A probiotic with improved vaginitis efficacy, and a composite micro-ecological preparation and application thereof

Abstract

The application discloses probiotics with the effect of improving vaginitis, a compound micro-ecological preparation and application thereof, and is characterized by comprising at least one of the Lactobacillus plantarum 22-9 strain with the preservation number of CGMCC No.33867, the Lactobacillus plantarum XA-9 strain with the preservation number of CGMCC No.33868 and the Lactobacillus pentosus B-1-35 strain with the preservation number of CGMCC No.33870, and the lactic acid bacteria culture of the Lactobacillus plantarum 22-9, the Lactobacillus plantarum XA-9 and the Lactobacillus pentosus B-1-35 is mixed in a volume ratio of 2:1:3, and the lactic acid bacteria fermenting agent is inoculated in a fermentation medium in a volume ratio of 1-5% to prepare the probiotic compound micro-ecological preparation, and the advantage is that the content of lactic acid bacteria in the vagina can be increased, the content of pathogenic bacteria can be reduced, the number of inflammatory cells in the vagina can be reduced, and the levels of inflammatory factors IL-1beta, IL-6 and TNF-alpha in serum can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a probiotic compound microecological preparation, in particular to a probiotic with the effect of improving vaginitis and a compound microecological preparation and application thereof. BACKGROUND

[0002] Vaginal itching, burning, irritation, pain, "fishy smell" vaginal odor and abnormal vaginal discharge are symptoms of vaginitis. Vaginal infection can affect reproductive health, increase the risk of sexually transmitted infections, and induce other gynecological inflammation, affect pregnancy, increase the risk of miscarriage, premature birth, infertility and ectopic pregnancy, and the infection rate is 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, the most common bacterial vaginosis (BV) and VVC are mainly caused by Gardnerella vaginalis and Candida albicans. After adhering and colonizing in the vaginal environment, they gather other pathogenic bacteria to form a biofilm, resist the host's immune defense environment, and exacerbate vaginal inflammation.

[0003] When the vagina is invaded by a large number of pathogens, the body can regulate through the self-regulation of the vagina. Mainly including: the vagina secretes acidic secretions to expel part of the pathogen; the vaginal innate immune cells can recognize and phagocytize the pathogen to initiate an immune response to prevent the spread of infection; the vaginal lactobacillus produces lactic acid, hydrogen peroxide and other substances to create and maintain the acidic environment of the vagina, thereby inhibiting the growth of other harmful bacteria. However, the body's self-regulation is limited and cannot completely eliminate the pathogens in the vagina, so treatment intervention is needed. At present, the treatment of vaginal infection is mainly divided into drug treatment and biological treatment. Drug treatment can inhibit pathogenic bacteria, but it has adverse effects on vaginal lactobacillus flora, which is prone to recurrence and chronic infection. Biological treatment such as lactobacillus treatment can inhibit the invasion and overgrowth of exogenous bacteria and opportunistic pathogens, and has obvious advantages such as low recurrence rate, high safety, small side effects, and no drug resistance. In order to maintain the health of the vaginal microecology, it is urgent to develop a probiotic compound microecological preparation with the effect of improving vaginitis. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a probiotic with the effect of improving vaginitis and a compound microecological preparation and application thereof, which can increase the content of lactobacillus 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 serum.

[0005] The present application adopts the technical solution for solving the above technical problems, which is characterized in that

[0006] The present application provides a probiotic with the effect of improving vaginitis, which comprises at least one of Lactiplantibacillus plantarum 22-9 strain with the preservation number of CGMCC No.33867, Lactiplantibacillus plantarum XA-9 strain with the preservation number of CGMCC No.33868 and Lactiplantibacillus pentosus B-1-35 strain with the preservation number of CGMCC No.33870.

[0007] The present application also provides a probiotic compound micro-ecological preparation with the effect of improving vaginitis, which comprises at least one of Lactiplantibacillus plantarum 22-9 strain with the preservation number of CGMCC No.33867, Lactiplantibacillus plantarum XA-9 strain with the preservation number of CGMCC No.33868 and Lactiplantibacillus pentosus B-1-35 strain with the preservation number of CGMCC No.33870, wherein the active ingredient of the probiotic compound micro-ecological preparation comprises the probiotic bacteria.

[0008] Further, the number of effective bacteria in the probiotic compound micro-ecological preparation is not less than 1×10 7 CFU / mL.

[0009] Further, the concentration of the Lactiplantibacillus plantarum 22-9 bacterial solution, the Lactiplantibacillus plantarum XA-9 bacterial solution and the Lactiplantibacillus pentosus B-1-35 bacterial solution is the same, and the mixed volume ratio is 2:1:3.

[0010] The present application also provides a preparation method of the probiotic compound micro-ecological preparation with the effect of improving vaginitis, which comprises the following steps:

[0011] (1) Lactobacillus plantarum 22-9, Lactobacillus plantarum XA-9 and Lactobacillus pentosus B-1-35 are inoculated into a fermentation medium at a ratio of 1-5% by volume, respectively, and cultured at 35-40℃ for 15-20h to obtain a lactic acid bacteria culture, and the lactic acid bacteria culture of Lactobacillus plantarum 22-9, Lactobacillus plantarum XA-9 and Lactobacillus pentosus B-1-35 is mixed at a ratio of (1-3):(1-3):(1-3) by volume to obtain a mixed starter culture, which is inoculated into a fermentation medium at a ratio of 2% by volume and cultured at 37℃ for 18h to obtain a mixed lactic acid bacteria culture;

[0012] (2) The lactic acid bacteria culture obtained in step (1) is centrifuged to obtain a lactic acid bacteria precipitate, and the bacterial concentration is adjusted to 1×10 7 -1×10 10 CFU / mL using sterile PBS buffer, an equal volume of freeze-drying protective agent is added, and the mixture is pre-frozen overnight in a -80℃ refrigerator, and then freeze-dried to obtain the probiotic compound microecological preparation.

[0013] Further, the lactic acid bacteria culture of Lactobacillus plantarum 22-9, Lactobacillus plantarum XA-9 and Lactobacillus pentosus B-1-35 in step (1) is mixed at a ratio of 2:1:3 by volume to obtain a mixed starter culture, and the bacterial concentration of each lactic acid bacteria culture is the same.

[0014] Further, the fermentation medium in step (1) is prepared by dissolving 1L of distilled water in MRS solid medium, sterilizing at 121℃ for 15min, and the formula of the MRS solid medium is as follows: 10g of proteose peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80mL of Tween 80, 0.5g of magnesium sulfate, 0.25g of manganese sulfate and 15g of agar powder.

[0015] Further, the formula of the freeze-drying protective agent in step (2) is as follows: 118.2g / L of trehalose, 17.1g / L of L-cysteine, 10.3g / L of sorbitol, 1.7g / L of sodium acetate and 120g / L of skimmed milk powder.

[0016] The application also provides the use of the above-mentioned probiotic bacteria in the preparation of a Gardnerella vaginalis and / or Candida albicans inhibitor.

[0017] The application also provides the use of the above-mentioned probiotic compound microecological preparation in the preparation of a drug for improving or treating vaginitis.

[0018] Compared with the prior art, the advantages of the probiotic with improved vaginitis efficacy, the composite microecological preparation and application thereof are that three strains of lactic acid bacteria with significant inhibitory effect on Gardnerella vaginalis and Candida albicans are screened, the microecological preparation is obtained by using the three kinds of lactic acid bacteria, the growth of pathogenic bacteria in the vagina can be greatly inhibited, the proportion of lactic acid bacteria in the vagina can be effectively increased, the number of inflammatory cells in the vagina can be reduced, the levels of inflammatory factors IL-1beta, IL-6 and TNF-alpha in serum can be reduced, excessive inflammatory reaction can be relieved, the mouse model of vaginitis can be obviously improved, and the blank of probiotic preparation products for relieving the symptoms of vaginitis in the market is made up.

[0019] The above-mentioned Lactiplantibacillus plantarum 22-9 strain has a preservation number of CGMCC No.33867 and was preserved at the China General Microbiological Culture Collection Center on March 19, 2025, at an address of No.3, Beichen West Road, Haidian District, Beijing.

[0020] The above-mentioned Lactiplantibacillus plantarum XA-9 strain has a preservation number of CGMCC No.33868 and was preserved at the China General Microbiological Culture Collection Center on March 19, 2025, at an address of No.3, Beichen West Road, Haidian District, Beijing.

[0021] The above-mentioned Lactiplantibacillus pentosus B-1-35 strain has a preservation number of CGMCC No.33870 and was preserved at the China General Microbiological Culture Collection Center on March 19, 2025, at an address of No.3, Beichen West Road, Haidian District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The inhibition rate of the lactic acid bacteria on Gardnerella vaginalis;

[0023] Figure 2 The inhibition rate of the lactic acid bacteria on Candida albicans;

[0024] Figure 3 The growth and acid production curve of the lactic acid bacteria;

[0025] Figure 4 The survival rate and OD value of the lactic acid bacteria at different pH values, wherein (A) is the OD value of the lactic acid bacteria at different pH values, (B) is the survival rate of the lactic acid bacteria at pH 3.0, and (C) is the survival rate of the lactic acid bacteria at pH 3.5;

[0026] Figure 5Survival rate and OD value of lactic acid bacteria under different bile salts, wherein (A) OD value of lactic acid bacteria under different bile salts, (B) survival rate of lactic acid bacteria under 0.3% bile salt; (C) survival rate of lactic acid bacteria under 0.6% bile salt; (D) survival rate of lactic acid bacteria under 0.9% bile salt;

[0027] Figure 6 Adhesion ability evaluation of lactic acid bacteria (A) ability of lactic acid bacteria to self-aggregate; (B) hydrophobicity of lactic acid bacteria;

[0028] Figure 7 Hemolysis experiment of 3 strains of lactic acid bacteria;

[0029] Figure 8 Transmission electron microscope graph of 30000x of cell morphology of 3 strains of lactic acid bacteria;

[0030] Figure 9 PCR product electrophoresis of 16S rDNA of 3 strains of lactic acid bacteria;

[0031] Figure 10 Development tree of 3 strains of lactic acid bacteria;

[0032] Figure 11 Inhibition ability of microecological preparation on Gardnerella vaginalis under different compounding ratios of lactic acid bacteria;

[0033] Figure 12 Inhibition ability of microecological preparation on Candida albicans under different compounding ratios of lactic acid bacteria;

[0034] Figure 13 Total number of colonies of undiluted microecological preparation before and after freeze-drying;

[0035] Figure 14 Total number of colonies of undiluted microecological preparation under different storage temperatures;

[0036] Figure 15 Gram staining of vaginal lavage fluid of mouse vaginitis model on Day 4 of administration period;

[0037] Figure 16 Vaginal uterine tissue of mouse vaginitis model after administration period;

[0038] Figure 17 HE x 400 staining result graph of mouse vaginal tissue (inflammatory cells are indicated by black arrows);

[0039] Figure 18 PAS x 200 staining result graph of mouse vaginal tissue (fungal positive is indicated by green arrow);

[0040] Figure 19The number of inflammatory cells and epithelial cells in the vagina of mice, wherein (A) is the number of inflammatory cells in the vagina, (B) is the number of epithelial cells in the vagina; the letters a-c in the figure represent significant differences (P<0.05) between different treatment groups;

[0041] Figure 20 The IL-1β, IL-6, and TNF-α levels in the serum of mice in each group, wherein A: IL-1β; B: IL-6; C:

[0042] TNF-α; the letters a-c in the figure represent significant differences (P<0.05) between different treatment groups;

[0043] Figure 21 The effect of the probiotic compound microecological preparation on the species relative abundance of intestinal bacterial phylum level;

[0044] Figure 22 The effect of the probiotic compound microecological preparation on the species relative abundance of intestinal bacterial genus level;

[0045] Figure 23 The effect of the probiotic compound microecological preparation on the species relative abundance of vaginal bacterial phylum level;

[0046] Figure 24 The effect of the probiotic compound microecological preparation on the species relative abundance of vaginal bacterial genus level;

[0047] Figure 25 The effect of the probiotic compound microecological preparation on the species relative abundance of vaginal fungal phylum level;

[0048] Figure 26 The effect of the probiotic compound microecological preparation on the species relative abundance of vaginal fungal genus level. DETAILED DESCRIPTION

[0049] The application will be further described in detail below with reference to the accompanying drawings.

[0050] I. Experimental methods

[0051] 1. Preparation of culture medium and solution

[0052] MRS liquid medium: Dissolve the purchased MRS liquid medium (MRS liquid medium formula: peptone 10 g, beef extract 10 g, yeast extract 5 g, diammonium citrate 2 g, sodium acetate 5 g, glucose 20 g, Tween 80 mL, magnesium sulfate 0.5 g, manganese sulfate 0.25 g, and agar powder 15 g) in 1 L distilled water, and sterilize at 121°C for 15 min.

[0053] MRS solid medium: MRS liquid medium was added with agar 15.0 g, sterilized at 121 ℃ for 15 min.

[0054] YM solid medium: YM solid medium (YM solid medium formula: glucose 20 g, peptone 5 g, yeast extract powder 3 g, potassium dihydrogen phosphate 1 g, magnesium sulfate 0.5 g, agar 15 g) purchased was dissolved with 1 L distilled water, sterilized at 121 ℃ for 15 min.

[0055] YPD liquid medium: YPD liquid medium (YPD liquid medium formula: yeast extract powder 10 g, peptone 20 g, glucose 20 g) purchased was dissolved with 1 L distilled water, sterilized at 121 ℃ for 15 min.

[0056] Selective Gardnerella vaginalis medium: Selective Gardnerella vaginalis medium purchased was dissolved with 1 L distilled water, sterilized at 121 ℃ for 15 min. When used, 5wt%-10wt% sterile defibrillated horse blood, 15-20 μg / ml naltrexone acid solution, 10-20 μg / ml gentamicin sulfate, and 2-5 μg / ml amphotericin B solution were added to the above medium when it was cooled to about 50 ℃.

[0057] Blood agar base medium: Blood agar base medium (blood agar base medium formula: peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 15 g) purchased was dissolved with 1 L distilled water, sterilized at 121 ℃ for 15 min. When used, 5wt%-10wt% sterile defibrillated sheep blood or rabbit blood was added to the above base medium when it was cooled to about 50 ℃ to provide special nutrients required for bacterial growth, and to help observation of hemolysis phenomenon of certain bacteria.

[0058] 2. Activation and preservation of strains

[0059] 2 mL of lactic acid bacteria strains preserved in a refrigerator at -80 ℃ were selected and inoculated into 50 mL of fermentation medium, and cultured at 37 ℃ for 12 h to obtain seed liquid. The seed liquid was subjected to spread culture, and the colony morphology was observed and inverted and stored in a refrigerator at -4 ℃ for standby use.

[0060] 3. Preparation of lactic acid bacteria liquid and pathogenic bacteria liquid

[0061] Lactic acid bacteria culture: The above preserved lactic acid bacteria seed liquid was inoculated into fermentation medium (MRS solid medium dissolved with 1 L distilled water and sterilized at 121 ℃ for 15 min) and cultured at 37 ℃ for 18 h, and activated for three generations to obtain lactic acid bacteria culture, and the OD 600 was adjusted to 1, and the bacterial liquid was used for standby.

[0062] Lactic acid bacteria cell-free supernatant (CFS): The obtained lactic acid bacteria culture was centrifuged at 8000 r / min for 10 min, and the fermentation supernatant was taken and filtered through a 0.22 μm PES filter membrane to obtain the cell-free supernatant (CFS).

[0063] Culture of Gardnerella vaginalis: Gardnerella vaginalis was inoculated on blood agar solid medium, and after two generations of single colony picking and activation, it was cultured in a selective Gardnerella vaginalis liquid medium at 37°C for 24-48 h under anaerobic conditions, and the bacterial liquid concentration was adjusted to 10 8 CFU / mL.

[0064] Culture of Candida albicans: Candida albicans was inoculated on YM solid medium, and after two generations of single colony picking and activation, it was cultured in YPD liquid medium at 30°C for 24 h, and the bacterial concentration was adjusted to 4×10 6 CFU / mL.

[0065] 4. Inhibition of Gardnerella vaginalis and Candida albicans

[0066] In a 96-well plate, 150 μL of 2% v / v inoculum of Candida albicans YPD liquid medium and 50 μL of lactic acid bacteria CFS were added and mixed and cultured in a biochemical incubator at 30°C for 24 h, and the OD 600 value was recorded as OD 处理组 ; 200 μL of 2% v / v inoculum of Candida albicans was added and mixed and cultured at 30°C for 24 h, and the OD 600 value was recorded as OD 对照组 .

[0067] In a 96-well plate, 150 μL of 2% v / v inoculum of Gardnerella vaginalis and 50 μL of lactic acid bacteria fermentation supernatant were added and mixed and cultured in an anaerobic incubator at 37°C for 24-48 h, and the OD 600 value was recorded as OD 处理组 ; 200 μL of 2% v / v inoculum of Gardnerella vaginalis was added and mixed and cultured at 37°C for 24-48 h, and the OD 600 value was recorded as OD 对照组 .

[0068] By comparing the inhibition rates of the two, the strain with better inhibition ability to Gardnerella vaginalis and Candida albicans was determined. The calculation formula is as follows:

[0069]

[0070] 5. Growth curve and acid production curve

[0071] After activating the seed culture for three generations, the culture was inoculated and cultured at an inoculum size of 2% (v / v). The pH value and absorbance at 600 nm of the fermentation broth were measured every 2 hours. The culture medium was thoroughly mixed before each measurement, and three replicates were performed.

[0072] 6. Determination of acid and bile salt resistance

[0073] Acid tolerance was assessed using MRS broth at pH 3.0 and 3.5, and bile tolerance was tested using MRS broth supplemented with 0.3%, 0.6%, and 0.9% (w / v) porcine bile salts. Five strains of lactic acid bacteria were inoculated into the above-mentioned medium at a 2% v / v inoculum and cultured at 37°C for 18 h. OD was then measured. 600 It is denoted as A1; it is inoculated into additive-free MRS broth and cultured at 37°C for 18 hours, and the OD is measured. 600 Let this be denoted as A0. The survival rate calculation formula is as follows:

[0074]

[0075] 7. Determination of self-aggregation ability and surface hydrophobicity

[0076] The bacterial culture was inoculated into MRS broth medium at a 2% v / v inoculum and cultured for 18 h. The bacterial cells were collected by centrifugation at 5000 rpm for 10 min at 4 °C, washed twice with PBS, and then resuspended in PBS. The OD was then measured. 600 And adjust the OD of the bacterial suspension. 600 To ensure the OD value was within the range of 0.25 ± 0.05, the initial OD was recorded as A0. 4 mL of bacterial suspension was transferred to an EP tube and incubated at 37°C for 20 h. Then, 3 mL of the supernatant was added to another centrifuge tube, and its OD value was measured. 600 Let A1 be the value of the self-aggregation rate. Calculate the self-aggregation rate using the following formula. Repeat the experiment three times independently and take the average value.

[0077]

[0078] Prepare a bacterial suspension as described above. Add 1 mL of xylene to 3 mL of the bacterial suspension, vortex to mix thoroughly, and let stand at 37°C for 1 h. Collect the aqueous phase and measure its OD. 600 Let this be labeled A. Calculate the hydrophobicity of the strain using the following formula. Each independent experiment was repeated three times, and the average value was taken.

[0079]

[0080] 8. Antibiotic susceptibility testing

[0081] Pour 10 mL MRS solid medium into the culture dish, after it solidifies, pour the soft agar containing 1% v / v lactic acid bacteria inoculation amount, after it solidifies, the drug sensitive paper sheet is flat pasted on the surface, and the group number is recorded. The drug sensitive paper sheets used are: vancomycin, tetracycline, streptomycin, penicillin, ampicillin, erythromycin, kanamycin, ceftazidime, gentamicin, doxycycline (doxycycline). The flat plate with the pasted drug paper sheet is placed at 37°C for 48h, three parallel plates are set up, and the size of the bacteriostatic ring produced by each drug sensitive paper sheet is measured and recorded.

[0082] 9. Transmission electron microscope (TEM)

[0083] After fermentation, the lactic acid bacteria liquid is centrifuged at 5000r / min for 10min, the bacterial precipitate is collected, and the residual medium is removed by washing with PBS buffer (pH 7.4) for 3 times. Then, the bacterial body is resuspended in PBS, and an appropriate amount of bacterial suspension is added dropwise to a 200-mesh copper mesh (carbon support film), and left to stand for 15min to allow the bacterial body to fully settle. After adsorption is completed, the excess liquid is absorbed with filter paper to avoid excessive thickness of the sample. Then, the copper mesh is immersed in 2%-5% uranyl acetate ethanol solution for 30s, and the staining is repeated 3 times to enhance the contrast. Finally, the bacterial body morphology and ultrastructure are observed under a transmission electron microscope (TEM).

[0084] 10. Hemolysis experiment

[0085] The lactic acid bacteria are inoculated on agar plates containing blood suitable for the growth of the bacterial strain, and the hemolytic ring is observed.

[0086] 11. 16S r DNA identification and evolution analysis of lactic acid bacteria

[0087] (1) Extraction of DNA

[0088] Fresh bacterial liquid is centrifuged at 8000rpm for 1min to obtain the bacterial body, and the total DNA of the strain is extracted by using the kit.

[0089] (2) PCR amplification and product detection

[0090] The universal primer is used to amplify the 16S r RNA gene, and the primer sequence is as follows:

[0091] Upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0092] Downstream primer 1495R: 5'-CTACGGCTACCTTGTTACGA-3';

[0093] PCR cycle parameters: 94℃ pre-denaturation 5 min, 94℃ denaturation 1 min, 64℃ annealing 1 min, 72℃ extension 2 min, 4℃ terminal extension 10 min, 34 cycles. The amplified PCR product was sent to Shengong Bioengineering Co., Ltd. for gene sequence detection.

[0094] (3) Phylogenetic tree

[0095] The obtained strain sequence was subjected to 16S rDNA gene fragment alignment on the NCBI website, and the 16S rDNA gene sequence of the strain with high similarity was extracted, and a phylogenetic tree was constructed using software MEGAX 64.

[0096] 12. Optimization of the compounding ratio of probiotic microecological preparations

[0097] After the three strains of lactic acid bacteria screened were activated, the OD 600 =1 (10 9 CFU / mL) was adjusted, and the compounding was performed according to the compounding ratios 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, and inoculated in 10 mL MRS broth at an inoculum of 2% v / v, and after 18 h of culture, centrifuged at 8000 r / min for 10 min, the fermentation supernatant was taken, filtered with a 0.22 μm PES filter membrane to obtain CFS.

[0098] In a 96-well plate, 150 μL of 2% v / v inoculum of Candida albicans YPD liquid culture medium and 50 μL of lactic acid bacteria CFS were added and mixed in a biochemical incubator at 30°C for 24 h of culture, and the OD 600 value was detected and recorded as OD 处理组 ; 200 μL of 2% inoculum of Candida albicans was added and mixed at 30°C for 24 h of culture, and the OD 600 value was detected and recorded as OD 对照组 .

[0099] 150 μL of 2% v / v inoculum of Gardnerella vaginalis and 50 μL of lactic acid bacteria fermentation supernatant were added and mixed in an anaerobic incubator at 37°C for 24-48 h of culture, and the OD 600 value was detected and recorded as OD 处理组 ; 200 μL of 2% v / v inoculum of Gardnerella vaginalis was added and mixed at 37°C for 24-48 h of culture, and the OD 600 value was detected and recorded as OD 对照组 .

[0100]

[0101] By comparing the inhibition rates of the two, the most potential for improving vaginitis is screened out.

[0102] 13. Detection of freeze-dried survival rate of probiotic microecological preparation

[0103] After the selected 3 strains of lactic acid bacteria are compounded according to the optimal compound ratio, inoculate 2% v / v into MRS broth, culture for 18 h, centrifuge at 8000 r / min for 10 min, and obtain bacterial slurry. Add 2 mL of freeze-drying protective agent to the bacterial slurry obtained by centrifugation of 50 mL of culture, freeze-dry in a freeze-drying machine, and obtain high-concentration lactic acid bacteria preparation.

[0104] The freeze-drying protective agent is the best formula optimized in the laboratory in the early stage: 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. The ten-fold gradient dilution method is used for coating count of bacterial liquid and bacterial powder before and after freeze-drying, and the freeze-drying survival rate is obtained by dividing the two.

[0105] 14. Detection of total number of colonies of probiotic microecological preparation during storage period

[0106] The bacterial powder obtained by freeze-drying is stored at 4°C, 37°C and room temperature (10°C-20°C), respectively, and on the 0th, 7th, 14th, 21st and 28th day of storage, the microecological preparation under different storage conditions is diluted and plated for counting.

[0107] 15. Data processing and statistical analysis

[0108] Each experiment is repeated 3 times, and the experimental data is statistically analyzed and plotted using GraphPad Prism 9.4.0 and SPSS19.0, and the experimental results are presented in the form of mean ± standard deviation (mean ± SEM). Multiple data is analyzed for significance by One-Way ANOVA single factor analysis of variance.

[0109] II. Screening of strains

[0110] 1. Screening of lactic acid bacteria

[0111] When the vaginal microecological balance is broken, harmful bacteria such as Gardnerella vaginalis and Candida albicans will proliferate. Lactic acid bacteria can produce lactic acid, H2O2 and bacteriocin and other substances, effectively inhibit the growth and reproduction of harmful bacteria, and help the vaginal microecological balance. In the process of applying lactic acid bacteria to the treatment of vaginitis, the bacteriostatic ability is the most critical indicator, which can be used as the primary standard for screening lactic acid bacteria with the potential to improve vaginitis. 32 strains of lactic acid bacteria were randomly selected from the strain preservation library of the Laboratory of Animal Product Processing of Ningbo University for bacteriostatic experiment. By detecting the inhibitory ability of lactic acid bacteria CFS on Gardnerella vaginalis and Candida albicans, 5 strains of lactic acid bacteria with the potential to improve vaginitis were screened out.

[0112] From Figure 1 It can be seen that the growth of Gardnerella vaginalis was inhibited by 57.22%, 53.96%, 52.58% and 50.87% by lactic acid bacteria B-1-35, 22-9, XA-9 and 20A-1, respectively. From Figure 2 It can be seen that the growth of Candida albicans was inhibited by 46.34%, 43.09%, 43.61%, 42.78%, 41.97% and 43.18% by B-1-35, 83A-58, H-1-46, XA-9, 54A-32 and 22-9, respectively. Based on the inhibitory ability of lactic acid bacteria on Gardnerella vaginalis and Candida albicans, lactic acid bacteria 22-9, 83A-58, B-1-35, 54A-32 and XA-9 were selected as candidate strains for compound microecological agent.

[0113] 2. Growth curve and acid production curve

[0114] By drawing the growth curve, the growth state of lactic acid bacteria at different culture stages can be determined, which helps to determine the optimal culture time of lactic acid bacteria and provides experimental basis for the production of microecological preparations. Acid production curve can reflect the ability and dynamic changes of lactic acid bacteria in the process of acid production, which can be used to determine the acid production ability of different lactic acid bacteria strains.

[0115] For example Figure 3As shown, when the culture temperature was 37℃, after adjusting the five lactic acid bacteria strains to the same concentration, they were inoculated into MRS liquid medium at 2% v / v. After 2 hours, they entered the logarithmic growth phase, and after 14 hours, they entered the plateau phase. The initial pH of the lactic acid bacteria fermentation broth was around 5.60, which rapidly decreased to around 3.80 within 2-10 hours of culture, and then slowly decreased until it stabilized after 14 hours. The growth curves and pH trends of the five lactic acid bacteria strains were consistent, with no significant differences. The experimental results indicate that all the above strains have good acid-producing capacity and can quickly enter the logarithmic phase, resulting in a rapid decrease in pH. This characteristic has a good effect on restoring the acidic environment of the vagina and inhibiting the growth of pathogenic bacteria.

[0116] 3. Determination of acid and bile salt resistance

[0117] The human gastrointestinal environment is complex, with gastric acid and bile being important components. Testing the acid and bile salt tolerance of lactic acid bacteria can simulate their survival conditions in the gastrointestinal tract, assessing whether they can maintain their activity and exert beneficial effects after traversing the acidic environment of the stomach and the bile salt environment of the small intestine. In the development of probiotic preparations, selecting lactic acid bacteria strains with strong acid and bile salt tolerance ensures that a sufficient number of live bacteria reach the intestines after oral administration, thereby improving the efficacy and stability of the lactic acid bacteria preparation.

[0118] like Figure 4 As shown in (A), when lactic acid bacteria are grown in an environment with a pH of 6.5 (control), the growth of each lactic acid bacteria is rapid, and the OD... 600 All were above 1.1. The growth capacity of each lactic acid bacteria changed as the pH value decreased. For example... Figure 4 As shown in Figure (C), at pH 3.5, the survival rates of strains B-1-35, 83A-58, 22-9, 54A-32, and XA-9 were 14.23%, 23.23%, 19.71%, 15.16%, and 24.12%, respectively. At this pH, XA-9 exhibited the highest survival rate, significantly (P<0.05) higher than the other strains. Figure 4 As shown in Figure (B), the survival rates of all lactic acid bacteria decreased significantly at pH 3, at 5.55%, 3.93%, 4.83%, 3.29%, and 4.03%, respectively. Furthermore, there were no significant differences among the five strains. These results indicate that the lactic acid bacteria exhibit a certain degree of tolerance to acidic environments.

[0119] like Figure 5 As shown in (A), lactic acid bacteria grow normally and reach their maximum growth value when the bile salt concentration is 0%. With increasing bile salt concentration, the growth capacity of each lactic acid bacteria changes. Figure 5As shown in Figure (B), at a bile salt concentration of 0.3%, the survival rates of B-1-35, 83A-58, 22-9, 54A-32, and XA-9 were 38.31%, 32.20%, 31.57%, 21.98%, and 15.28%, respectively. At this concentration, B-1-35 had the highest survival rate, significantly (P<0.05) higher than 54A-32 and XA-9. Figure 5 As shown in Figure (C), at a bile salt concentration of 0.6%, the survival rates of the various lactic acid bacteria were 21.46%, 22.85%, 27.43%, 21.08%, and 14.28%, respectively; among them, 54A-32 and XA-9 showed almost no change, while 22-9 showed a smaller decrease. Figure 5 As shown in Figure (D), at a bile salt concentration of 0.9%, the survival rates of the various lactic acid bacteria were 18.89%, 17.88%, 10.42%, 15.55%, and 14.21%, respectively; the survival rates of the lactic acid bacteria did not decrease significantly. In summary, the results indicate that the above lactic acid bacteria all exhibit a certain degree of tolerance to the bile salt environment.

[0120] 4. Determination of self-aggregation ability and surface hydrophobicity

[0121] Self-aggregation ability helps lactic acid bacteria build communities, better resist antimicrobial substances, and maintain high density in the local environment. Simultaneously, the self-aggregation ability of lactic acid bacteria is closely related to their adhesion characteristics; lactic acid bacteria with strong self-aggregation ability are more likely to colonize in the vagina. Hydrophobicity affects interactions with other substances in the surrounding environment; lactic acid bacteria with suitable hydrophobicity can better bind specifically or non-specifically to epithelial cells and other microbial cells, which is beneficial for their colonization in the human body. A self-aggregation rate of approximately 16-35% indicates low self-aggregation ability, 36-50% indicates moderate self-aggregation ability, and above 51% indicates high self-aggregation ability. The cell adhesion ability of lactic acid bacteria plays a crucial role in both oral and vaginal administration. Using self-aggregation ability and surface hydrophobicity as secondary screening criteria, combined with the acid and bile salt resistance characteristics of lactic acid bacteria, three strains were selected from five candidate strains for the preparation of probiotic microecological preparations.

[0122] according to Figure 6 From (A), the self-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 exhibiting high self-aggregation ability; 83A-58 exhibits moderate self-aggregation ability. According to... Figure 6The XA-9, B-1-35, and 22-9 are moderately hydrophobic, and the 54A-32 and 83A-58 are non-hydrophobic. Auto-aggregation and surface hydrophobicity are important indicators for evaluating the adhesion ability of lactic acid bacteria, and therefore, according to the two indicators, combined with the acid and bile salt resistance, the strains for the microecological preparation are screened. Finally, the XA-9, B-1-35, and 22-9 are selected as the strains for the microecological preparation.

[0123] 5. Antibiotic sensitivity test

[0124] The purpose of detecting the antibiotic sensitivity of lactic acid bacteria is to avoid the transfer of drug resistance genes and to avoid interference in subsequent combination therapy with drugs. Drug-resistant lactic acid bacteria may carry drug resistance genes and have the potential to be transmitted to some pathogenic bacteria that harm the human body. Detecting antibiotic sensitivity helps to discover and control the spread of drug-resistant strains in a timely manner and prevent the spread of drug resistance genes in microbial communities.

[0125] Meanwhile, in the treatment of vaginitis, the method of combination therapy with antibiotics and lactic acid bacteria is often used. By detecting the antibiotic sensitivity of lactic acid bacteria, it is helpful to select drugs that are effective against pathogenic bacteria and have less impact on lactic acid bacteria when combined with drugs, improve the safety and effectiveness of treatment, and avoid interference with lactic acid bacteria treatment by antibiotic therapy.

[0126] Table 1: Results of lactic acid bacteria drug sensitivity test

[0127]

[0128] Note: Determination criteria (inhibition zone diameter): S for sensitive, I for intermediate, and R for resistant.

[0129] As can be seen from Table 1, the antibiotic sensitivity of the lactic acid bacteria 22-9, B-1-35, and XA-9 is almost completely consistent, and they are all resistant to vancomycin, streptomycin, penicillin, and kanamycin; sensitive to tetracycline, ampicillin, erythromycin, ceftazidime, and doxycycline; and only differ in gentamicin, with 22-9 being resistant, B-1-35 being intermediate, and XA-9 being sensitive.

[0130] 7. Hemolysis test

[0131] Hemolytic ability is an important indicator for measuring the potential pathogenicity of bacteria. Some lactic acid bacteria with hemolytic properties may cause damage to host cells, trigger inflammatory responses, and even cause diseases. Through hemolysis test detection, it can be preliminarily judged whether the lactic acid bacteria have potential pathogenicity. The experimental results are as follows Figure 7 It can be seen that the experimental strains have no hemolysis phenomenon on the blood plate.

[0132] 8. Transmission electron microscopy (TEM)

[0133] The cell morphology and structure of different lactic acid bacteria are different. Transmission electron microscopy can clearly see the fine structure and overall shape of the cell wall and cell membrane, which can assist in classification and identification. Morphology reflects physiological state. Lactic acid bacteria have different morphologies at different growth stages and under different culture conditions, which can help us speculate their metabolic, growth and stress response functions.

[0134] As shown in Figure 8 , the three strains are bacilli. XA-9 is in the division period and appears dumbbell-shaped, with a new cell wall and membrane structure in the middle connecting the daughter cells. B-1-35 is short and compact, with clear boundaries and no signs of division, or it is in the logarithmic growth phase, with stable cell state and complete structure. 22-9 is connected by two cells, which may have just divided and not completely separated.

[0135] 9. Identification results of lactic acid bacteria strains

[0136] (1) Amplification of 16S rDNA of lactic acid bacteria isolates

[0137] The gel electrophoresis results of PCR amplification products are shown in Figure 9 . The products have good integrity without degradation or tailing. The left side of the picture is DNA marker, which is 8000 bp. The other three columns from left to right are strains 22-9, XA-9 and B-1-35.

[0138] (2) Phylogenetic tree and homology analysis

[0139] The phylogenetic tree of strains 22-9, XA-9 and B-1-35 is shown in Figure 10 . From the figure, it can be seen that strains 22-9 and XA-9 are Lactiplantibacillus plantarum, and B-1-35 is Lactiplantibacillus pentosus.

[0140] In summary, 32 strains of lactic acid bacteria were randomly selected from Ningbo Bacteria Culture Collection for screening, and 5 strains of lactic acid bacteria strains were obtained which significantly inhibited the growth of Gardnerella vaginalis and Candida albicans. On this basis, the self-aggregation ability and surface hydrophobicity index were used to evaluate the adhesion ability of lactic acid bacteria, and three strains of lactic acid bacteria with excellent antibacterial performance were determined, which were 22-9, XA-9 and B-1-35.

[0141] The growth curve and acid production curve show that the three strains can grow rapidly and stably produce acid, enter the exponential growth phase after 2h and have a long stationary phase, and have good growth stability. The above three strains have excellent tolerance to acidic and bile salt environments, and strain XA-9 has the best tolerance, with a growth rate of 24.12% in a pH 3.5 environment. At the same time, the self-aggregation rates of XA-9, B-1-35 and 22-9 are 52.52%, 55.14% and 51.42%, respectively, with high self-aggregation capacity and moderate hydrophobicity. The in vitro safety evaluation shows that the three strains meet the safety evaluation and can be used as a composite strain of probiotic microecological preparation. Through 16S rDNA sequencing identification, it is determined that strains XA-9 and 22-9 are Lactiplantibacillus plantarum, and B-1-35 is Lactiplantibacillus pentosus.

[0142] The above-mentioned Lactiplantibacillus plantarum strain 22-9 has a preservation number of CGMCC No. 33867 and was preserved at the China General Microbiological Culture Collection Center on March 19, 2025, at an address of No. 3, Beichen West Road, Haidian District, Beijing.

[0143] The above-mentioned Lactiplantibacillus plantarum strain XA-9 has a preservation number of CGMCC No. 33868 and was preserved at the China General Microbiological Culture Collection Center on March 19, 2025, at an address of No. 3, Beichen West Road, Haidian District, Beijing.

[0144] The above-mentioned Lactiplantibacillus pentosus strain B-1-35 has a preservation number of CGMCC No. 33870 and was preserved at the China General Microbiological Culture Collection Center on March 19, 2025, at an address of No. 3, Beichen West Road, Haidian District, Beijing.

[0145] III. Determination of optimal proportion of probiotic microecological preparation strains and quality detection

[0146] 1. Effect of different fermentation strains and proportions on inhibition ability of Gardnerella vaginalis and Candida albicans

[0147] Developing a microecological preparation composed of three lactic acid bacteria offers several advantages over single-strain preparations: It boasts diverse functions, containing multiple bacteria with varying metabolites and functions, allowing for synergistic effects; it exhibits high stability, with complementary growth and metabolism among the lactic acid bacteria, enabling them to coexist and adapt to complex environments, facilitating colonization; and it has a wider applicability, targeting various vaginitis pathogens, thus having a broader scope of application than single-strain preparations. Furthermore, probiotic microecological preparations are safer than live bacteria, as freeze-drying induces dormancy in the lactic acid bacteria, reducing the risk of harm due to bacterial death or mutation; they are also more durable, slowly reviving, colonizing, and multiplying under suitable in vivo conditions, resulting in a longer-lasting effect, whereas live bacteria are easily eliminated and difficult to colonize long-term.

[0148] Depend on Figure 11 The inhibition rates of 22-9, XA-9, and B-1-35 against Gardnerella vaginalis at the same concentration were 53.83%, 52.58%, and 57.23%, respectively. When the ratios of equal amounts of 22-9, XA-9, and B-1-35 were 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 were 67.52%, 66.11%, 67.82%, 69.13%, 70.35%, 68.10%, and 72.59%, respectively, demonstrating significantly higher antibacterial activity than single bacterial species.

[0149] Depend on Figure 12 The inhibition rates of 22-9, XA-9, and B-1-35 against Candida albicans were 42.33%, 44.46%, and 43.58%, respectively. When the ratio of 22-9, XA-9, and B-1-35 was 1:0:1, 3:1:1, 2:1:3, 3:2:1, and 2:1:1, the inhibition rates were 56.73%, 56.51%, 54.59%, 54.08%, and 54.33%, respectively, and their antibacterial ability was much higher than that of a single strain.

[0150] In summary, when the compound ratio of 22-9, XA-9, and B-1-35 is 3:2:1 or 2:1:3, the inhibitory effect of the compound probiotic preparation on Candida albicans and Gardnerella vaginalis is much higher than that of the single species. Therefore, 2:1:3 was selected as the final ratio for the probiotic preparation.

[0151] 2. Detection of the survival rate of freeze-dried probiotic microecological preparations

[0152] Detecting the survival rate of freeze-dried probiotic preparations helps to intuitively understand the impact of freeze-drying on lactic acid bacteria and judge the effectiveness of freeze-drying. At the same time, the survival rate is related to the quality and efficacy of probiotic preparations. Only when enough live bacteria survive can the probiotics be effectively regulated, harmful bacteria inhibited, and the expected results achieved.

[0153] Depend on Figure 13 It can be seen that the total bacterial count of the undiluted probiotic preparation before and after freeze-drying is within 10. 17The CFU / mL level achieved a freeze-drying survival rate of 94.80%.

[0154] 3. Detection of total bacterial count during storage of probiotic microecological preparations

[0155] The purpose of detecting the total bacterial count during the storage period of probiotic preparations is to control quality. The total bacterial count directly reflects the number of microorganisms. An abnormal increase in the total bacterial count suggests that the product may be contaminated by other bacteria or that the growth of microorganisms is out of control, which in turn disrupts the microecological balance and affects the quality. At the same time, it can also assess stability. Regular testing can be used to judge the stability of the product based on changes in the total bacterial count and obtain the best storage conditions.

[0156] Depend on Figure 14 It can be seen that the total bacterial count of undiluted probiotic preparations remained essentially unchanged when stored at 4°C, showed a slight decreasing trend when stored at room temperature (10-20°C), and decreased significantly at 37°C. Even so, the total bacterial count of lactic acid bacteria in the probiotic preparations was still very high, and it is uncertain whether the activity of dormant lactic acid bacteria will be affected. Storage at 4°C is recommended.

[0157] IV. Example of Product Application 1. Specific Implementation Examples

[0159] Example 1

[0160] A method for preparing a probiotic compound microecological preparation with the effect of improving vaginitis includes the following steps:

[0161] (1) Lactic acid bacteria fermentation: The lactic acid bacteria cultures of Lactobacillus plantarum 22-9, Lactobacillus plantarum XA-9 and Lactobacillus pentosus B-1-35 were mixed in a volume ratio of 2:1:3 to obtain a mixed fermentation agent. The mixed fermentation agent was inoculated into the fermentation medium at a volume ratio of 2% and cultured at 37℃ for 18h to obtain a mixed culture of lactic acid bacteria.

[0162] (2) Freeze-drying: The obtained mixed culture of lactic acid bacteria was centrifuged at 8000 rpm and 4℃ for 15 min. The lactic acid bacteria pellet was collected, and the bacterial concentration was adjusted to 1×10⁻⁶ using sterile PBS buffer. 9 CFU / mL, add an equal volume of lyophilization protectant to sterile PBS buffer, mix well, and pre-freeze overnight at -80℃. Then freeze-dry the pre-frozen sample at -49℃, 9 Pa for 48 hours to obtain the probiotic compound microecological preparation. The lyophilization protectant formulation consists of 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.

[0163] (3) Administration method: vaginal administration.

[0164] The finally prepared composite microecological preparation is named as high-dose vaginal administration group (HD).

[0165] Example 2

[0166] The same as Example 1 above, except that the administration mode in step (3) is oral and vaginal administration. The finally prepared composite microecological preparation is named as high-dose combined administration (oral + vaginal administration) group (HH).

[0167] Example 3

[0168] The same as Example 1 above, except that the concentration of the lactic acid bacteria in the lactic acid bacteria precipitate is adjusted to 1×10 8 CFU / mL using sterile PBS buffer. The finally prepared composite microecological preparation is named as medium-dose vaginal administration group (MD).

[0169] Example 4

[0170] The same as Example 3 above, except that the administration mode in step (3) is oral and vaginal administration. The finally prepared composite microecological preparation is named as medium-dose combined administration (oral + vaginal administration) group (MH).

[0171] Example 5

[0172] The same as Example 1 above, except that the concentration of the lactic acid bacteria in the lactic acid bacteria precipitate is adjusted to 1×10 7 CFU / mL using sterile PBS buffer. The finally prepared composite microecological preparation is named as low-dose vaginal administration group (LD).

[0173] Example 6

[0174] The same as Example 5 above, except that the administration mode in step (3) is oral and vaginal administration. The finally prepared composite microecological preparation is named as low-dose combined administration (oral + vaginal administration) group (LH).

[0175] 2. Improvement effect of probiotic composite microecological preparation on mouse vaginitis model

[0176] 1) Experimental materials: SPF healthy female Kunming mice (KM mice), weighing about 35.00±2 g per mouse. Provided by Sibeifu (Suzhou) Biotechnology Co., Ltd., production license: SCXK (Su) 2022-0006, this batch of qualified certificate number: A202412190019.

[0177] 2) Experimental environment and ethics: Conducted in the ABSL-2 laboratory of Leading Biotechnology (Kunshan) Co., Ltd., experimental animal use license: SYXK(Su)2023-0083; Suzhou Animal Pathogen Microorganism Laboratory Record No. Su Kun Dynamic Laboratory

[2024] 320583012; Ethics Approval IACUC No. 20241216-0002-01.

[0178] Housing conditions: Animals were housed for 3 days in the experimental environment before starting the experiment. Animals were housed in IVC (independent ventilation system) cages (5 animals per cage) in the ABSL-2 animal room. All cages, bedding, and drinking water must be sterilized before use. All personnel operating in the animal room should wear protective clothing and latex gloves. The animal information card in each cage should indicate the number of animals in the cage, gender, strain, date of receipt, project number, and group. Feed and drinking water are replaced once a week. During the feeding period, animals have free access to feed and water. Temperature: 20-26°C; Humidity: 40-70%; Light cycle: 12h light, 12h dark.

[0179] Feed ingredients: The feed meets the experimental animal food identification standards and comes from Kaoxie Cooperation (Tianjin) Feed Co., Ltd. with a feed production license number of Tianjin Inspection and Certification (2020) 01005 and an execution standard of GB14924.3-2010. The maximum content of contaminants is within the controllable range and is subject to regular inspection by the manufacturer. Drinking water is produced by reverse osmosis filtration.

[0180] 3) Experimental protocol: 88 mice were randomly divided into 8 groups: they were randomly assigned to 11 experimental groups, each containing 8 mice. Two different administration methods were used in the experimental design: single vaginal route administration (vaginal administration) and combined administration of two routes (vaginal administration combined with oral administration). The specific grouping is as follows: blank control group (CK), estradiol control group (CKC), model vaginal administration group (MDD), model combined administration group (MDH), commercially available lactobacillus 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).

[0181] The entire experimental process can be divided into three stages: induction of estrus period (Day 1-6), modeling period (Day 7-11), and administration period (Day 12-19).

[0182] During the induction of estrus period (Day 1-6), except for the CK group, each mouse was subcutaneously injected with benzoic acid estradiol (2mg / mL) 0.05mL, once every other day, until the end of the experiment.

[0183] At the modeling period (Day 7-11), 20 μL (10 6 CFU / mL) mixed bacteria suspension of Candida albicans and Gardnerella vaginalis was inoculated into the vagina of the mice, and the mouse vaginal orifice was gently pinched with a pair of tweezers for 30 s to prevent the bacteria solution from leaking out. The CK group and the CKC group were given the same amount of normal saline, and the inoculation was performed for 5 consecutive days. The appearance of the vaginal orifice was observed, and the vaginal lavage fluid was taken to detect whether the modeling was successful according to the Gram staining method. After verifying the success of the modeling, the drug administration period was entered.

[0184] At the drug administration period (Day 12-19), the drug administration scheme (including the administration route and the dose) of each group is shown in Table 2.

[0185] Table 2 Administration route and dose

[0186]

[0187]

[0188] The vaginal and uterine tissues of the mice were observed by using a kit, and the vaginal tissue was subjected to HE and PAS staining. The IL-1β, IL-6, and TNF-α levels in the serum of the mice were determined, and the relative abundance of intestinal bacteria, vaginal bacteria, and fungi of the mice was determined.

[0189] 4) Experimental results: On the 4th day after drug administration, the vaginal lavage fluid was observed under a microscope. The results are shown in Table 3. Figure 15 It was found that compared with the CK group and the CKC group, a large number of Candida albicans was observed in the MDD group and the MDH group. Compared with the MDD group and the MDH group, the number of bacteria colonies in each drug administration group was reduced, and the dose was negatively correlated. There was no significant difference between the vaginal administration and the combined administration.

[0190] It was found that compared with the CK group and the CKC group, a large number of Candida albicans was observed in the MDD group and the MDH group. Compared with the MDD group and the MDH group, the number of bacteria colonies in each drug administration group was reduced, and the dose was negatively correlated. There was no significant difference between the vaginal administration and the combined administration. Figure 16 It was found that compared with the CK group and the CKC group, a large number of Candida albicans was observed in the MDD group and the MDH group. Compared with the MDD group and the MDH group, the number of bacteria colonies in each drug administration group was reduced, and the dose was negatively correlated. There was no significant difference between the vaginal administration and the combined administration.

[0191] Representative pathological pictures are shown in Figs. 1-4. Figure 17 , Figure 18In the mouse vaginitis model study, HE staining and PAS staining have significant effects. HE staining, as a commonly used staining method, can clearly present the cell structure and morphology of mouse vaginal tissue, such as the number of epithelial cell layers, arrangement, blood vessels, connective tissue and immune cell distribution in the lamina propria, helping to understand the impact of vaginitis on tissue morphology. It can also identify the types and number 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 find pathological changes such as epithelial cell degeneration, necrosis, lamina propria edema, hyperemia and fibrous tissue hyperplasia, helping to explore the pathological mechanism of vaginitis.

[0192] PAS (periodic acid-Schiff) staining detects fungi based on its specific staining reaction to polysaccharide substances. Fungal cell walls contain abundant polysaccharide components such as chitin and cellulose. Periodic acid can oxidize the glycol group in the polysaccharide of the fungal cell wall to aldehyde group, and the aldehyde group can combine with the colorless magenta in Schiff's reagent to form a purple red or red complex, so that the fungi show obvious color under the microscope, which is convenient for identification.

[0193] After careful microscopic observation of the tissue of the submitted sample, it is found that:

[0194] In the CK group and the CKC group, the vaginal tissue structure is normal, and mucosal epithelium can be seen micro-abscess, which is a common normal change during the diestrum, mainly neutrophils, and no other obvious pathological changes are found; a small amount of inflammatory cells infiltrate the mucosa in the CK group; some mice in the CKC group are in the proestrus stage, and there are mucous cells, which are positive for PAS staining.

[0195] In the MDD group and the MDH group, PAS staining (red) positive fungi can be seen in the vagina, which should be Candida albicans according to the experiment. Mucosal epithelium can be seen micro-abscess, which is a common normal change during the diestrum, mainly neutrophils. A large number of inflammatory cells infiltrate the mucosa, and edema is occasionally seen.

[0196] In the CG group, the LD group, the LH group, the MD group, the MH group, the HD group and the HH group, PAS staining (red) positive fungi can be seen in the vagina, which should be Candida albicans according to the experiment. Mucosal epithelium can be seen micro-abscess, which is a common normal change during the diestrum, mainly neutrophils. A small amount of inflammatory cells infiltrate the mucosa.

[0197] The number of inflammatory cells (neutrophils, lymphocytes, macrophages, etc.) directly reflects the degree of inflammation, which can be used to judge the acute and chronic inflammation and the development process; the abnormal increase or decrease of epithelial horn cell number indicates the overgrowth or damage of epithelial cells, which can be used to judge the degree of epithelial tissue damage; after drug administration, the number of inflammatory cells decreases and the number of epithelial horn cells returns to normal, which represents improvement in most cases.

[0198] As shown in Table 3 andFigure 19 As shown, the number of inflammatory cells in the lamina propria of each group ( Figure 19 (A) and the number of epithelial keratinocytes ( Figure 19 The number of inflammatory cells in the model group was increased compared with the control group (B). Compared with the model group, the number of inflammatory cells decreased in each treatment group, with the HD group showing the most significant decrease (P<0.05). There was no significant difference in the number of epithelial keratinocytes among the groups (P>0.05).

[0199] Table 3. Histopathological Examination Record Sheet

[0200]

[0201]

[0202] like Figure 20 As shown in Figure A, in terms of IL-1β level comparison, the serum IL-1β levels in the MDD and MDH groups were significantly higher (P<0.01) than those in the treated group and the blank group. After treatment, the decrease in IL-1β increased with the increase in the dose of the probiotic. There were no significant differences among the LD, LH, and MH groups, and among the LH, MH, and HD groups (P>0.05), indicating that the dose has a relatively small effect on IL-1β to a certain extent, but the effect becomes extremely significant after exceeding a certain dose (P<0.01). In the combination and vaginal administration, only the high-dose group showed better combined administration. The commercially available lactic acid bacteria preparation had a higher ability to reduce IL-1β than the low and medium dose groups, but lower than the high-dose group. The IL-1β level in the HH group decreased to a level that was not significantly different from that in the control group (P>0.05), showing excellent ability to reduce IL-1β levels.

[0203] like Figure 20 As shown in Figure B, the serum IL-6 levels in the MDD and MDH groups were significantly higher than those in the control group (P<0.01). After drug administration, the concentration of IL-6 showed a trend of first increasing and then decreasing with increasing dose of the probiotic preparation, with the low-dose and high-dose groups showing better effects. There were no significant differences among the LD, MH, and HD groups (P>0.05), indicating that the dose has a relatively small effect on IL-6 to a certain extent, but the effect becomes significant after exceeding a certain dose (P<0.05). In combined administration and vaginal administration, the combined administration of medium and high doses was more effective. The commercially available lactic acid bacteria preparation had a higher ability to reduce IL-6 than the other drug administration groups, with no significant difference compared to the control group (P>0.05). The HH group showed a better ability to reduce IL-6 levels.

[0204] like Figure 20As shown in Fig. C, the content of TNF-α in the serum of the MDD group and the MDH group was significantly higher than that of the blank group (P<0.01) in the comparison of TNF-α level. After the treatment, with the increase of the dose of the microecological preparation, the TNF-α showed a downward trend. There was no significant difference (P>0.05) among the MDD group, the MDH group, the LD group and the MD group, indicating that the low-dose and medium-dose vaginal administration had little effect on TNF-α. When reaching the high-dose group, the effect was significant (P<0.05). In the combined administration and vaginal administration, all doses of the combined administration were better than those of the vaginal administration. The ability of the commercial 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 was better in reducing the level of TNF-α.

[0205] By 16S rRNA gene sequencing analysis, it was found that Figure 21 、 Figure 22 、 Figure 23 、 Figure 24 、 Figure 25 and Figure 26 With the increase of the dose of the administration, the relative abundance of Bacteroidetes in the intestinal tract of the mice increased, and the relative abundance of Firmicutes decreased. In the HD group, the relative abundance of beneficial bacteria such as Lactobacillus and Ligilactobacillus accounted for nearly 30.6%, which was significantly higher than that of the model group (P<0.05). At the same time, the abundance of Firmicutes in the vagina of the mice increased, the abundance of Proteobacteria showed a trend of first increasing and then decreasing, and the relative abundance of Proteobacteria in the HD and HH groups was lower than that in the other administration groups. The relative abundance of Streptococcus decreased significantly. In the vagina of the mice, the relative abundance of Ascomycota in the HD group decreased, and the relative abundance of Candida in the CG, MH and HD groups decreased. The HD and HH groups could improve the community species richness and diversity of the intestinal tract and vagina of the mice to a certain extent, and the colony structure gradually approached to that of the CK group.

[0206] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled persons within the essential scope of the present application shall also fall within the protection scope of the present application.

Claims

1. A probiotic having improved efficacy against vaginitis, characterized in that: The probiotics mentioned include *Lactobacillus plantarum* with the preservation number CGMCC No. 33867. Lactiplantibacillus plantarum Lactobacillus plantarum strain 22-9, with preservation number CGMCC No. 33868 ( Lactiplantibacillus plantarum Strain XA-9 and *Lactobacillus pentosaceus* with accession number CGMCC No. 33870 ( Lactiplantibacillus pentosus B-1-35 strain.

2. A probiotic complex microecological preparation with improved efficacy against vaginitis, characterized in that it contains: The probiotic composite micro-ecological preparation comprises Lactobacillus plantarum (Lactobacillus plantarum) with the preservation number of CGMCC No.33867 Lactiplantibacillus plantarum Lactobacillus plantarum (Lactobacillus plantarum) with the preservation number of CGMCC No.33868 Lactiplantibacillus plantarum Lactobacillus pentosus (Lactobacillus pentosus) with the preservation number of CGMCC No.33870 Lactiplantibacillus pentosus Lactobacillus pentosus (Lactobacillus pentosus) with the preservation number of CGMCC No.33870 The active ingredients of the probiotic composite micro-ecological preparation comprise bacterial cells.

3. The probiotic composite microecological preparation with improved efficacy against vaginitis according to claim 2, characterized in that: The effective bacteria quantity in the probiotic compound micro-ecological preparation is not less than 1×10 7 CFU / mL.

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

3.

5. A method for preparing the probiotic composite microecological preparation with improved efficacy against vaginitis according to claim 2, characterized in that The method comprises the following steps: (1) inoculating the Lactiplantibacillus plantarum 22-9, the Lactiplantibacillus plantarum XA-9 and the Lactiplantibacillus pentosus B-1-35 into a fermentation medium at a volume ratio of 1-5%, and culturing at 35-40 ℃ for 15-20 h to obtain lactic acid bacterial cultures, mixing the lactic acid bacterial cultures of the Lactiplantibacillus plantarum 22-9, the Lactiplantibacillus plantarum XA-9 and the Lactiplantibacillus pentosus B-1-35 at a volume ratio of (1-3):(1-3):(1-3) to obtain a mixed starter, and inoculating the mixed starter into a fermentation medium at a volume ratio of 2% and culturing at 37 ℃ for 18 h to obtain a mixed lactic acid bacterial culture; (2) Centrifuge the lactic acid bacteria culture obtained in step (1) to obtain lactic acid bacteria precipitate, and adjust the concentration of the bacteria to 1x10 7 -1x10 10 CFU / mL using sterile PBS buffer. Add an equal volume of freeze-drying protective agent to the bacteria, mix well, and pre-freeze in a -80°C refrigerator overnight. Freeze-dry the pre-frozen sample to obtain the probiotic composite microecological preparation.

6. The preparation method of a probiotic compound microecological preparation with the effect of improving vaginitis according to claim 5, characterized in that: The lactic acid bacterial cultures of the Lactiplantibacillus plantarum 22-9, the Lactiplantibacillus plantarum XA-9 and the 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 the lactic acid bacterial cultures are the same.

7. The preparation method of a probiotic compound microecological preparation with the effect of improving vaginitis according to claim 5, characterized in that: The fermentation medium in step (1) is prepared by dissolving MRS solid medium in 1 L of distilled water, sterilizing at 121 ℃ for 15 min, and the formula of the MRS solid medium is as follows: 10 g of proteose 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 compound microecological preparation with the effect of improving vaginitis according to claim 5, characterized in that: The formula of the freeze-drying protectant in step (2) is as follows: 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 skimmed milk powder.

9. Use of the probiotic bacteria of claim 1 in the preparation of a Gardnerella vaginalis and / or Candida albicans inhibitor.

10. Use of the probiotic bacteria complex microecological preparation of claim 2 in the preparation of a drug for improving or treating vaginitis.

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