Lactobacillus salivarius LS100 for inhibiting pathogenic bacteria of vaginitis and preventing and treating vaginitis and application of lactobacillus salivarius LS100

Abstract

The invention relates to combined lactobacillus salivarius LS100 for inhibiting vaginitis pathogenic bacteria and preventing and treating vaginitis and application of the combined lactobacillus salivarius LS100. The combined lactobacillus salivarius LS100 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.32135. The combined lactobacillus salivarius LS100 has good hydrogen peroxide production capacity and adhesion capacity, and can be used for preparing medicines for preventing and treating vaginitis. The traditional Chinese medicine composition has a relatively strong inhibition effect on main vaginitis pathogenic bacteria, is effective on nutritional pathogenic bacteria, and can also reduce form transformation and biological membrane formation of the pathogenic bacteria and prevent generation of drug resistance; besides, the fermentation product can reduce the content of pathogenic bacteria metabolites, regulate the secretion of cell factors and play a role in relieving inflammation, so that the fermentation product can be used for preparing products for preventing and treating genital tract pathogenic bacteria infection and / or preventing and treating vaginitis.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and specifically relates to a saliva-combined lactobacillus LS100 for inhibiting vaginitis pathogens and preventing and treating vaginitis, and an application thereof. Background Art

[0002] Destruction of the reproductive tract microecological environment can lead to various infections, such as vaginitis, chronic pelvic inflammatory disease, cervicitis, endometritis, fallopian tube obstruction, etc.

[0003] At present, antibiotics or antifungal drugs are usually used to treat female reproductive tract infections and vaginal inflammation. Although the initial treatment effect is good, the recurrence rate is high due to the formation of pathogenic bacteria biofilm and the development of antibiotic resistance. Lactobacillus is usually the most abundant taxon in the vaginal microbiome (VMB), producing lactic acid and bacteriocins to inhibit microorganisms related to dysbiosis, maintain homeostasis, and reduce the risk of disease. Therefore, screening lactobacillus strains that can reduce female reproductive tract infections and reduce the occurrence of vaginal inflammation, and are used to improve the vaginal microenvironment and reduce the recurrence of vaginal inflammation, which is crucial for maintaining female reproductive tract health and preventing and treating reproductive tract diseases. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide a saliva-combined lactobacillus LS100 for inhibiting vaginitis pathogens and preventing and treating vaginitis and its application, which can effectively prevent and treat genital tract pathogenic bacteria infection and vaginitis.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A saliva-associated Lactobacillus Ls100, classified and named as saliva-associated Lactobacillus (Ligilactobacillus salivarius), is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCCNO.32135. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is September 30, 2024.

[0007] As a further technical solution, the saliva combined with Lactobacillus Ls100 is derived from the reproductive tract secretions of healthy women.

[0008] A microbial agent comprises one or more of the live bacteria of saliva-associated Lactobacillus Ls100, the inactivated bacteria of saliva-associated Lactobacillus Ls100, and the fermentation product of saliva-associated Lactobacillus Ls100.

[0009] The saliva combined with Lactobacillus Ls100 or the microbial agent is used in preparing products for preventing and treating genital tract pathogenic bacteria infection and / or preventing and treating vaginitis.

[0010] The reproductive tract pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, hemolytic Staphylococcus, Streptococcus angina pectoris, Candida glabrata, Candida albicans, and Candida diverticula.

[0011] As a further technical solution, the saliva combined with Lactobacillus Ls100 or microbial agent has one or more of the following effects:

[0012] 1) Saliva combined with Lactobacillus Ls100 or microbial agents can produce hydrogen peroxide;

[0013] 2) Saliva combined with Lactobacillus Ls100 or microbial agents have an antibacterial effect on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, Staphylococcus hemolyticus, Streptococcus angina pectoris, Candida glabrata, Candida albicans, and Candida diverticulatus;

[0014] 3) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae;

[0015] 4) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the biofilm formation of vaginal Gardnerella and / or Candida albicans at different stages;

[0016] 5) Saliva combined with Lactobacillus Ls100 or microbial agents can reduce the hydrophobicity of Candida albicans;

[0017] 6) Saliva combined with Lactobacillus Ls100 or microbial agents can chelate iron ions;

[0018] 7) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage;

[0019] 8) Saliva combined with Lactobacillus Ls100 or microbial preparations can reduce the secretion of inflammatory factors IL-6 and IL-8;

[0020] 9) Saliva combined with Lactobacillus Ls100 or microbial agents have a good degradation effect on biogenic amines.

[0021] As a further technical solution, the product includes an oral product or an external product;

[0022] As a further technical solution, the oral product includes food or health care products; such as powders, granules, tablets, yogurt, candy, beverages, etc.

[0023] As a further technical solution, the external-use products include one or more of external-use medicines, sanitary products, and family planning products.

[0024] The sanitary products include sanitary napkins, panty liners, wet wipes, tampons, and antibacterial / antibacterial lotions; family planning products include condoms and lubricants.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) The saliva-associated Lactobacillus Ls100 isolated and screened by the present invention is derived from the secretions of the reproductive tract of healthy women. Compared with drugs such as antibiotics in traditional technologies, it is safe and healthy and has almost no side effects.

[0027] 2) The saliva-associated lactobacillus Ls100 separated and screened by the present invention can produce hydrogen peroxide, and has a strong inhibitory effect on common vaginal pathogens such as Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, and Candida albicans, and has an inhibitory effect on the original pathogens of vaginitis, such as Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, hemolytic Staphylococcus, Streptococcus angina pectoris, Candida glabrata, Candida albicans, and Candida diverticulata, and can be used to prevent and treat female reproductive tract pathogenic bacteria infection.

[0028] 3) Saliva combined with Lactobacillus Ls100 can inhibit the formation of Candida albicans hyphae, reduce the number and length of hyphae, and have a significant effect on female vaginal diseases caused by Candida albicans.

[0029] 4) Saliva combined with Lactobacillus Ls100 can inhibit the formation of biofilms by vaginal Gardnerella and Candida albicans at different stages, and it has strong hydrophobicity, which can reduce the hydrophobicity of Candida albicans and thus reduce its adhesion effect, reduce the probability of biofilm formation, and prevent the occurrence of Candida albicans resistance.

[0030] 5) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage;

[0031] 6) Saliva combined with Lactobacillus Ls100 can chelate iron ions;

[0032] 7) Saliva combined with Lactobacillus Ls100 and its fermentation products can reduce the secretion of inflammatory factors IL-6 and IL-8, thereby achieving the purpose of preventing and treating vaginal inflammation;

[0033] 8) Saliva combined with Lactobacillus Ls100 has a good degradation effect on biogenic amines.

[0034] 9) The saliva combined with Lactobacillus Ls100 of the present invention is non-toxic and harmless, and can be used to prepare a wide range of products, including oral products such as food, medicine, health products, beverages, candies, etc., as well as external medicines for reproductive tract use, and can even be used in sanitary products and family planning products, such as sanitary napkins, female antibacterial / antibacterial lotions, condoms, etc.

[0035] In summary, the saliva-associated Lactobacillus Ls100 screened by the present invention has good hydrogen peroxide production and adhesion capabilities, and has a strong inhibitory effect on major vaginitis pathogens. It is not only effective against vegetative pathogens, but also can reduce their morphological transformation and biofilm formation, and prevent the generation of drug resistance; in addition, its fermentation product can also reduce the content of pathogenic bacterial metabolites, regulate the secretion of cytokines, and relieve inflammation, so that it can be used to prepare products for preventing and treating genital tract pathogenic bacteria infections and preventing and treating vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a diagram showing the effect of saliva combined with Lactobacillus Ls100 in producing hydrogen peroxide;

[0037] Figure 2 The chelation effect of saliva and Lactobacillus Ls100 on iron ions;

[0038] Figure 3 The degradation effect of saliva combined with Lactobacillus Ls100 on biogenic amines. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0041] Example 1: Isolation, screening and identification of saliva-associated Lactobacillus Ls100

[0042] Separation: Take a sample of secretions from a healthy woman, place it in an MRS medium for anaerobic culture, take 1 mL of the culture solution and dilute it with 9 mL of sterile saline, then apply it to the MRS solid medium and culture it at 37°C for 72 hours. Select an MRS plate with a single colony, pick colonies of different sizes, colors, and states, perform Gram staining, inoculate Gram-positive bacteria into the MRS liquid medium, and culture at 37°C until turbidity. Streak the bacterial liquid in the MRS solid medium, repeat this process for more than 3 times, and obtain a number of strains with consistent colony and bacterial morphology, which are then preserved.

[0043] Screening: All the isolated colonies were milky white, raised, with neat edges and smooth surfaces, and were rod-shaped, Gram-positive, and catalase-negative under a microscope. The strains with strong inhibitory effects on Escherichia coli, Staphylococcus aureus, Candida albicans, and Gardnerella vaginalis, as well as strong adhesion and hydrogen peroxide production capabilities were screened and named Ls100;

[0044] Identification: The strain Ls100 obtained by screening was identified by 16S rDNA gene, and the strain was identified as Ligilactobacillus salivarius; the 16S rRNA nucleotide sequence of Ligilactobacillus salivariusus100 is shown in SEN ID NO.1.

[0045] Among them, the performance and efficacy of strain Ls100 are studied in the following examples.

[0046] Example 2: Experiment on the ability of saliva combined with Lactobacillus Ls100 to produce hydrogen peroxide and its antibacterial effect on common vaginal bacteria

[0047] 1. Hydrogen peroxide production capacity test

[0048] 1.1 Experimental methods

[0049] (1) Prepare TMB-HRP-MRS plate. Cool solid MRS culture medium to 40-50°C, add 3,3',5,5'-tetramethylbenzidine TMB 25 mg / 100 mL (TMB is insoluble in water and needs to be dissolved in a small amount of dimethyl sulfoxide in advance) and horseradish peroxidase HPR 5 mg / 100 mL, mix well, pour into a culture dish, cool and solidify, and store at 4°C for later use.

[0050] (2) Streak each lactic acid bacteria liquid on a TMB-HRP-MRS plate and culture it anaerobically at 37°C for 48 hours. Then expose the culture plate to air for 30 minutes. The colonies producing hydrogen peroxide will turn blue. The lactic acid bacteria are graded according to the intensity of the blue color. They are divided into four types: strong positive (+++), positive (++), weak positive (+), and negative (-). The results are shown in Figure 1 .

[0051] 1.2 Experimental Results

[0052] Table 1: Hydrogen peroxide production capacity of each strain

[0053] strain Hydrogen peroxide production LG19 (Lactobacillus gasseri) — LP20 (Lactobacillus plantarum) — LC21 (Lactobacillus crispatus) — Ls100 (Lactobacillus salivarius) +++ LP23 (Lactobacillus plantarum) — LP24 (Lactobacillus plantarum) —

[0054] Hydrogen peroxide has a strong antibacterial effect and can inhibit Gram-negative bacteria and catalase-positive bacteria such as Pseudomonas, Escherichia coli and Salmonella. 3,3',5,5'-Tetramethylbenzidine (TMB) is oxidized by hydrogen peroxide and horseradish peroxidase (HRP) to form a blue product. The higher the hydrogen peroxide content, the darker the blue color. Figure 1 The results showed that the ability of saliva combined with Lactobacillus Ls100 to produce hydrogen peroxide was strong and positive.

[0055] 2. Test on the inhibitory ability against Escherichia coli and Staphylococcus aureus

[0056] 2.1 Test methods

[0057] (1) Using the double-layer plate antagonism method, 10 mL of 2% agar was added to the culture dish and placed in an Oxford cup after solidification;

[0058] (2) Take 1 mL of activated Escherichia coli or Staphylococcus aureus and add it to 100 mL of nutrient agar (containing 0.8% agar concentration) medium and mix well. Take 10 mL and add it to a culture dish. After the medium solidifies, take out the Oxford cup to form an Oxford cup hole.

[0059] (3) The selected saliva-associated Lactobacillus Ls100 was inoculated into MRS liquid culture medium at 3% and cultured at 37°C for 24 h. The fermentation broth was centrifuged at 8000 rpm for 10 min to collect the supernatant. Then 0.1 mL of the supernatant was added to each well and cultured at 37°C overnight. The diameter of the inhibition zone was measured. The test results are shown in Table 2.

[0060] 2.2 Test results

[0061] Table 2 Antibacterial results of strain Ls100 against Escherichia coli and Staphylococcus aureus

[0062]

[0063]

[0064] Note: Since the hole of the Oxford cup has a diameter of 8mm, only the antibacterial zone diameter ≥ 8mm indicates an antibacterial effect.

[0065] The present invention adopts the double-layer agar diffusion method to compare the antibacterial ability of the fermentation supernatant of the test strain on Escherichia coli and Staphylococcus aureus. The data results in Table 2 show that Ls100 has good antibacterial effect on both Escherichia coli and Staphylococcus aureus, and MRS as a control has no inhibitory effect.

[0066] 3. Test on the inhibitory ability of Candida albicans

[0067] 3.1 Experimental methods

[0068] (1) The Ls100 fermentation broth cultured for 16 h was centrifuged, the supernatant (without the strain) was collected, 5 mL of the fermentation supernatant and 5 mL of the Candida albicans culture were mixed in equal volumes, and the viable bacteria of Ca were counted at 0 h, 12 h, and 24 h, respectively, using selective YPD medium;

[0069] (2) Mix equal volumes of pH 6.8 modified MRS medium and pH 4.0 modified MRS medium with Candida albicans culture medium, respectively, and repeat the above test to eliminate the interference of MRS itself on Candida albicans.

[0070] (3) Calculate the inhibitory ability of lactic acid bacteria on Candida albicans. The results are shown in Table 3:

[0071] Candida albicans inhibition rate A=(A0-A t ) / A0×100%, where A t represents the number of Ca viable bacteria in each experimental group after 12 h and 24 h of culture, and A0 represents the number of Ca viable bacteria after 0 h;

[0072] 3.2 Experimental Results

[0073] Table 3: Antibacterial effect on Candida albicans

[0074] strain 12h inhibition rate% 24h inhibition rate% Saliva combined with Lactobacillus Ls100 65.39 54.50 pH4.0MRS 0.00±0.00 0.00±0.00 pH6.8MRS 0.00±0.00 0.00±0.00

[0075] Candida albicans is the main pathogen of candidal vaginitis. The results in Table 3 show that saliva combined with Lactobacillus Ls100 has a good inhibitory effect on Candida albicans, while MRS liquid culture medium at pH 4.0 and pH 6.8 cannot inhibit the growth of Candida albicans.

[0076] 4. Test of the inhibitory ability to vaginal Gardnerella

[0077] 4.1 Experimental methods

[0078] (1) The lactic acid bacteria strain selected in Example 1 was activated for three generations, the third generation was cultured at 37° C. for 24 h, and the supernatant was collected by centrifugation at 8000 rpm / m for 10 min;

[0079] (2) Using the agar diffusion method, pour 20 mL of TSA (containing 5% defibrinated sheep blood) solid culture medium into a sterile plate. After solidification, use a sterile cotton swab to dip into vaginal Gardnerella liquid and evenly spread it on the TSA solid culture medium;

[0080] (3) Place a sterilized Oxford cup on the above culture medium and let it stand for 10 min. Add 0.1 mL of lactic acid bacteria fermentation supernatant into the Oxford cup and culture it at 37°C for 20 h. Use a vernier caliper to measure the diameter of the inhibition zone. Set up two parallel groups for each group. The results are shown in Table 4.

[0081] 4.2 Experimental Results

[0082] Table 4: Antibacterial effect on vaginal Gardnerella

[0083] strain Inhibition zone diameter Saliva combined with Lactobacillus Ls100 9.20 MRS 8.00

[0084] Gardnerella vaginalis is the main pathogen of bacterial vaginitis. The results in Table 4 show that saliva combined with Lactobacillus Ls100 has a good inhibitory effect on Gardnerella vaginalis, while MRS as a control has no inhibitory effect on Gardnerella vaginalis.

[0085] Example 3: Hydrophobicity detection of saliva combined with Lactobacillus Ls100

[0086] 1. Experimental Methods

[0087] The overnight cultured saliva-associated Lactobacillus Ls100 was centrifuged at 5000rpm for 3min, the bacterial sludge was collected, washed once with PBS, and then resuspended in PBS. The OD value was measured at a wavelength of 600nm, and was 0.7-0.8, recorded as A0. The Ls100 bacterial suspension was mixed with xylene, chloroform, and ethyl acetate in a ratio of 3:1, respectively, and thoroughly shaken and mixed for 30s, allowed to stand for 10s, shaken and mixed for 30s, allowed to stand for 30min, and after the aqueous phase and the organic phase were separated, the OD value of the aqueous phase at 600nm was measured, recorded as A, and the hydrophobicity of the saliva-associated Lactobacillus Ls100 cell surface was calculated. The results are shown in Table 5.

[0088] Cell surface hydrophobicity % = (1-A / A0) × 100%;

[0089] Hydrophobicity standard: more than 50% is highly hydrophobic, between 20%-50% is moderately hydrophobic, and less than 20% is non-hydrophobic.

[0090] 2. Experimental Results

[0091] Table 5: Hydrophobicity of saliva combined with Lactobacillus Ls100

[0092] Organic phase Hydrophobicity Xylene 81.81％ Chloroform 79.80％ Ethyl acetate 45.55％

[0093] An important prerequisite for lactic acid bacteria to produce physiological effects on the host is that they can effectively adhere to the host epithelial cells. This adhesion can be divided into nonspecific adhesion and specific adhesion. Nonspecific adhesion is mainly related to the electrostatic effect, hydrophobicity and self-aggregation ability of the lactic acid bacteria surface. The results in Table 5 show that saliva-combined lactobacillus Ls100 has strong hydrophobicity, and the hydrophobicity rate of xylene and chloroform is greater than 50%, reaching a high hydrophobicity level, and the hydrophobicity rate of ethyl acetate reaches 45%, reaching a moderate hydrophobicity level, which can effectively adhere to the host epithelial cells.

[0094] Example 4: Effect of saliva combined with Lactobacillus Ls100 on the hydrophobicity of Candida albicans

[0095] 1. Test methods

[0096] (1) The overnight culture of Candida albicans was centrifuged at 5000 r / min for 10 min, the cells were collected, washed once with PBS (pH 6.8) and centrifuged, and the cells were resuspended in PBS (pH 6.8) and the concentration of the bacterial solution was adjusted to an absorbance of about 1.000 at 560 nm.

[0097] (2) Take 4.5 ml of the Candida albicans solution, add 4.5 ml of saliva combined with Lactobacillus Ls100 fermentation supernatant, mix well and culture at 37°C for 24 hours, then measure the hydrophobicity. Add 4.5 ml of MRS broth medium instead of lactic acid bacteria fermentation supernatant as a control.

[0098] (3) The bacterial suspensions of the experimental and control groups were adjusted to a concentration at 560 nm, with an absorbance of about 1.000, recorded as A0. 3.0 mL of the bacterial suspension was mixed with 0.6 mL of xylene, ethyl acetate, and chloroform, respectively, and shaken for 30 s, allowed to stand for 10 s, shaken for 30 s, allowed to stand for 20 min, and after the two phases were separated, the absorbance of the aqueous phase was measured at 560 nm, recorded as A, and the hydrophobicity was calculated. The results are shown in Table 6; 3 parallel experiments were performed for each group, and the average value was taken.

[0099] Hydrophobicity (CSH%) = (1-A / A0) × 100%;

[0100] The specific standards for the hydrophobicity are as follows: CSH%>50% is highly hydrophobic, CSH% between 20% and 50% is moderately hydrophobic, and CSH%<20% is non-hydrophobic.

[0101] 2. Test results

[0102] Table 6 Effect of saliva combined with Lactobacillus Ls100 on the hydrophobicity of Candida albicans

[0103] Xylene Ethyl acetate Chloroform Experimental group (Ls100+Candida albicans) 58.77％ 23.14％ 29.57％ Control group (MRS + Candida albicans) 79.39％ 47.57％ 43.04％

[0104] Hydrophobicity is a nonspecific adhesion mechanism and is related to the ability of strains to adhere and colonize in the reproductive tract. By comparing the data in Table 6, it can be seen that the fermentation supernatant of saliva combined with Lactobacillus Ls100 can reduce the hydrophobicity of Candida albicans, indicating that its fermentation products can reduce the ability of Candida albicans to nonspecifically adhere to the reproductive tract and significantly affect the colonization of Candida albicans in the reproductive tract.

[0105] Example 5: Study on the inhibitory effect of saliva combined with Lactobacillus Ls100 on pathogenic bacteria biofilm

[0106] 1. Test methods

[0107] (1) 100 μL of Candida albicans or Gardnerella vaginalis was added to a 96-well plate. After 0, 12, and 24 h of culture, 100 μL of saliva combined with Lactobacillus Ls100 fermentation supernatant (bacteria-free) was added and cultured at 37°C for 24 h. 100 μL of MRS was used as a negative control. 10 μL of chlorhexidine acetate (0.1%) was added to MRS as a positive control.

[0108] (2) Remove the bacterial solution in the well, wash twice with 200 μL PBS and dry, add 200 μL 0.1% crystal violet to stain for 15 min, wash three times with PBS and dry;

[0109] (3) Decolorize with 200 μL of anhydrous ethanol for 15 min, measure the OD value at a wavelength of 570 nm, and calculate the inhibition rate of pathogenic bacteria biofilm. The results are shown in Table 7;

[0110] Inhibition rate of pathogenic bacteria biofilm % = (ODc-ODt) / ODc×100%;

[0111] Among them, ODc: OD value of negative control group; ODt: OD value of experimental group.

[0112] 2. Test results

[0113] Table 7 Inhibition of pathogenic bacterial biofilm

[0114]

[0115] Biofilm is composed of microbial communities and their coated extracellular polymers and matrix networks. It is a multicellular, multi-layered three-dimensional structure that adheres to each other or to the surface of tissues or objects. Biofilm is closely related to the formation of microbial resistance, gene transfer, and persistent infection of diseases. Biofilm is highly resistant to antimicrobial agents and host defense. The physical structure of biofilm helps pathogens escape many negative stimuli and improves tolerance to antimicrobial substances (such as H2O2, lactic acid and bacteriocins). The vaginal biofilm of healthy women is thin and loose, while women with vaginitis often have thick biofilms, so the difficulty of treating them increases dramatically. During conventional antibiotic treatment of vaginitis, the patient's clinical symptoms disappear in a short period of time, but after stopping the drug, dense and active biofilms quickly appear on the vaginal mucosa, leading to persistent infection or recurrence of vaginitis.

[0116] The formation of biofilm includes several key time points: initial adhesion of microorganisms at 0h, early biofilm formation at 12h, and biofilm maturity at 24h. Therefore, the present invention adds lactic acid bacteria fermentation supernatant for mediation at these key time points, and detects the biofilm formation amount and biofilm reduction rate after mediation at different periods.

[0117] From the data results in Table 7, it can be seen that: 1) In the initial adhesion stage and early formation stage of vaginal Gardnerella, saliva combined with Lactobacillus Ls100 can significantly inhibit the formation of vaginal Gardnerella biofilm, with inhibition rates of 51.5% and 48.6% respectively. In the mature stage of biofilm, the inhibition rate of Ls100 on vaginal Gardnerella biofilm is 38.9%. 2) In the initial adhesion stage, formation stage and mature stage of Candida albicans, saliva combined with Lactobacillus Ls100 can significantly inhibit the formation of Candida albicans biofilm, with inhibition rates of 76.8%, 78.2% and 54.8% respectively. Therefore, it can be seen that the present invention can utilize the destructive effect of saliva combined with Lactobacillus Ls100 on the biofilm formed by pathogenic bacteria and reduce the continued formation of biofilm by planktonic pathogenic bacteria, thereby achieving the effect of preventing and reducing the recurrence of vaginitis.

[0118] Example 6: Chelation of iron ions by saliva combined with Lactobacillus Ls100

[0119] 1. Experimental Methods

[0120] (1) Saliva was combined with Lactobacillus spp. Ls100 and cultured overnight. The supernatant was collected by centrifugation at 8000 rpm for 10 min. The cells were washed once with PBS and resuspended in PBS. The cell count was adjusted to 1×10 9 CFU / mL, inactivated bacteria were heat inactivated in a water bath at 80°C for 30 min, and the bacterial count was adjusted to 1×10 9 Cells / mL;

[0121] (2) 0.1 mL of 1% ascorbic acid, 0.1 mL of 2% FeSO4, and 1 mL of 0.2 mol / L NaOH were mixed thoroughly, and 0.5 mL of lactic acid bacteria sample was added. The mixture was kept at 37°C for 120 min to chelate the iron ions. 0.5 mL of 10% TCA was added to precipitate protein. The mixture was centrifuged at 8000 rpm for 5 min. 0.4 mL of supernatant was mixed with 4 mL of 0.1% o-phenanthroline, and the mixture was kept at 37°C for 10 min. The absorbance at 510 nm was measured. The blank group was replaced with MRS medium or PBS, which was A0. The test results are shown in Table 1. Figure 2 .

[0122] Fe 2+ Chelation rate % = (1-A / A0) × 100%;

[0123] 2. Experimental Results

[0124] The proliferation of vaginal Gardnerella depends on exogenous iron. Lactobacilli can reduce the content of free iron ions by chelating iron ions, thereby limiting the proliferation of BV bacteria that require iron. Figure 2 The results showed that the fermentation broth (containing bacteria), supernatant (without bacteria), live bacteria (without fermentation products) and inactivated bacteria (without fermentation products) of saliva combined with Lactobacillus Ls100 all have the ability to chelate iron ions. The chelation ability is in the order of live bacteria > fermentation broth > supernatant > inactivated bacteria. The chelation of iron ions by saliva combined with Lactobacillus Ls100 reduces the content of free iron ions, helping to reduce the proliferation of BV pathogenic bacteria in the body.

[0125] Example 7: Antibacterial effect of saliva combined with Lactobacillus Ls100 on the original pathogenic bacteria of vaginitis

[0126] 1. Experimental Methods

[0127] 1. Antibacterial methods for pathogenic bacteria

[0128] (1) Using the double-layer plate antagonism method, 10 mL of 2% agar was added to the culture dish and placed in an Oxford cup after solidification;

[0129] (2) Add the activated clinical pathogenic bacteria to the nutrient agar (containing 0.8% agar concentration) medium and mix well. Take 10 mL and add it to the culture dish. After the medium solidifies, take out the Oxford cup to form the Oxford cup hole.

[0130] (3) Saliva combined with Lactobacillus Ls100 was inoculated into MRS liquid culture medium at 3% and cultured at 37°C for 24 h. The fermentation broth was centrifuged at 8000 rpm for 10 min to collect the supernatant. Then 0.1 mL of the supernatant was added to each well and cultured at 37°C overnight. The diameter of the inhibition zone was measured. The results are shown in Table 8.

[0131] 2. Antibacterial methods for pathogenic fungi

[0132] (1) Using the double-layer plate antagonism method, 10 μL of saliva-associated Lactobacillus Ls100 bacterial solution was inoculated into MRS solid culture medium and cultured at 37° C. for 48 h until saliva-associated Lactobacillus Ls100 colonies were formed;

[0133] (2) A layer of YPD medium containing 0.8% agar of pathogenic fungi was spread on the culture medium. After solidification, the culture medium was inverted and cultured overnight. The antibacterial zone was observed around the colony of Lactobacillus saliva Ls100. The diameter of the antibacterial zone and the diameter of the colony of Lactobacillus saliva Ls100 were measured with a vernier caliper. The difference between the two was the antibacterial effect. The results are shown in Table 9.

[0134] 2. Experimental Results

[0135] 1. Antibacterial results against pathogenic bacteria

[0136] Table 8 Antibacterial results of saliva combined with Lactobacillus Ls100 on pathogenic bacteria of origin

[0137]

[0138] The data results in Table 8 show that saliva combined with Lactobacillus Ls100 has an inhibitory effect on 6 strains of pathogenic bacteria of female vaginitis, including Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, Staphylococcus hemolyticus, and Streptococcus angina pectoris.

[0139] 2. Antibacterial results against pathogenic fungi

[0140] Table 9 The results of antibacterial activity of saliva combined with Lactobacillus Ls100 against pathogenic fungi of origin

[0141]

[0142] The data results in Table 9 show that saliva combined with Lactobacillus Ls100 has an inhibitory effect on 5 strains of native pathogenic fungi, including Candida glabrata, Candida albicans, and Candida metastases.

[0143] In summary, saliva combined with Lactobacillus Ls100 has an inhibitory effect on these original pathogens of vaginitis, reducing the growth and reproduction of these pathogens, thereby alleviating vaginal inflammation.

[0144] Example 8: Study on the activity of sialidase by saliva combined with Lactobacillus Ls100

[0145] 1. Experimental Methods

[0146] (1) Vaginal Gardnerella culture

[0147] 1 mL of BHI medium containing 10% fetal bovine serum was added to a 24-well plate, and vaginal Gardnerella was inoculated at 10% and cultured for 24 h.

[0148] (2) Saliva combined with Lactobacillus s100 intervention

[0149] 1 mL of saliva combined with Lactobacillus Ls100 fermentation supernatant was added to the above 24-well plate, cultured for 24 hours, the culture solution was centrifuged at 8000 rpm for 5 minutes, and the supernatant was taken for sialidase activity detection. MRS medium and BHI medium were used as controls instead of saliva combined with Lactobacillus Ls100 fermentation supernatant.

[0150] (3) Sialidase detection

[0151] The samples were processed according to the instructions of the kit.

[0152] The sialidase activity was expressed as the concentration of sialidase. The results are shown in Table 10.

[0153] 2. Experimental Results

[0154] Table 10 Effect of saliva combined with Lactobacillus Ls100 on sialidase activity

[0155] Group Sialidase concentration ng / mL MRS control 504.34 BHI control 514.55 Ls100 (Lactobacillus salivarius) 368.58 LG19 (Lactobacillus gasseri) 421.48 LP20 (Lactobacillus plantarum) 459.90 LC21 (Lactobacillus crispatus) 427.10 LP23 (Lactobacillus plantarum) 426.01 LP24 (Lactobacillus plantarum) 447.31

[0156] A characteristic of bacterial vaginosis (BV) is that the activity of sialidase in vaginal lavage fluid is higher than that in healthy women. This enzyme is generally believed to play a key role in the development of BV. It destroys the vaginal epithelial mucus layer, a process that may increase the adhesion of pathogens to the vaginal epithelium and promote the formation of biofilm.

[0157] The results in Table 10 show that vaginal Gardnerella can secrete sialidase, and its sialidase content reaches 500 ng / mL after 48 hours of culture. Compared with the control group, the fermentation supernatant of saliva combined with Lactobacillus Ls100 can inhibit the secretion of sialidase by vaginal Gardnerella, and the content of sialidase in the sample is only 368.58 ng / mL, which can reduce its activity and thus play a role in slowing down the damage to the vaginal epithelial mucosa.

[0158] Example 9: Saliva combined with Lactobacillus Ls100 interferes with the morphological transformation ability of Candida albicans

[0159] 1. Experimental Methods

[0160] (1) Centrifuge the Candida albicans culture, remove the supernatant, wash once with PBS, and then resuspend in fresh YPD medium. Take 1 mL and add it to a 24-well plate;

[0161] (2) The saliva-Lactobacillus Ls100 fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. 1 mL was added to the above 24-well plate and mixed;

[0162] (3) 0.1% streptomycin solution was added to the above mixture, and 1 mL of MRS was used as a positive control instead of the supernatant of saliva combined with Lactobacillus Ls100 fermentation broth;

[0163] (4) Incubate at 37°C for 3 h and 6 h, take 10 μL and place on a glass slide, observe the growth of Candida albicans hyphae under a microscope, count the number of hyphae and yeast phases within the field of view, calculate the proportion of hyphae phase, and count the hyphae length in μm. The results are shown in Table 11;

[0164] 2. Experimental Results

[0165] Table 11 Effect of saliva combined with Lactobacillus Ls100 on the hyphae formation ability of Candida albicans

[0166]

[0167] The results in Table 11 show that when saliva combined with Lactobacillus Ls100 and Candida albicans are co-cultured for 3 hours, the formation of Candida albicans hyphae can be inhibited and the hyphae percentage can be reduced, but there is no obvious inhibitory effect on the hyphae length; when saliva combined with Lactobacillus Ls100 and Candida albicans are co-cultured for 6 hours, saliva combined with Lactobacillus Ls100 can inhibit the formation of Candida albicans hyphae and reduce the hyphae length of Candida albicans.

[0168] Example 10: Regulatory effect of saliva combined with Lactobacillus Ls100 on inflammatory factors in Hela cells

[0169] 1. Experimental Methods

[0170] 1. Strain activation, enrichment and cell culture

[0171] (1) Preparation of live bacterial suspension

[0172] The fermentation liquid of the test strain was centrifuged to collect the bacterial sludge, washed twice with PBS, and resuspended with MEM culture medium to adjust the bacterial liquid concentration to 1.5×10 8 CFU / mL, and the live bacterial suspension was obtained. The concentration of Candida albicans suspension was 1.5×10 8 CFU / mL.

[0173] (2) Preparation of inactivated bacterial suspension

[0174] The inactivated bacteria were collected as above, resuspended, and placed in an 80°C water bath for 30 min to inactivate the bacteria. The concentration of the bacterial solution was adjusted to 1.5×10 8 Cells / mL to obtain inactivated bacterial suspension.

[0175] (3) Cell culture

[0176] Hela cells were revived in MEM complete culture medium containing 10% fetal bovine serum, incubated in a cell culture incubator at 37°C and 5% CO2, and subcultured when the cells grew well and the density reached about 80%. Subcultured for 3 times before conducting subsequent experiments.

[0177] 2. Determination of the effect of strains on the secretion of cellular immune factors

[0178] Hela cells were collected at 1.5×10 5 The cells were inoculated in a 24-well plate at a concentration of 100 / mL. After the cells adhered to the wall, the supernatant was removed, and the cells were washed once with PBS to remove the non-adherent cells. Then the test samples were added and incubated, with the live bacteria incubated for 4 hours and the inactivated bacteria incubated for 24 hours.

[0179] The experimental groups are as follows:

[0180] 1) Negative control group (MEM): MEM was added to the wells and incubated for 4 h and 24 h respectively;

[0181] 2) Positive control group (Ca): Candida albicans was added to the wells and incubated for 4 h and 24 h respectively;

[0182] 3) Ls100 test group: 1 mL of live bacterial suspension or inactivated bacterial suspension of strain Ls100 was added to the wells, and the live bacterial group was incubated for 4 h, and the inactivated group was incubated for 12 h;

[0183] 4) Ls100 competition group (strain Ls100+Ca): Ca and live bacterial suspension or inactivated bacterial suspension of strain Ls100 were added to each well at the same time, and the live bacterial group was incubated for 4 hours, and the inactivated group was incubated for 12 hours;

[0184] 5) Ls100 treatment group (C.a+ strain Ls100): Ca was first added to each well and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria), and then live bacterial suspension or inactivated bacterial suspension of strain Ls100 was added. The live bacteria group was incubated for 4 h, and the inactivated group was incubated for 12 h;

[0185] 6) Ls100 prevention group (strain Ls100+Ca): strain Ls100 suspension was first added to each well, the live bacteria group was incubated for 4 h, the inactivated group was incubated for 12 h, and then Ca was added and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria);

[0186] The cell culture supernatant was collected, centrifuged at 8000 r / min for 10 min, and then dispensed into sterile centrifuge tubes. The secretion levels of IL-6 and IL-8 cytokines in the above different groups were determined according to the instructions of the ELISA kit. The results are shown in Tables 12-13.

[0187] 2. Test results

[0188] 1. Effect on IL-6:

[0189] Table 12 Effect of saliva combined with live and dead Lactobacillus Ls100 on IL-6 secretion by Hela cells

[0190]

[0191]

[0192] IL-6 can regulate the growth and differentiation of various cells, regulate immune response, acute phase response and hematopoiesis, and play an important role in the body's anti-infection immune response. IL-6 has obvious changes in various diseases, and its dysregulated expression can cause many diseases. The clinical manifestation of vaginitis patients is mainly the increase of IL-6 level at the onset of the disease.

[0193] From the data in Table 12, it can be seen that the test results of the live bacteria group show that in the positive control group, Candida albicans Ca can stimulate Hela cells to secrete a large amount of IL-6; compared with the negative control group, the Ls100 live bacteria group co-incubated with cells can stimulate the secretion of cytokine IL-6, causing a physiological inflammatory response. Co-incubating Ls100 live bacteria and Candida albicans Ca with cells at the same time can reduce the secretion of IL-6 compared with the positive control. Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then lactic acid live bacteria were added for incubation. The IL-6 secretion of Ls100 live bacteria was lower than the secretion of Candida albicans Ca alone incubated with cells. Compared with the positive control, Ls100 live bacteria were first co-incubated with cells, and then Candida albicans Ca was added for incubation, which can reduce IL-6 secretion.

[0194] The results of the inactivated bacteria group test showed that compared with the negative control group, the inactivated bacteria Ls100 co-incubated with cells can stimulate the secretion of cytokine IL-6 and cause a physiological inflammatory response. When the inactivated bacteria and Candida albicans Ca were co-incubated with cells at the same time, Ls100 could reduce the secretion of IL-6 compared with the positive control. Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then inactivated bacteria Ls100 was added for incubation. The IL-6 secretion was lower than the secretion when Candida albicans Ca was incubated with cells alone. Ls100 inactivated bacteria was first incubated with cells, and then Candida albicans Ca was added for incubation. The IL-6 secretion was lower than the secretion when Candida albicans Ca was incubated with cells alone.

[0195] In summary, Candida albicans Ca can promote the secretion of IL-6 by Hela cells, leading to cellular inflammation, while intervention with live or inactivated Ls100 bacteria can reduce the secretion of IL-6 and improve the inflammatory state of cells.

[0196] 2. Effect on IL-8:

[0197] Table 13 Results of saliva combined with live and dead Lactobacillus Ls100 on the secretion of IL-8 by Hela cells

[0198]

[0199]

[0200] IL-8 is a common inflammatory chemokine. IL-8 can activate and aggregate inflammatory cells, thereby promoting inflammatory response. It is an important influencing factor in the occurrence and development of inflammation and can reflect the level of inflammation to a certain extent. The increased expression level of IL-8 in vaginal secretions is closely related to local vaginal microbial infection. Under various vaginal inflammatory infection conditions, the balance of vaginal microecology is destroyed, the local specific immune function of the vagina changes, and the expression level of inflammatory factors increases, which participates in the occurrence and development of vaginal inflammation. By observing the changes in IL-8 expression levels, the occurrence and development process and severity of inflammation can be inferred, and this can be used as one of the outcome indicators to judge the anti-inflammatory effect of drugs.

[0201] The data in Table 13 show that the test results of the live bacteria group show that in the positive control group, Candida albicans Ca can stimulate Hela cells to secrete IL-8; compared with the negative control group, the saliva combined with the live bacteria group of Lactobacillus Ls100 can promote the secretion of cytokine IL-8 and cause a physiological inflammatory response. Saliva combined with live Lactobacillus Ls100 and Candida albicans Ca were incubated with cells at the same time, which can reduce the secretion of IL-8 compared with the positive control. Candida albicans Ca was first incubated with cells to create a cell inflammation model, and then lactic acid live bacteria were added for incubation. The IL-8 secretion of saliva combined with live Lactobacillus Ls100 was lower than the secretion of Candida albicans Ca alone. Saliva combined with live Lactobacillus Ls100 was first incubated with cells, and then Candida albicans Ca was added for incubation, which can reduce the secretion of IL-8 compared with the positive control.

[0202] The results of the inactivated bacteria group test showed that compared with the negative control group, the inactivated bacteria of saliva combined with Lactobacillus Ls100 and cells co-incubated can promote the secretion of cytokine IL-8 and cause physiological inflammatory response. When inactivated bacteria and Candida albicans Ca were co-incubated with cells at the same time, compared with the positive control, the inactivated bacteria of saliva combined with Lactobacillus Ls100 could reduce the secretion of IL-8. Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then inactivated bacteria of saliva combined with Lactobacillus Ls100 were added for incubation. The secretion of IL-8 was lower than that of Candida albicans Ca alone. The inactivated bacteria of saliva combined with Lactobacillus Ls100 were first co-incubated with cells, and then Candida albicans Ca was added for incubation. Compared with the positive control, the secretion of IL-8 was lower than that of Candida albicans Ca alone.

[0203] In summary, Candida albicans Ca can promote the secretion of IL-8 by Hela cells, leading to cellular inflammation, while intervention with live or inactivated Ls100 bacteria can reduce the secretion of IL-8 and improve the inflammatory state of cells.

[0204] Example 11: Degradation of biogenic amines by saliva combined with Lactobacillus Ls100

[0205] 1. Experimental Methods

[0206] 1. Growth of biogenic amines

[0207] To prepare vaginal simulated fluid SVF, cadaverine, putrescine and tyramine were added respectively, with the concentration of each biogenic amine being 100 mg / L. Saliva combined with Lactobacillus Ls100 was added at 5% to the SVF containing the three biogenic amines and cultured at 37°C. The OD value, pH value and viable count were measured at 0h, 12h and 24h, respectively. The results are shown in Table 15.

[0208] 2. Degradation of biogenic amines

[0209] 1) Preparation of standard curve of biogenic amines

[0210] Accurately weigh 10 mg of each of putrescine, cadaverine, and tyramine and dissolve them in 0.1 mol / L HCl solution. After diluting to 10 mL, obtain standard stock solutions of various biogenic amines with a mass concentration of 1000 mg / L, and store them at -20°C. Take 1 mL of each of the above biogenic amine standard stock solutions and mix them in a 10 mL brown volumetric flask, dilute them with HCl solution of the same concentration to a mixed standard solution with a mass concentration of 100 mg / L, and dilute them to mixed standard working solutions with concentrations of 50.0, 25.0, 15.0, 10.0, 5.0, and 2.5 mg / L, respectively, and store them at 4°C for use. Take 1 mL of the mixed standard solution of biogenic amines and perform pre-column derivatization according to the method described below. The derivatized product is determined by high performance liquid chromatography (HPLC). The standard curves of the three biogenic amines are plotted with the peak area (Y) as the ordinate and the biogenic amine concentration (X, mg / L) as the abscissa. The regression equations are as follows: the regression equation of putrescine is y=35.167x+87.393, R2=0.9994; the regression equation of cadaverine is y=16.676x+91.729, R2=0.9965; the regression equation of tyramine is y=36.702x-43.263, R2=0.9985.

[0211] 2) Pre-column derivatization of biogenic amines

[0212] Take 1mL of the mixed standard solution of biogenic amines / the sample to be tested and place it in a 5mL brown volumetric flask, add 200μL of 2mol / L sodium hydroxide solution to adjust the pH to alkaline, add 300μL of saturated sodium bicarbonate buffer and 2mL of 10mg / mL dansyl chloride derivative (prepared in acetone), place the volumetric flask in a 40℃ water bath under light-proof conditions and react for 45min. Finally, add 100μL of ammonia water to terminate the reaction, dilute to 5mL with acetonitrile, mix well and filter through a 0.22μm filter membrane, place the filtrate in a 2mL brown bottle and wait for analysis.

[0213] 3) HPLC conditions

[0214] Chromatographic separation conditions: chromatographic column: SHIMADZU C18-AQ (5 μm, 250 mm×4.6 mm); mobile phase A: water; mobile phase B: acetonitrile; injection volume: 20 μL; column temperature: 35°C; UV detection wavelength: 254 nm, gradient elution program: see Table 14 below.

[0215] Table 14: HPLC gradient elution program

[0216] Time (min) A(％) B(％) Flow rate (mL / min) 0 30 70 0.80 15 25 75 1.00 22 15 85 1.00 25 10 90 1.00 28 10 90 1.00 35 40 60 1.00

[0217] 4) Degradation of biogenic amines by saliva combined with Lactobacillus spp. Ls100

[0218] Saliva combined with Lactobacillus Ls100 was added to SVF containing three biogenic amines (each biogenic amine concentration was 100 mg / L) at 5%, and the one with MRS added was used as the control group. The culture was carried out at 37°C for 24 hours, and the bacterial suspension was centrifuged at 8000rpm for 10 minutes. 1mL of the supernatant was collected and added with 4mL of 0.1mol / L HCl to dissolve the biogenic amines as the sample treatment solution, and then derivatized and stored at -20°C in the dark for the determination of the content of biogenic amines and the calculation of the degradation rate of biogenic amines. The results are shown in Figure 3 .

[0219] Degradation effect of biogenic amines: biogenic amine degradation rate % = (W-W1) / W×100%, W is the biogenic amine content of the control group, and W1 is the biogenic amine content of the experimental group.

[0220] 2. Test results

[0221] 1. Growth of saliva-associated Lactobacillus spp. Ls100 in SVF containing biogenic amines

[0222] Table 15: Growth of saliva-associated Lactobacillus Ls100 in SVF containing biogenic amines

[0223] Cultivation time OD value pH Viable bacteria count CFU / mL 0h 0.130 5.39 4.72E+07 12h 0.320 4.21 1.45E+08 24h 0.351 3.92 2.01E+08

[0224] From the data in Table 15, it can be seen that saliva-associated Lactobacillus Ls100 has a strong survival ability in SVF containing three biogenic amines: cadaverine, putrescine, and tyramine. It can use its nutrients to maintain its own growth and reproduction, and produce acidic substances to reduce the increase in environmental pH caused by biogenic amines and maintain an acidic environment.

[0225] 2. Degradation of biogenic amines by saliva combined with Lactobacillus spp. Ls100

[0226] Figure 3 The results showed that saliva combined with Lactobacillus Ls100 had different degrees of degradation effects on putrescine, cadaverine and tyramine, three biogenic amines. By reducing the content of these three biogenic amines, the production of odor was reduced, and the pH increase caused by biogenic amines was reduced, thereby alleviating the symptoms of vaginitis caused by biogenic amines.

[0227] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A saliva-associated lactobacillus Ls100, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the accession number CGMCC NO.32135.

2. The saliva-associated Lactobacillus Ls100 according to claim 1, characterized in that: The saliva-lactobacillus Ls100 is derived from the reproductive tract secretions of healthy women.

3. A microbial agent, characterized in that: The invention comprises one or more of the live bacteria of saliva-associated Lactobacillus Ls100, the inactivated bacteria of saliva-associated Lactobacillus Ls100, and the fermentation product of saliva-associated Lactobacillus Ls100 as claimed in claim 1.

4. Use of the saliva-combined lactobacillus Ls100 as claimed in claim 1 or the microbial agent as claimed in claim 2 in preparing a product for preventing and treating genital tract pathogenic bacteria infection and / or preventing and treating vaginitis.

5. The use according to claim 4, characterized in that: The reproductive tract pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, hemolytic Staphylococcus, Streptococcus angina pectoris, Candida glabrata, Candida albicans, and Candida diverticula.

6. The use according to claim 4, characterized in that: The saliva combined with Lactobacillus Ls100 or microbial agent has one or more of the following effects: 1) Saliva combined with Lactobacillus Ls100 or microbial agents can produce hydrogen peroxide; 2) Saliva combined with Lactobacillus Ls100 or microbial agents have an antibacterial effect on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, Staphylococcus hemolyticus, Streptococcus angina pectoris, Candida glabrata, Candida albicans, and Candida diverticulatus; 3) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae; 4) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the biofilm formation of vaginal Gardnerella and / or Candida albicans at different stages; 5) Saliva combined with Lactobacillus Ls100 or microbial agents can reduce the hydrophobicity of Candida albicans; 6) Saliva combined with Lactobacillus Ls100 or microbial agents can chelate iron ions; 7) Saliva combined with Lactobacillus Ls100 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage; 8) Saliva combined with Lactobacillus Ls100 or microbial preparations can reduce the secretion of inflammatory factors IL-6 and IL-8; 9) Saliva combined with Lactobacillus Ls100 or microbial agents have a good degradation effect on biogenic amines.

7. The use according to claim 4, characterized in that: The products include oral products or external products.

8. The use according to claim 4, characterized in that: The oral product includes food or health care product; The external-use products include one or more of external-use medicines, sanitary products, and family planning products.

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