells and epithelial keratinocytes in mouse vaginal tissue, where (A) is the number of inflammatory cells and (B) is the number of epithelial keratinocytes; letters a-c in the figure: different letters indicate significant differences between different treatment groups ( P <0.05); Figure 20 The levels of IL-1β, IL-6, and TNF-α in the sera of mice in each group, where A: IL-1β; B: IL-6; C: TNF-α; letters a-c in the figure: different letters indicate significant differences between different treatment groups ( P <0.05); Figure 21 The effects of the probiotic complex microecological preparation on the relative abundances of species at the phylum level of intestinal bacteria; Figure 22 The effects of the probiotic complex microecological preparation on the relative abundances of species at the genus level of intestinal bacteria; Figure 23 The effects of the probiotic complex microecological preparation on the relative abundances of species at the phylum level of vaginal bacteria; Figure 24 The effects of the probiotic complex microecological preparation on the relative abundances of species at the genus level of vaginal bacteria; Figure 25 The effects of the probiotic complex microecological preparation on the relative abundances of species at the phylum level of vaginal fungi; Figure 26 The effects of the probiotic complex microecological preparation on the relative abundances of species at the genus level of vaginal fungi. Detailed implementation methods

[0020] The present invention will be further described in detail below in conjunction with the embodiments with reference to the accompanying drawings.

[0021] I. Experimental methods 1. Preparation of culture media and solutions MRS liquid medium: Dissolve the purchased MRS liquid medium (the formula of MRS liquid medium is 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 80 mL of Tween, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder) in 1 L of distilled water, and sterilize at 121 °C for 15 min.

[0022] MRS solid medium: Add 15.0 g of agar to the MRS liquid medium, and sterilize at 121 °C for 15 min.

[0023] YM solid medium: Dissolve the purchased YM solid medium (the formula of YM solid medium is 20 g of glucose, 5 g of peptone, 3 g of yeast extract powder, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 15 g of agar) in 1 L of distilled water, and sterilize at 121 °C for 15 min.

[0024] YPD liquid medium: Dissolve the purchased YPD liquid medium (the formula of YPD liquid medium is 10 g of yeast extract powder, 20 g of peptone, 20 g of glucose) in 1 L of distilled water, and sterilize at 121 °C for 15 min.

[0025] Selective Gardnerella vaginalis medium: Dissolve the purchased selective Gardnerella vaginalis medium in 1 L of distilled water, and sterilize at 121 °C for 15 min. When in use, when the above medium is cooled to about 50 °C, add 5 wt% - 10 wt% sterile defibrinated horse blood, 15 - 20 μg / ml nalidixic acid solution, 10 - 20 μg / ml gentamicin sulfate, 2 - 5 μg / ml amphotericin B solution.

[0026] Blood agar base medium: Dissolve the purchased blood agar base medium (the formula of blood agar base medium is 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, 15 g of agar) in 1 L of distilled water, and sterilize at 121 °C for 15 min. When in use, when the above base medium is cooled to about 50 °C, add 5 wt% - 10 wt% sterile defibrinated sheep blood or rabbit blood to provide special nutrients required for bacterial growth and help observe the hemolysis phenomenon of some bacteria.

[0027] 2. Activation and preservation of strains Select 2 mL of the lactic acid bacteria strain stored in a -80 °C refrigerator, inoculate it into 50 mL of fermentation medium, and culture it at 37 °C for 12 h to obtain the seed solution. Spread and culture the seed solution, observe the colony morphology, and place it upside down in a -4 °C refrigerator for standby.

[0028] 3. Preparation of lactic acid bacteria liquid and pathogenic bacteria liquid Cultivation of lactic acid bacteria: Inoculate the above-preserved lactic acid bacteria seed solution into the fermentation medium (dissolve the MRS solid medium in 1 L of distilled water and sterilize it at 121 °C for 15 min), culture it at 37 °C for 18 h, and activate it for three generations to obtain the lactic acid bacteria culture, and adjust its OD 600 to 1, and reserve the bacterial liquid.

[0029] Cell-free supernatant (CFS) of lactic acid bacteria: Centrifuge the obtained lactic acid bacteria culture at 8000 r / min for 10 min, take the fermentation supernatant, and filter it through a 0.22 μm PES filter membrane to obtain the cell-free supernatant (CFS).

[0030] Cultivation of Gardnerella vaginalis: Inoculate Gardnerella vaginalis on blood agar solid medium, pick a single colony and activate it for two generations, then culture it anaerobically at 37 °C for 24 - 48 h in selective Gardnerella vaginalis liquid medium, and adjust the bacterial liquid concentration to 10 8 CFU / mL.

[0031] Cultivation of Candida albicans: Inoculate Candida albicans on YM solid medium, pick a single colony and activate it for two generations, then culture it at 30 °C for 24 h in YPD liquid medium, and adjust its cell concentration to 4×10 6 CFU / mL.

[0032] 4. Determination of the ability to inhibit Gardnerella vaginalis and Candida albicans In a 96-well plate, add 150 μL of YPD liquid medium of Candida albicans with an inoculation amount of 2% v / v and 50 μL of lactic acid bacteria CFS, and mix and culture them at 30 °C in a biochemical incubator for 24 h, and detect its OD 600 value and record it as OD 处理组 ; add 200 μL of Candida albicans with an inoculation amount of 2% v / v, mix and culture them at 30 °C for 24 h, and detect its OD 600 value and record it as OD 对照组 .

[0033] Add 150 μL of Gardnerella vaginalis with an inoculation amount of 2% v / v and 50 μL of lactic acid bacteria fermentation supernatant, and mix and culture them at 37 °C in an anaerobic incubator for 24 - 48 h, and detect its OD 600The value is denoted as OD 处理组 ; Add 200 μL of Gardnerella vaginalis with an inoculum size of 2% v / v, and incubate at 37°C for 24 - 48 h with mixing, then measure its OD 600 The value is denoted as OD 对照组 .

[0034] By comparing the inhibition rates of the two, determine the strains with better inhibitory ability against Gardnerella vaginalis and Candida albicans. The calculation formula is as follows: …………(1).

[0035] 5. Growth curve and acid production curve After activating the seed liquid for three generations, inoculate and culture it at an inoculum size of 2% (v / v). Every 2 h, measure the pH value of the fermentation broth and the absorbance value at 600 nm. Before each measurement, shake the culture broth well and measure three replicates.

[0036] 6. Determination of acid and bile salt tolerance Evaluate acid tolerance using MRS broth with pH 3.0 and 3.5, and test bile tolerance using MRS broth supplemented with 0.3%, 0.6%, 0.9% (w / v) porcine bile salts. Inoculate 5 strains of lactic acid bacteria at an inoculum size of 2% v / v into the above media and culture at 37°C for 18 h, then measure OD 600 , denoted as A1; inoculate into MRS broth without additives and culture at 37°C for 18 h, then measure OD 600 , denoted as A0. The survival rate calculation formula is as follows: ………………(2).

[0037] 7. Determination of auto - aggregation ability and surface hydrophobicity Inoculate the bacterial solution into MRS broth medium at an inoculum size of 2% v / v and culture for 18 h. Centrifuge at 4°C, 5000 r / min for 10 min to collect the bacteria, wash twice with PBS, and then resuspend in PBS to measure OD 600 , and adjust the OD 600 of the bacterial suspension so that its range is 0.25 ± 0.05, and record the initial OD as A0. Pipette 4 mL of the bacterial suspension into an EP tube, let it stand at 37°C for 20 h, then take 3 mL of the supernatant and add it to another centrifuge tube to measure its OD 600 , denoted as A1. Calculate the auto - aggregation rate according to the following formula. Repeat 3 independent experiments and take the average value.

[0038] ………………(3); Prepare the bacterial suspension according to the above method. Add 1 mL of xylene to 3 mL of the bacterial suspension, mix well on a vortex oscillator, let it stand for 1 h at 37°C, take the aqueous phase, and measure the OD of the aqueous phase. 600 Record it as A. Calculate the hydrophobicity rate of the strain using the following formula. Repeat the independent experiment 3 times and take the average value.

[0039] ……………… (4).

[0040] 8. Antibiotic Sensitivity Assay Pour 10 mL of MRS solid medium into a petri dish. After it solidifies, pour soft agar containing a 1% v / v lactic acid bacteria inoculum. After it solidifies, place the drug sensitivity test paper flat on the surface, group and number it for recording. The drug sensitivity test papers used are: vancomycin, tetracycline, streptomycin, penicillin, ampicillin, erythromycin, kanamycin, ceftazidime, gentamicin, doxycycline. Incubate the plate with the drug papers at 37°C for 48 h, set up three parallels, and measure and record the size of the inhibition zones produced by each drug sensitivity test paper.

[0041] 9. Transmission Electron Microscope (TEM) Centrifuge the fermented lactic acid bacteria liquid at 5000 r / min for 10 min, collect the cell precipitate, and wash it 3 times with PBS buffer (pH 7.4) to remove residual medium. Subsequently, resuspend the cells in PBS, take an appropriate amount of the cell suspension and drop it onto a 200-mesh copper grid (carbon support film), let it stand for 15 min to allow the cells to settle fully. After adsorption, use filter paper to absorb the excess liquid to avoid the sample being too thick. Then, immerse the copper grid in a 2%-5% uranyl acetate ethanol solution for 30 s and repeat the staining 3 times to enhance the contrast. Finally, observe the cell morphology and ultrastructure under a transmission electron microscope (TEM).

[0042] 10. Hemolysis Experiment Inoculate lactic acid bacteria on a blood-containing agar plate suitable for the growth of the strain, and culture and observe the hemolysis zone.

[0043] 11. 16S rDNA Identification and Phylogenetic Analysis of Lactic Acid Bacteria (1) DNA Extraction Take fresh bacterial liquid, centrifuge at 8000 rpm for 1 min to obtain the cells, and extract the total DNA of the strain using a kit.

[0044] (2) PCR Amplification and Product Detection Use universal primers to amplify the 16S rRNA gene. The primer sequences are as follows: Forward primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; Downstream primer 1495R: 5′-CTACGGCTACCTTGTTACGA-3′; The PCR cycling parameters were as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 64 °C for 1 min, extension at 72 °C for 2 min, final extension at 4 °C for 10 min, for 34 cycles. The amplified PCR products were sent to Sangon Biotech Co., Ltd. for gene sequence detection.

[0045] (3)Phylogenetic tree The obtained strain sequences were aligned with the 16S rDNA gene fragments on the NCBI website, and the 16S rDNA gene sequences of the strains with high similarity were extracted. The phylogenetic tree was constructed using the software MEGAX 64.

[0046] 12. Optimization of the compounding ratio of probiotic microecological preparations After activating the 3 selected lactic acid bacteria strains, adjust the OD 600 = 1 (10 9 CFU / mL), and compound them according to the compounding ratios of 1:1:1, 1:1:2, 2:2:3, 3:2:1, 1:3:2, 1:1:0, 2:1:1, 3:1:1, 3:1:2, 3:2:2, 2:1:3, 1:0:1, 1:2:0, 2:0:1, 1:2:3, 1:2:1, 1:3:1, 1:1:3, 2:3:2, 2:3:1, 0:1:1, 0:1:2, 1:0:2, 0:2:1, 2:1:0. Inoculate them into 10 mL of MRS broth at an inoculation amount of 2% v / v. After culturing for 18 h, centrifuge at 8000 r / min for 10 min. Take the fermentation supernatant and filter it through a 0.22 μm PES filter membrane to obtain CFS.

[0047] In a 96-well plate, add 150 μL of YPD liquid medium of Candida albicans with an inoculation amount of 2% v / v and 50 μL of lactic acid bacteria CFS, and mix and culture them in a biochemical incubator at 30 °C for 24 h. Detect its OD 600 value and record it as OD 处理组 ; Add 200 μL of Candida albicans with an inoculation amount of 2%, mix and culture them at 30 °C for 24 h, and detect its OD 600 value and record it as OD 对照组 .

[0048] Add 150 μL of Gardnerella vaginalis with an inoculation amount of 2% v / v and 50 μL of lactic acid bacteria fermentation supernatant, and mix and culture them in an anaerobic incubator at 37 °C for 24 - 48 h. Detect its OD 600 value and record it as OD 处理组; Add 200 μL of Gardnerella vaginalis with an inoculation amount of 2% v / v, and incubate at 37 °C for 24 - 48 h, then measure its OD 600 value, denoted as OD 对照组 .

[0049] (1), By comparing the inhibition rates of the two, screen out the compounding ratio with the greatest potential to improve vaginitis.

[0050] 13. Detection of the freeze-dried survival rate of probiotic microecological preparations After compounding the three selected strains of lactic acid bacteria according to the optimal compounding ratio, inoculate them into MRS broth at an inoculation amount of 2% v / v. After culturing for 18 h, centrifuge at 8000 r / min for 10 min to obtain bacterial sludge. Add 2 mL of freeze-drying protectant to the bacterial sludge obtained by centrifuging every 50 mL of culture, and freeze-dry with a freeze-dryer to obtain a high-concentration lactic acid bacteria preparation.

[0051] The freeze-drying protectant is the optimal formula optimized in the early stage of the laboratory: 118.2 g / L trehalose, 17.1 g / L L-cysteine, 10.3 g / L sorbitol, 1.7 g / L sodium acetate, and 120 g / L skim milk powder. Use the ten-fold gradient dilution method to coat and count the bacterial liquid and bacterial powder before and after freeze-drying, and divide the two to obtain the freeze-dried survival rate.

[0052] 14. Detection of the total number of colonies of probiotic microecological preparations during the storage period Store the freeze-dried bacterial powder at 4 °C, 37 °C, and room temperature (10 °C - 20 °C) respectively. At the 0th, 7th, 14th, 21st, and 28th days of storage, dilute and coat and count the microecological preparations under different storage conditions.

[0053] 15. Data processing and statistical analysis Each experiment was repeated 3 times. GraphPad Prism 9.4.0 and SPSS 19.0 were used to statistically analyze the experimental data and draw graphs. The experimental results were presented in the form of mean ± standard deviation (mean ± SEM). One-Way ANOVA was used for significant analysis of multiple groups of data.

[0054] II. Screening of strains 1. Screening of lactic acid bacteria When the vaginal microecological balance is disrupted, Gardnerella vaginalis , Candida albicansA large number of harmful bacteria such as Gardnerella vaginalis , Candida albicans will breed. Lactobacilli can produce substances such as lactic acid, H2O2, and bacteriocins, effectively inhibiting the growth and reproduction of harmful bacteria and helping to restore the balance of the vaginal microecology. In the process of applying lactobacilli to the treatment of vaginitis, the antibacterial ability is the most crucial indicator and can be used as the primary criterion for screening lactobacilli strains with the potential to improve vaginitis. 32 lactobacilli strains were randomly selected from the strain collection of the Laboratory of Livestock Product Processing at Ningbo University for antibacterial experiments. By detecting the inhibitory ability of the cell-free supernatant (CFS) of lactobacilli against

[0055] from Figure 1 , it can be obtained that Gardnerella vaginalis the growth of Figure 2 is inhibited by Lactobacillus B-1-35, 22-9, XA-9, and 20A-1 by 57.22%, 53.96%, 52.58%, and 50.87% respectively. From Candida albicans , it can be obtained that Gardnerella vaginalis , Candida albicans the growth of

[0056] is best inhibited by B-1-35, 83A-58, H-1-46, XA-9, 54A-32, and 22-9, with inhibition rates of 46.34%, 43.09%, 43.61%, 42.78%, 41.97%, and 43.18% respectively. Considering the inhibitory ability of lactobacilli against By plotting the growth curve, the growth state of lactobacilli at different culture stages can be determined, which helps to determine the optimal culture time of lactobacilli and provides an experimental basis for the production of microecological preparations. The acid production curve can reflect the acid production ability and dynamic changes of lactobacilli during growth and can be used to determine the acid production ability of different lactobacilli strains.

[0057] As Figure 3 shown, when the culture temperature is 37 °C, after adjusting the 5 lactobacilli strains to the same concentration and inoculating them into MRS liquid medium at 2% v / v, they enter the logarithmic growth phase after 2 h and the growth plateau phase after 14 h; the initial pH value of the lactobacilli fermentation broth is about 5.60, rapidly decreases to about 3.80 within 2 - 10 h of culture, then slowly decreases, and tends to be unchanged after 14 h of inoculation; the growth curves and pH change trends of the 5 lactobacilli strains are consistent and there are no significant differences. The experimental results show that the above strains all have good acid production ability and can enter the logarithmic phase relatively quickly, causing the pH value to drop rapidly, and this characteristic has a good effect on restoring the acidic environment of the vagina and inhibiting the growth of pathogenic bacteria.

[0058] 3. Determination of Acid and Bile Salt Tolerance The human gastrointestinal environment is complex, and gastric acid and bile are important components. Detecting the acid and bile salt tolerance of lactic acid bacteria can simulate their survival conditions in the gastrointestinal tract and evaluate whether lactic acid bacteria can remain active and play a beneficial role after passing through the acidic environment of the stomach and the bile salt environment of the small intestine. In the development of microecological preparations, selecting lactic acid bacteria strains with strong acid and bile salt tolerance can ensure that a sufficient number of live bacteria reach the intestine after oral administration, thereby improving the efficacy and stability of lactic acid bacteria preparations.

[0059] As Figure 4 shown in (A) below, when lactic acid bacteria grow in an environment with a pH of 6.5 (control), each lactic acid bacteria grows rapidly, and the OD 600 is above 1.1. As the pH value decreases, the growth ability of each lactic acid bacteria changes. As Figure 4 shown in (C) below, at pH 3.5, the survival rates of B-1-35, 83A-58, 22-9, 54A-32, and XA-9 are 14.23%, 23.23%, 19.71%, 15.16%, and 24.12% respectively. At this time, the survival rate of XA-9 is the highest, significantly ( P <0.05) higher than that of the other strains. As Figure 4 shown in (B) below, at pH 3, the survival rates of each lactic acid bacteria decrease significantly, which are 5.55%, 3.93%, 4.83%, 3.29%, and 4.03% respectively. And there is no significant difference among the five strains. The above results show that each lactic acid bacteria has a certain tolerance to the acidic environment.

[0060] As Figure 5 shown in (A) below, when the bile salt concentration is 0%, each lactic acid bacteria grows normally, and the growth value reaches the maximum. As the bile salt concentration increases, the growth ability of each lactic acid bacteria changes. As Figure 5 shown in (B) below, at a bile salt concentration of 0.3%, the survival rates of B-1-35, 83A-58, 22-9, 54A-32, and XA-9 are 38.31%, 32.20%, 31.57%, 21.98%, and 15.28% respectively; at this time, the survival rate of B-1-35 is the highest, significantly ( P <0.05) higher than that of 54A-32 and XA-9. As Figure 5 shown in (C) below, at a bile salt concentration of 0.6%, the survival rates of each lactic acid bacteria are 21.46%, 22.85%, 27.43%, 21.08%, and 14.28% respectively; among them, 54A-32 and XA-9 hardly change, and the decline of 22-9 is relatively small. As Figure 5As shown in (D), when the bile salt concentration is 0.9%, the survival rates of each lactic acid bacterium are 18.89%, 17.88%, 10.42%, 15.55%, and 14.21% respectively; the survival rate of lactic acid bacteria does not decrease significantly. The above results show that the above lactic acid bacteria have a certain tolerance to the bile salt environment.

[0061] 4. Determination of auto-aggregation ability and surface hydrophobicity The auto-aggregation ability helps lactic acid bacteria to form communities, better resist antibacterial substances, and is beneficial for maintaining a high density in the local environment. At the same time, the auto-aggregation ability of lactic acid bacteria is closely related to its adhesion characteristics. Lactic acid bacteria with strong auto-aggregation ability are more likely to colonize in the vagina. Hydrophobicity affects the interaction with other substances in the surrounding environment. Lactic acid bacteria with appropriate hydrophobicity can better specifically or non-specifically bind to epithelial cells, other microbial cells, etc., which is beneficial for the colonization of lactic acid bacteria in the human body. When the auto-aggregation of lactic acid bacteria is about 16 - 35%, it has low auto-aggregation ability; when it is 36 - 50%, it has medium auto-aggregation ability; when it is above 51%, it has high auto-aggregation ability. The cell adhesion ability of lactic acid bacteria plays a very important role whether in oral administration or vaginal administration. Using the auto-aggregation ability and surface hydrophobicity as the second screening criteria, and combining the characteristics of lactic acid bacteria's acid and bile salt tolerance, 3 strains were screened out from 5 alternative strains for the preparation of probiotic microecological preparations.

[0062] According to Figure 6 in (A), the auto-aggregation rates of XA-9, B-1-35, 22-9, and 54A-32 are 52.52%, 55.14%, 51.42%, and 53.09% respectively, all of which have high auto-aggregation ability, and 83A-58 has medium auto-aggregation ability. According to Figure 6 in (B), XA-9, B-1-35, and 22-9 are moderately hydrophobic, while 54A-32 and 83A-58 are non-hydrophobic. Auto-aggregation and surface hydrophobicity are important indicators for evaluating the cell adhesion ability of lactic acid bacteria. Therefore, based on these two aspects and combined with the acid and bile salt tolerance ability, the component strains for the microecological preparation are screened. Finally, XA-9, B-1-35, and 22-9 are selected as the component strains of the microecological preparation.

[0063] 5. Antibiotic sensitivity determination The purpose of detecting the antibiotic sensitivity of lactic acid bacteria is to avoid the transfer of drug-resistant genes and avoid interference during subsequent combined treatment with drugs. Drug-resistant lactic acid bacteria may carry drug-resistant genes and may be transmitted to some pathogenic bacteria that are harmful to the human body. Detecting antibiotic sensitivity helps to timely detect and control the spread of drug-resistant strains and prevent the diffusion of drug-resistant genes in the microbial community.

[0064] Meanwhile, in the treatment of vaginitis, the combined treatment method of antibiotics and lactic acid bacteria is mostly adopted. By detecting the antibiotic sensitivity of lactic acid bacteria, it helps to select drugs that are effective against pathogenic bacteria and have less impact on lactic acid bacteria during combined drug administration, improve the safety and effectiveness of treatment, and avoid interference of antibiotic treatment on lactic acid bacteria treatment.

[0065] Table 1 Results of drug sensitivity test of lactic acid bacteria

[0066] Note: Judgment criteria (diameter of inhibition zone): S is sensitive, I is intermediate, and R is resistant.

[0067] It can be seen from Table 1 that the sensitivities of Lactobacillus 22-9, B-1-35, and XA-9 to antibiotics are almost completely the same. They are all resistant to vancomycin, streptomycin, penicillin, and kanamycin; sensitive to tetracycline, ampicillin, erythromycin, ceftazidime, and doxycycline; only different in gentamicin. 22-9 is resistant to it, B-1-35 is intermediate to it, and XA-9 is sensitive to it.

[0068] 7. Hemolysis experiment Hemolytic ability is an important indicator to measure the potential pathogenicity of bacteria. Some lactic acid bacteria with hemolytic characteristics may damage host cells, trigger inflammatory reactions, and even cause diseases. Through hemolysis experiment detection, it can be preliminarily judged whether lactic acid bacteria have potential pathogenicity. The experimental results are as Figure 7 It can be seen that there is no hemolysis phenomenon of the experimental strains on the blood agar plate.

[0069] 8. Transmission electron microscope (TEM) The cell morphologies and structures of different lactic acid bacteria are different. By using a transmission electron microscope to see the fine structures such as cell walls and cell membranes and the overall shape, it can assist in classification and identification. Morphology reflects the physiological state. Under different growth stages and culture conditions, the morphologies of lactic acid bacteria are different. Through this, the functional changes such as metabolism, growth, and stress response can be inferred.

[0070] As Figure 8 shown, all three strains are rod-shaped strains. XA-9 seems to be in the division stage, showing a dumbbell shape. In the middle may be the new cell wall and membrane structures, connecting the daughter cells; B-1-35 is short and compact, with clear boundaries and no signs of division. It may be in the logarithmic growth phase, with stable cell state and complete structure; 22-9 has two cells connected, probably just divided and not completely separated.

[0071] 9. Identification results of lactic acid bacteria strains (1) Amplification of 16S rDNA of lactic acid bacteria isolates The gel electrophoresis results of PCR amplification products are as Figure 9As shown, there is no degradation or tailing of the product, and its integrity is good, meeting the requirements for submission. On the left side of the picture is the DNA marker with a maximum of 8000bp, and the other three columns from left to right are strains 22-9, XA-9, and B-1-35 respectively.

[0072] (2)Phylogenetic tree and homology analysis The phylogenetic trees of strains 22-9, XA-9, and B-1-35 are shown in Figure 10 , and it can be seen from the figure that strains 22-9 and XA-9 are Lactiplantibacillus plantarum ), while B-1-35 is Lactiplantibacillus pentosus .

[0073] In summary, 32 lactic acid bacteria strains were randomly selected from the Ningbo School Culture Collection for screening, and 5 lactic acid bacteria strains with significant inhibitory effects on the growth of Gardnerella vaginalis and Candida albicans were obtained. On this basis, using the self-aggregation ability and surface hydrophobicity index to evaluate the adhesion ability of lactic acid bacteria, 3 lactic acid bacteria strains with excellent antibacterial properties were determined, namely 22-9, XA-9, and B-1-35.

[0074] The growth curves and acid production curves showed that all 3 strains could grow rapidly and produce acid stably. They could enter the exponential growth phase after 2 h and had a long stationary phase, with good growth stability. The above 3 strains had excellent tolerance to acidic and bile salt environments. Among them, strain XA-9 had the best tolerance, and its growth rate reached 24.12% in the pH 3.5 environment. At the same time, the self-aggregation rates of XA-9, B-1-35, and 22-9 were 52.52%, 55.14%, and 51.42% respectively, with high self-aggregation ability and were all moderately hydrophobic. The in vitro safety evaluation showed that the 3 strains met the safety evaluation and could be used as composite strains of probiotic microecological preparations. Through 16S rDNA sequencing identification, it was determined that strains XA-9 and 22-9 were Lactiplantibacillus plantarum ), while B-1-35 was Lactiplantibacillus pentosus .

[0075] The above-mentioned Lactiplantibacillus plantarum strain 22-9 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 19, 2025, with the deposit number CGMCC No. 33867, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0076] The above-mentioned Lactiplantibacillus plantarum)Strain XA-9, with the preservation number of CGMCC No. 33868, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 19, 2025, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0077] The above Lactiplantibacillus pentosus ( Lactiplantibacillus pentosus )Strain B-1-35, with the preservation number of CGMCC No. 33870, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 19, 2025, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0078] III. Determination of the optimal ratio of probiotic microecological preparation strains and quality detection 1. Effects of different fermentation strains and ratios on the inhibitory ability against Gardnerella vaginalis and Candida albicans Developing a microecological preparation composed of a compound of 3 strains of lactic acid bacteria compared with single bacteria: it has diverse functions, contains multiple bacteria, and their metabolites and functions are different, and they can play multiple roles synergistically; it has strong stability, the growth and metabolism of lactic acid bacteria are complementary to each other, and multiple bacteria coexist and adapt to complex environments, making it easier to colonize; it has a wide range of applications, and multiple bacteria can target different vaginitis pathogens, and the applicable range is larger than that of single bacteria. And the probiotic microecological preparation is safer than live bacteria. After freeze-drying, the lactic acid bacteria are in a dormant state, reducing the risk of harm caused by the death and mutation of lactic acid bacteria; it is more persistent, slowly revives, colonizes, and multiplies under suitable conditions in the body, with a long-lasting effect, and live bacteria are easily cleared and difficult to colonize for a long time.

[0079] It can be seen from Figure 11 that the inhibition rates of 22-9, XA-9, and B-1-35 against Gardnerella vaginalis at the same concentration are 53.83%, 52.58%, and 57.23% respectively. When the equal-dose compounding ratios of 22-9, XA-9, and B-1-35 are 3:2:1, 2:1:3, 1:2:3, 1:2:1, 1:3:1, 2:3:2, and 2:3:1, the inhibition rates are 67.52%, 66.11%, 67.82%, 69.13%, 70.35%, 68.10%, and 72.59% respectively, and the antibacterial ability is much higher than that of single strains.

[0080] It can be seen from Figure 12 that the inhibition rates of 22-9, XA-9, and B-1-35 against Candida albicans are 42.33%, 44.46%, and 43.58% respectively. When the compounding ratios of 22-9, XA-9, and B-1-35 are 1:0:1, 3:1:1, 2:1:3, 3:2:1, and 2:1:1, the inhibition rates are 56.73%, 56.51%, 54.59%, 54.08%, and 54.33% respectively, and the antibacterial ability is much higher than that of single strains.

[0081] In summary, when the compounding ratios of 22-9, XA-9, and B-1-35 are 3:2:1 and 2:1:3, the inhibitory ability of the compounded probiotic preparation against Candida albicans and Gardnerella vaginalis is much higher than that against a single strain. Therefore, 2:1:3 is selected as the final ratio of the probiotic preparation through comparison.

[0082] 2. Detection of the freeze-dried survival rate of the probiotic preparation Detecting the freeze-dried survival rate of the probiotic preparation helps to intuitively understand the impact of freeze-drying on lactic acid bacteria and judge the effect of freeze-drying. At the same time, the survival rate is related to the quality and efficacy of the probiotic preparation. Only when enough live bacteria survive can the microecology be effectively regulated, harmful bacteria be inhibited, and the expected effect be achieved.

[0083] It can be obtained from Figure 13 that the total number of colonies of the undiluted probiotic preparation before and after freeze-drying is at the order of 10 17 CFU / mL, and the freeze-dried survival rate reaches 94.80%.

[0084] 3. Detection of the total number of colonies of the probiotic preparation during storage Detecting the total number of colonies of the probiotic preparation during storage aims to control the quality. The total number of microorganisms can be directly reflected by the total number of colonies. If the total number of colonies increases abnormally, it implies that the product may be contaminated by miscellaneous bacteria or the growth of microorganisms is out of control, which will further disrupt the microecological balance and affect the quality. At the same time, it can also evaluate the stability. By regularly detecting the change of the total number of colonies, the stability of the product can be judged, and the best storage conditions can be obtained.

[0085] It can be obtained from Figure 14 that for the undiluted probiotic preparation, the total number of colonies basically remains unchanged when stored at 4 °C, shows a slight downward trend when stored at room temperature (10-20 °C), and significantly decreases at 37 °C. Even so, the total number of colonies of lactic acid bacteria in the probiotic preparation is still very high, and it is uncertain whether the activity of lactic acid bacteria in the dormant state will be affected. It is recommended to store at 4 °C.

[0086] IV. Product Application Example 1 1. Specific implementation example Example 1 A preparation method of a probiotic compound microecological preparation with the efficacy of improving vaginitis includes the following steps: (1) Lactic acid bacteria fermentation: A mixed starter is obtained by mixing the lactic acid bacteria cultures of Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 in a volume ratio of 2:1:3, and inoculated into the fermentation medium at a volume ratio of 2%. Then, it is cultured at 37 °C for 18 h to obtain a mixed lactic acid bacteria culture.

[0087] (2)Freeze-drying: The obtained lactic acid bacteria mixed culture was centrifuged at 8000 rpm and 4 °C for 15 min. The lactic acid bacteria precipitate was taken, and the cell concentration was adjusted to 1×10 9 CFU / mL using sterile PBS buffer. Freeze-drying protectant with the same volume as the sterile PBS buffer was added, mixed well and placed in a -80 °C refrigerator overnight for pre-freezing. The pre-frozen sample was freeze-dried at -49 °C and 9 Pa for 48 h to obtain the probiotic complex microecological preparation. The formula of the freeze-drying protectant was 118.2 g / L trehalose, 17.1 g / L L-cysteine, 10.3 g / L sorbitol, 1.7 g / L sodium acetate and 120 g / L skim milk powder; (3)Administration method: Vaginal administration.

[0088] The finally prepared complex microecological preparation was named the high-dose vaginal administration group (HD).

[0089] Example 2 Same as Example 1 above, the difference is that: in step (3), the administration method was oral and vaginal administration. The finally prepared complex microecological preparation was named the high-dose combined administration (oral + vaginal administration) group (HH).

[0090] Example 3 Same as Example 1 above, the difference is that: in step (2), the lactic acid bacteria precipitate was taken and the cell concentration was adjusted to 1×10 8 CFU / mL using sterile PBS buffer. The finally prepared complex microecological preparation was named the medium-dose vaginal administration group (MD).

[0091] Example 4 Same as Example 3 above, the difference is that: in step (3), the administration method was oral and vaginal administration. The finally prepared complex microecological preparation was named the medium-dose combined administration (oral + vaginal administration) group (MH).

[0092] Example 5 Same as Example 1 above, the difference is that: in step (2), the lactic acid bacteria precipitate was taken and the cell concentration was adjusted to 1×10 7 CFU / mL using sterile PBS buffer. The finally prepared complex microecological preparation was named the low-dose vaginal administration group (LD).

[0093] Example 6 Same as Example 5 above, the difference is that: in step (3), the administration method was oral and vaginal administration. The finally prepared complex microecological preparation was named the low-dose combined administration (oral + vaginal administration) group (LH).

[0094] 2. Improvement effect of probiotic complex microecological preparation on mouse vaginitis model 1) Experimental materials: SPF-grade healthy female Kunming mice (KM mice), with a body weight of about 35.00 ± 2 g per mouse. Provided by Spiff (Suzhou) Biotechnology Co., Ltd., production license: SCXK (Su) 2022-0006, batch certificate number: A202412190019.

[0095] 2) Experimental environment and ethics: Conducted in the ABSL-2 laboratory of Lead Biological Technology (Kunshan) Co., Ltd., experimental animal use license: SYXK (Su) 2023-0083; record number of Suzhou animal pathogenic microorganism laboratory: Su Kun Dong Laboratory Preparation

[2024] No. 320583012; IACUC number of ethical approval letter: 20241216-0002-01.

[0096] Feeding conditions: After the animals arrived, they were raised in the experimental environment for 3 days before the experiment. The animals were raised in IVC (independent air supply system) cages (5 per cage) in the ABSL-2 animal house. All cages, bedding, and drinking water need to be sterilized before use. All experimental personnel should wear protective clothing and latex gloves when operating in the animal house. The information card for each cage of animals should indicate the number of animals, gender, strain, reception date, project number, and group in the cage. Feed and drinking water are changed once a week. During the feeding period, the animals were allowed to freely ingest feed and drinking water. Temperature: 20-26°C; humidity: 40-70%; light cycle: 12 h of light and 12 h of darkness.

[0097] Feed composition: The feed meets the experimental animal food identification standard, from Keao Xieli (Tianjin) Feed Co., Ltd., feed production license number: Jin Diao Zheng (2020) 01005, implementation standard: GB14924.3-2010; the highest content of pollutants is within the controllable range and is subject to routine inspection by the manufacturer; the drinking water is produced by reverse osmosis filtration.

[0098] 3) Experimental protocol: Eighty-eight mice were randomly divided into 8 groups: They were randomly assigned to 11 experimental groups, with 8 mice in each group. The experimental design adopted two different administration methods: single vaginal route administration (vaginal administration) and dual route combined administration (vaginal administration combined with intragastric administration). The specific grouping settings are as follows: blank control group (CK), estradiol control group (CKC), model vaginal administration group (MDD), model combined administration group (MDH), commercially available lactic acid bacteria preparation group (CG), low-dose vaginal administration group (LD), low-dose combined administration group (LH), medium-dose vaginal administration group (MD), medium-dose combined administration group (MH), high-dose vaginal administration group (HD), and high-dose combined administration group (HH).

[0099] The whole experimental process can be mainly divided into three stages: the estrus induction period (Day 1 - 6), the model establishment period (Day 7 - 11), and the drug administration period (Day 12 - 19).

[0100] During the estrus induction period (Day 1 - 6), except for the CK group, each mouse was subcutaneously injected with 0.05 mL of estradiol benzoate (2 mg / mL) every other day until the end of the experiment.

[0101] During the model establishment period (Day 7 - 11), 20 μL (10 6 CFU / mL) Candida albicans and Gardnerella vaginalis of the mixed bacterial suspension was inoculated into the vagina of the mice. The vaginal orifice of the mice was gently pinched with small forceps for 30 s to prevent the leakage of the bacterial solution. The CK group and the CKC group were given an equal amount of normal saline. The inoculation was carried out continuously for 5 d. The appearance of the vaginal orifice was observed, and the vaginal lavage fluid was taken to detect whether the model was successfully established according to the Gram staining method. After verifying the successful establishment of the model, the drug administration period was entered.

[0102] During the drug administration period (Day 12 - 19), the drug administration regimens (including the administration route and dose) of each group are shown in Table 2.

[0103] Table 2 Administration Route and Dose

[0104] Using the kit, the vaginal and uterine tissues of the mice were observed, and the vaginal tissues were stained with HE and PAS; the levels of IL-1β, IL-6, and TNF-α in the serum of the mice were measured; the relative abundances of intestinal bacteria, vaginal bacteria, and fungi in the mice were measured.

[0105] 4) Experimental results: On the 4th day after drug administration, the vaginal lavage fluid was examined under a microscope. The results were as Figure 15 , and it was found that compared with the CK group and the CKC group, a large number of Candida albicans were visible in the MDD group and the MDH group. Compared with the MDD group and the MDH group, the colony numbers in each drug administration group decreased, showing a negative correlation with the drug administration dose; there was no obvious difference between vaginal administration and combined administration.

[0106] It can be Figure 16 observed that obvious pathological characteristics such as swelling, ulceration, and congestion appeared in the tissues of the model group. The size of the vaginal tissues in the drug administration group was smaller, and the swelling condition was significantly improved, with the high-dose group showing the most obvious effect. In the high-dose group, there was no obvious difference between vaginal administration and combined administration (vaginal administration and gavage).

[0107] Representative pathological pictures are as Figure 17 , Figure 18. In the study of the mouse vaginitis model, HE staining and PAS staining showed significant effects. As a commonly used staining method, HE staining can clearly present the cell structures and morphologies of the mouse vaginal tissue, such as the number of epithelial cell layers, their arrangement, the distribution of blood vessels, connective tissues, and immune cells in the lamina propria, helping to understand the impact of vaginitis on tissue morphology. It can also be used to identify the types and numbers of inflammatory cells such as neutrophils and lymphocytes, and judge the severity of inflammation based on their aggregation and distribution, evaluate the modeling effect, and detect pathological changes such as epithelial cell degeneration, necrosis, lamina propria edema, hyperemia, and fibrous tissue hyperplasia, helping to explore the pathological mechanism of vaginitis.

[0108] PAS (periodic acid-Schiff) staining for detecting fungi is based on its specific staining reaction to polysaccharide substances. The fungal cell wall contains abundant polysaccharide components such as chitin and cellulose. Periodic acid can oxidize the glycol groups in the polysaccharides of the fungal cell wall into aldehyde groups, and the aldehyde groups then combine with the colorless magenta in the Schiff reagent to form a purple-red or red complex, thus making the fungi appear with obvious colors under the microscope for easy identification.

[0109] After careful microscopic observation of the tissues of the submitted samples, it was found that: In the CK group and the CKC group, the vaginal tissue structure was normal, and microabscesses were visible in the mucosal epithelium, which are common normal changes during the interestrus period, mainly neutrophils, and no other obvious pathological changes were seen; there was a small amount of inflammatory cell infiltration in the lamina propria of the CK group; in the CKC group, some mice were in the proestrus stage, with mucus cells, and the PAS staining was positive.

[0110] In the MDD group and the MDH group, fungi with positive PAS staining (red) were visible in the vagina, which should be Candida albicans according to the experiment. Microabscesses were visible in the mucosal epithelium, which are common normal changes during the interestrus period, mainly neutrophils. There was more inflammatory cell infiltration in the lamina propria, and occasional edema was seen.

[0111] In the CG group, LD group, LH group, MD group, MH group, HD group, and HH group, fungi with positive PAS staining (red) were visible in the vagina, which should be Candida albicans according to the experiment. Microabscesses were visible in the mucosal epithelium, which are common normal changes during the interestrus period, mainly neutrophils. There was a small amount of inflammatory cell infiltration in the lamina propria.

[0112] The number of inflammatory cells (neutrophils, lymphocytes, macrophages, etc.) can directly reflect the activity of inflammation, and can judge the acute and chronic nature and development process of inflammation; abnormal increase or decrease in the number of epithelial keratinocytes respectively indicates excessive epithelial cell hyperplasia or damage, assisting in judging the degree of damage to the epithelial tissue; after drug administration, the decrease in inflammatory cells and the restoration of the number of epithelial keratinocytes to normal mostly represent effective improvement.

[0113] As shown in Table 3 and Figure 19As shown, the number of lamina propria inflammatory cells in each group ( Figure 19 A in Figure 19 ) and the number of epithelial keratinocytes ( P B in P ) were counted. The results showed that compared with the blank group, the number of inflammatory cells in the model group increased; compared with the model group, the number of inflammatory cells in each administration group decreased, and the decrease in the HD group was the most significant ( <0.05); there was no significant difference in the number of epithelial keratinocytes among the groups (

[0114] >0.05).

[0115] Figure 20 As shown in P A, in terms of the comparison of IL-1β levels, the content of IL-1β in the serum of the MDD group and the MDH group was significantly ( P <0.01) higher than that of the administration group and the blank group; after the administration treatment, as the dose of the probiotic preparation increased, the decline range of IL-1β showed an increasing trend; there was no significant difference among the LD group, the LH group, the MH group, and the three groups of the LH group, the MH group, and the HD group ( P >0.05), indicating that to a certain extent, the dose had little effect on IL-1β, but after exceeding a certain dose, the effect was extremely significant ( P <0.01); in combined administration and vaginal administration, only the high-dose combined administration had a better effect; the ability of the commercially available lactic acid bacteria preparation to reduce IL-1β was higher than that of the low- and medium-dose groups and lower than that of the high-dose group; the IL-1β level of the HH group decreased to no significant difference from that of the control group (

[0116] >0.05), and it was excellent in the ability to reduce the IL-1β level. Figure 20 As shown in P B, in terms of the comparison of IL-6 levels, the content of IL-6 in the serum of the MDD group and the MDH group was significantly ( P <0.01) higher than that of the blank group; after the administration treatment, as the dose of the probiotic preparation increased, the concentration of IL-6 showed a trend of first increasing and then decreasing, and the low-dose group and the high-dose group had better effects; there was no significant difference among the LD group, the MH group, the HD group, and the three groups ( P >0.05), indicating that to a certain extent, the dose had little effect on IL-6, but after exceeding a certain dose, the effect was significant ( P <0.05); in combined administration and vaginal administration, the medium- and high-dose combined administration had a better effect; the ability of the commercially available lactic acid bacteria preparation to reduce IL-6 was higher than that of the other administration groups and had no significant difference from that of the control group (

[0117] AsFigure 20 As shown in C, in the comparison of TNF-α levels, the content of TNF-α in the serum of the MDD group and the MDH group was significantly ( P <0.01) higher than that of the blank group; after drug administration, with the increase of the dose of the probiotic preparation, TNF-α showed a downward trend; there was no significant difference among the four groups of MDD, MDH, LD, and MD groups ( P >0.05), indicating that vaginal administration of low and medium doses had little effect on TNF-α, and after reaching the high-dose group, the effect was significant ( P <0.05); in combined administration and vaginal administration, all doses of combined administration were superior to vaginal administration; the ability of the commercially available lactic acid bacteria preparation to reduce TNF-α was similar to that of the LH group and lower than that of the HH group; the HH group had a better ability to reduce the level of TNF-α.

[0118] Through 16S rRNA gene sequencing analysis, from Figure 21 , Figure 22 , Figure 23 , Figure 24 , Figure 25 and Figure 26 it can be seen that with the increase of the administration dose, the relative abundance of Bacteroidetes in the mouse intestine increased and that of Firmicutes decreased; in the HD group, the relative abundance of beneficial bacteria genera such as Lactobacillus ( Lactobacillus ) and Ligilactobacillus ( Ligilactobacillus ) accounted for nearly 30.6%, and the relative abundance was significantly higher than that of the model group ( P <0.05). At the same time, the abundance of Firmicutes in the mouse vagina increased, and the abundance of Proteobacteria showed a trend of first increasing and then decreasing. The relative abundance of Proteobacteria in the HD and HH groups was lower than that of the other drug administration groups; the relative abundance of Streptococcus ( Streptococcus ) decreased significantly. In the mouse vagina, the relative abundance of Ascomycota in the HD group decreased; the relative abundance of Candida ( Candida ) in the CG, MH, and HD groups decreased. To a certain extent, both the HD and HH groups could improve the community species richness and diversity of the mouse intestine and vagina, and the colony structure gradually approached that of the CK group.

[0119] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A probiotic with the efficacy of improving vaginitis, characterized in that: The probiotics described above include at least one of Lactiplantibacillus plantarum strain 22-9 with the preservation number of CGMCC No. 33867, Lactiplantibacillus plantarum strain XA-9 with the preservation number of CGMCC No. 33868, and Lactiplantibacillus pentosus strain B-1-35 with the preservation number of CGMCC No. 33870.

2. A probiotic complex microecological preparation with the efficacy of improving vaginitis, characterized in that: The probiotic complex microecological preparation described above includes at least one strain of Lactiplantibacillus plantarum strain 22-9 with the preservation number of CGMCC No. 33867, Lactiplantibacillus plantarum strain XA-9 with the preservation number of CGMCC No. 33868, and Lactiplantibacillus pentosus strain B-1-35 with the preservation number of CGMCC No. 33870. The active ingredient of the probiotic complex microecological preparation includes the probiotic cells described above.

3. A probiotic complex microecological preparation with the efficacy of improving vaginitis according to claim 2, characterized in that: The number of effective bacteria in the probiotic complex microecological preparation is not less than 1×10 7 CFU / mL.

4. The probiotic complex microecological preparation with the efficacy of improving vaginitis according to claim 2, characterized in that: The concentrations of the Lactiplantibacillus plantarum 22-9 bacterial liquid, the Lactiplantibacillus plantarum XA-9 bacterial liquid, and the Lactiplantibacillus pentosus B-1-35 bacterial liquid are the same, and the mixing volume ratio is 2:1:

3.

5. A preparation method of the probiotic complex microecological preparation with the efficacy of improving vaginitis as described in claim 2, characterized in that It includes the following steps: (1) Inoculate Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 into the fermentation medium at a volume ratio of 1-5% respectively, and culture at 35-40 °C for 15-20 h to obtain lactic acid bacteria cultures. Mix the lactic acid bacteria cultures of Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 at a volume ratio of (1-3):(1-3):(1-3) to obtain a mixed starter. Inoculate the fermentation medium at a volume ratio of 2% and culture at 37 °C for 18 h to obtain a mixed lactic acid bacteria culture; (2) Centrifuge the lactic acid bacteria culture obtained in step (1) to obtain lactic acid bacteria precipitate, and adjust the cell concentration to 1×10 7 -1×10 10 CFU / mL. Add a freeze-drying protectant with the same volume as the sterile PBS buffer, mix well, place it in a -80°C refrigerator for overnight pre-freezing, and lyophilize the pre-frozen sample to obtain the probiotic complex microecological preparation.

6. The preparation method of a probiotic complex microecological preparation with the efficacy of improving vaginitis according to claim 5, characterized in that: The lactic acid bacteria cultures of Lactiplantibacillus plantarum 22-9, Lactiplantibacillus plantarum XA-9, and Lactiplantibacillus pentosus B-1-35 are mixed at a volume ratio of 2:1:3 to obtain a mixed starter, and the cell concentrations of each lactic acid bacteria culture are the same.

7. The preparation method of a probiotic complex microecological preparation with the efficacy of improving vaginitis according to claim 5, characterized in that: The fermentation medium described in step (1) is prepared by dissolving the MRS solid medium in 1 L of distilled water and sterilizing it at 121 °C for 15 min. The formula of the MRS solid medium is 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 80 mL of Tween 80, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate, and 15 g of agar powder.

8. The preparation method of a probiotic complex microecological preparation with the efficacy of improving vaginitis according to claim 5, characterized in that: The formula of the lyoprotectant described in step (2) is 118.2 g / L of trehalose, 17.1 g / L of L-cysteine, 10.3 g / L of sorbitol, 1.7 g / L of sodium acetate, and 120 g / L of skim milk powder.

9. Application of the probiotics according to claim 1 in the preparation of an inhibitor of Gardnerella vaginalis and / or Candida albicans.

10. Application of the probiotic complex microecological preparation according to claim 2 in the preparation of a drug for improving or treating vaginitis symptoms.

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