Lactobacillus crispatus LC30 for preventing and treating female genital tract infection and application thereof

Abstract

The invention relates to lactobacillus crispatus LC30 for preventing and treating female genital tract infection and application of the lactobacillus crispatus LC30. The lactobacillus crispatus LC30 is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO.31923. The lactobacillus crispatus LC30 has good hydrogen peroxide production capacity and adhesion capacity, has a strong inhibition effect on main vaginitis pathogenic bacteria, and can be used for preventing and treating female genital tract infection. The composition is effective to nutritional pathogenic bacteria, and can also reduce form transformation and biological membrane formation of the pathogenic bacteria and prevent and treat drug resistance; besides, the fermentation product can reduce the content of pathogenic bacteria metabolites, regulate the secretion of cytokines and achieve the effect of relieving inflammation, so that the fermentation product can be used for preparing products for preventing and treating genital tract pathogenic bacteria infection, preventing and treating vaginitis and / or removing pudendum peculiar smell.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to Lactobacillus crispatus LC30 for preventing and treating female reproductive tract infection and application thereof. Background Art

[0002] Currently, more than 1 billion women worldwide suffer from urinary and reproductive tract infections every year, and vaginitis is the most common reproductive tract disease. According to a survey of Chinese women by the World Health Organization, 41% of women of childbearing age suffer from gynecological inflammation to varying degrees. According to statistics, the incidence rate among married women is as high as 70%.

[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 taxonomic group 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, it is crucial to screen lactobacillus strains that can prevent and treat female reproductive tract infections and vaginal inflammation, avoid the production of biofilms and drug resistance by pathogenic bacteria, utilize microbial growth metabolism and the competition and synergy between microorganisms, improve the vaginal microenvironment, inhibit vaginal pathogenic bacteria infection and recurrence, and prevent and treat vaginal inflammation, in order to maintain the health of the female reproductive tract and prevent and treat 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 Lactobacillus crispatus LC30 and its application, which can not only effectively prevent and treat genital tract pathogenic bacteria infection and vaginitis, but also remove vaginal odor.

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

[0006] A Lactobacillus crispatus LC30, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.31923, the deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the deposit time is September 10, 2024.

[0007] As a further technical solution, the Lactobacillus crispatus LC30 is derived from the reproductive tract secretions of healthy women.

[0008] A microbial agent comprises one or more of the live bacteria of Lactobacillus crispatus LC30, the inactivated bacteria of Lactobacillus crispatus LC30, and the fermentation product of Lactobacillus crispatus LC30.

[0009] The Lactobacillus crispatus LC30 or the microbial agent is used in preparing products for preventing and treating female reproductive tract pathogenic bacteria infection, preventing and treating vaginitis, and / or removing vaginal odor.

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

[0011] As a further technical solution, the Lactobacillus crispatus LC30 or microbial agent has one or more of the following effects:

[0012] 1) Lactobacillus crispatus LC30 or microbial agents can produce hydrogen peroxide;

[0013] 2) Lactobacillus crispatus LC30 or microbial agents have antibacterial effects on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Staphylococcus hominis, Klebsiella pneumoniae, Streptococcus agalactiae, Corynebacterium aureus, Streptococcus anginae, and Staphylococcus pyogenes;

[0014] 3) Lactobacillus crispatus LC30 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae;

[0015] 4) Lactobacillus crispatus LC30 or microbial agents can inhibit the biofilm formation of Gardnerella vaginalis and / or Candida albicans at different stages;

[0016] 5) Lactobacillus crispatus LC30 or microbial agents can reduce the hydrophobicity of Candida albicans, reduce the adhesion of Candida albicans, and inhibit the formation of biofilm;

[0017] 6) Lactobacillus crispatus LC30 or microbial agents can chelate iron ions;

[0018] 7) Lactobacillus crispatus LC30 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage;

[0019] 9) Lactobacillus crispatus LC30 or microbial agents can grow in an environment containing biogenic amines and can degrade biogenic amines;

[0020] 9) Lactobacillus crispatus LC30 or microbial agents can reduce the secretion of inflammatory factors IL-6 and IL-8.

[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, antibacterial / antibacterial lotions; family planning products include condoms, lubricants

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

[0026] 1) The Lactobacillus crispatus LC30 isolated and screened in 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 Lactobacillus crispatus LC30 isolated and screened by the present invention can produce hydrogen peroxide, 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 Staphylococcus hominis, Klebsiella pneumoniae, Streptococcus agalactiae, Corynebacterium aureus, Streptococcus angina pectoris, and hemolytic Staphylococcus, and can be used to prevent and treat female reproductive tract pathogenic bacteria infection.

[0028] 3) Lactobacillus crispatus LC30 can inhibit the formation of Candida albicans hyphae, reduce the number and length of hyphae, and has a significant effect on female vaginal diseases caused by Candida albicans.

[0029] 4) Lactobacillus crispatus LC30 can inhibit the formation of biofilm by vaginal Gardnerella and Candida albicans at different stages, and it has strong hydrophobicity and adhesion ability, and can reduce the hydrophobicity of Candida albicans, thereby reducing its adhesion effect and reducing the probability of biofilm formation, thereby preventing the occurrence of Candida albicans resistance.

[0030] 5) The proliferation of vaginal Gardnerella depends on exogenous iron, and Lactobacillus crispatus LC30 can reduce the content of free iron ions by chelating iron ions, thereby limiting the proliferation of vaginal Gardnerella that requires iron.

[0031] 6) Lactobacillus crispatus LC30 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage;

[0032] 7) Biogenic amines are the main source of vaginal odor. Lactobacillus crispatus LC30 can grow and reproduce using biogenic amines as a nitrogen source and has the ability to degrade biogenic amines, thereby achieving the effect of removing vaginal odor.

[0033] 8) Live and inactivated Lactobacillus crispatus LC30 can reduce the secretion of inflammatory factors IL-6 and IL-8, thereby achieving the purpose of preventing and treating vaginal inflammation.

[0034] 9) The Lactobacillus crispatus LC30 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 genitals, 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 Lactobacillus crispatus LC30 screened by the present invention has good hydrogen peroxide production ability and adhesion ability, has a strong inhibitory effect on the main vaginitis pathogens, is not only effective for vegetative pathogens, but also can reduce their morphological transformation and biofilm formation, and prevent the generation of drug resistance; in addition, its strains and fermentation products 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 female reproductive tract pathogenic bacteria infection, preventing and treating vaginitis, and / or removing vulva odor. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effect of Lactobacillus crispatus LC30 on the hydrophobicity of Candida albicans;

[0037] Figure 2 The effect of Lactobacillus crispatus LC30 on competitive adhesion of Candida albicans;

[0038] exist Figure 2 In the figure, a: blank cells; b: Lactobacillus crispatus LC30; c: competitive adhesion with Candida albicans; d: preventive adhesion with Candida albicans; e: therapeutic adhesion with Candida albicans;

[0039] Figure 3 is the chelating ability of different components of Lactobacillus crispatus LC30 on iron ions;

[0040] Figure 4 The inhibitory effect of Lactobacillus crispatus LC30 on vaginal Gardnerella and Candida albicans biofilm;

[0041] exist Figure 4 Middle, a: Gardnerella vaginalis biofilm; b: Candida albicans biofilm;

[0042] Figure 5 This is the degradation effect of Lactobacillus crispatus LC30 on biogenic amines. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] Example 1: Isolation of Lactobacillus crispatus LC30

[0046] 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.

[0047] Example 2, strain screening

[0048] All the isolated lactic acid bacteria strains with milky white, raised colonies, neat edges, smooth surfaces, rod-shaped, Gram-positive, and catalase-negative under a microscope were subjected to a screening test for antibacterial properties. A strain with strong inhibitory effects on Escherichia coli, Staphylococcus aureus, Candida albicans, and Gardnerella vaginalis, as well as strong adhesion and hydrogen peroxide production capabilities was selected and named LC30.

[0049] 1. Test on the inhibitory ability against Escherichia coli and Staphylococcus aureus

[0050] 1.1 Test methods

[0051] (1) The lactic acid bacteria strains screened in Example 1 were 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;

[0052] (2) Cool the 2.0% plain agar medium to about 50°C, pour 10 mL into a sterile plate, and dry it on a clean bench;

[0053] (3) Use sterile forceps to take the sterilized Oxford cup and place it on a plate filled with agar and let it stand for 10 min.

[0054] (4) Add 1 mL of Escherichia coli (or Staphylococcus aureus) to 100 mL of nutrient agar medium cooled to 50°C and mix thoroughly;

[0055] (5) Pour 10 mL of the above nutrient agar medium onto the plate with the Oxford cup placed on it, and after solidification, remove the Oxford cup by clipping it out to form an Oxford cup hole;

[0056] (6) 0.1 mL of the fermentation supernatant of each lactic acid bacteria was injected into each well of the Oxford cup and incubated at 37°C for 16-18 h. The diameter of the inhibition zone was measured with a vernier caliper. Two parallels were set for each group. The test results are shown in Table 1.

[0057] 1.2 Test results

[0058] Table 1 Antibacterial results of various lactic acid bacteria on Escherichia coli and Staphylococcus aureus

[0059] strain Escherichia coli inhibition zone (mm) Staphylococcus aureus inhibition zone (mm) LP01(Lactobacillus plantarum) 14.87±1.80 12.80±0.35 LC01 (Lactobacillus crispatus) 13.70±0.68 9.63±0.71 LC30 (Lactobacillus crispatus) 14.53±2.10 13.13±0.82 LG05 (Lactobacillus gasseri) 13.13±0.23 11.90±0.17 LG14 (Lactobacillus gasseri) 12.53±0.50 10.80±0.35 LR09 (Lactobacillus rhamnosus) 13.83±0.29 12.63±0.35 LP03 (Lactobacillus paracasei) 12.83±0.76 12.70±0.26 MRS 8.00±0.00 8.00±0.00

[0060] Note: Since the hole of the Oxford cup has a diameter of 8mm, only the antibacterial zone diameter ≥ 8mm indicates an antibacterial effect.

[0061] The data results in Table 1 show that strain LC30 has good inhibitory ability against Escherichia coli and Staphylococcus aureus, and its inhibitory effect is better than other strains. MRS as a control has no inhibitory effect.

[0062] 2. Test of the inhibitory ability to vaginal Gardnerella

[0063] 2.1 Test methods

[0064] (1) The lactic acid bacteria strains screened in Example 1 were 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;

[0065] (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;

[0066] (3) Place a sterilized Oxford cup on the above culture medium and let it stand for 10 min. Add 0.1 mL of the fermentation supernatant of each lactic acid bacteria 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 2.

[0067] 2.2 Test results

[0068] Table 2 Antibacterial effect on vaginal Gardnerella

[0069] strain Diameter of inhibition zone mm LP01(Lactobacillus plantarum) 11.33±0.31 LC01 (Lactobacillus crispatus) 10.70±0.75 LC30 (Lactobacillus crispatus) 10.73±1.33 LG05 (Lactobacillus gasseri) 10.50±0.10 LG14 (Lactobacillus gasseri) 10.33±0.58 LR09 (Lactobacillus rhamnosus) 9.00±0.57 LP03 (Lactobacillus paracasei) 10.43±0.40 MRS 8.00±0.00

[0070] Gardnerella vaginalis is the main pathogen of bacterial vaginitis. The results in Table 1 show that LS360 has a strong inhibitory ability against Gardnerella vaginalis, with an inhibitory diameter of 10.73 mm, and a better inhibitory effect. MRS as a control has no inhibitory effect on Gardnerella vaginalis.

[0071] 3. Test on the inhibitory ability of Candida albicans

[0072] 3.1 Test methods

[0073] (1) Centrifuge the fermented broth of each lactic acid bacteria cultured for 16 h, collect the supernatant (without the strain), mix 5 mL of the lactic acid bacteria fermented supernatant and 5 mL of the Candida albicans culture in equal volumes, and count the viable bacteria of Ca at 0 h and 24 h, respectively, using a selective YPD medium;

[0074] (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.

[0075] (3) Calculate the inhibitory ability of lactic acid bacteria on Candida albicans. The results are shown in Table 3:

[0076] 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 24 h of culture, and A0 represents the number of Ca viable bacteria after 0 h;

[0077] 3.2 Test results

[0078] Table 3: Inhibitory ability against Candida albicans

[0079]

[0080]

[0081] Candida albicans is the main pathogen of candidal vaginitis. The results in Table 3 show that the fermentation supernatant of strain LC30 has a strong 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.

[0082] 4. Hydrogen peroxide production capacity test

[0083] 4.1 Test methods

[0084] (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.

[0085] (2) Each lactic acid bacteria liquid was streaked on a TMB-HRP-MRS plate and cultured anaerobically at 37°C for 48 hours. The culture plate was then exposed to air for 30 minutes. The colonies producing hydrogen peroxide would turn blue. The lactic acid bacteria were graded according to the intensity of the blue color. They were divided into four types: strong positive (+++), positive (++), weak positive (+), and negative (-). The results are shown in Table 4.

[0086] 4.2 Test results

[0087] Table 4: Hydrogen peroxide production capacity of each strain

[0088] strain Hydrogen peroxide production LP01(Lactobacillus plantarum) — LC01 (Lactobacillus crispatus) ++ LC30 (Lactobacillus crispatus) +++ LG05 (Lactobacillus gasseri) + LG14 (Lactobacillus gasseri) ++ LR09 (Lactobacillus rhamnosus) — LP03 (Lactobacillus paracasei) —

[0089] From the results in Table 4, it can be seen that the blue intensity of strain LC30 is the deepest and its ability to produce hydrogen peroxide is the strongest.

[0090] 5. Hydrophobicity test of lactic acid bacteria

[0091] 5.1 Test methods

[0092] Each lactic acid bacteria cultured overnight was centrifuged at 5000rpm for 3min, the bacterial sludge was collected and washed once with PBS, then resuspended in PBS, and the OD value was measured at a wavelength of 600nm, 0.7-0.8, recorded as A0, and each bacterial suspension was mixed with xylene, chloroform, and ethyl acetate in a ratio of 3:1, and fully shaken for 30s, allowed to stand for 10s, shaken for 30s, and allowed to stand for 30min. 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 lactic acid bacteria cell surface was calculated. The results are shown in Table 5.

[0093] Lactic acid bacteria cell surface hydrophobicity % = (1-A / A0) × 100%

[0094] Hydrophobicity standard: more than 50% is highly hydrophobic, between 20%-50% is moderately hydrophobic, and less than 20% is non-hydrophobic.

[0095] 5.2 Test results

[0096] Table 5: Hydrophobicity of each strain

[0097] strain Xylene Chloroform Ethyl acetate LP01(Lactobacillus plantarum) 43.91％±2.29 57.46％±13.31 26.75％±0.84 LC01 (Lactobacillus crispatus) 79.28％±0.32 83.19％±0.29 49.30％±5.94 LC30 (Lactobacillus crispatus) 82.86％±8.21 85.58％±6.74 54.72％±0.67 LG05 (Lactobacillus gasseri) 77.29％±4.51 80.07％±1.12 41.38％±1.11 LG14 (Lactobacillus gasseri) 40.80％±0.65 42.31％±0.45 22.10％±0.27 LR09 (Lactobacillus rhamnosus) 0.87％±0.06 0.00±0.00 0.00±0.00 LP03 (Lactobacillus paracasei) 26.28％±4.75 25.49％±2.70 0.00±0.00

[0098] 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 surface of lactic acid bacteria. The results in Table 5 show that strain LC30 has strong hydrophobicity, and the hydrophobicity rates of xylene, chloroform and ethyl acetate are all greater than 50%, reaching a high hydrophobicity level, and it can effectively adhere to the host epithelial cells.

[0099] Based on the antibacterial results against Escherichia coli, Staphylococcus aureus, Candida albicans, and Gardnerella vaginalis, as well as the hydrogen peroxide production capacity and hydrophobicity results, a comprehensive evaluation of the above strains revealed that strain LC30 had the best overall effect.

[0100] Example 3: Identification of strains

[0101] The 16S rRNA gene of the strain LC30 obtained by screening was identified, and the nucleotide sequence was shown in SENIDNO.1. It was identified that the strain belonged to Lactobacillus crispatus; therefore, it was named Lactobacillus crispatus LC30.

[0102] Example 4: Study on the effect of Lactobacillus crispatus LC30 on the hydrophobicity of Candida albicans

[0103] 1. Test methods

[0104] (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.

[0105] (2) Take 4.5 ml of the above Candida albicans liquid, add 4.5 mL of lactic acid bacteria fermentation supernatant, mix well and culture at 37°C for 24 h as the experimental group. 4.5 mL of LMRS broth medium instead of lactic acid bacteria fermentation supernatant is used as the control group.

[0106] (3) The concentration of the bacterial suspension in the experimental group and the control group was adjusted to an absorbance of about 1.000 at 560 nm, which was 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 seconds, allowed to stand for 10 seconds, shaken for 30 seconds, allowed to stand for 20 minutes, and after the two phases were separated, the aqueous phase was aspirated to measure the absorbance at 560 nm, which was recorded as A. Three parallel experiments were performed for each group, and the average value was taken.

[0107] Hydrophobicity (CSH%) = (1-A / A0) × 100%

[0108] 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.

[0109] 2. Test results

[0110] Figure 1 The results showed that the hydrophobicity of Candida albicans in the control group was 79.39%. After co-culturing the fermentation supernatant of Lactobacillus crispatus LC30 with Candida albicans in the experimental group, the hydrophobicity of Candida albicans was reduced to 64.42%. At the same time, the acid-base charge characteristics of the Candida albicans surface were changed, affecting the adhesion ability of Candida albicans, thereby reducing the colonization of Candida albicans in the reproductive tract.

[0111] Example 5: Lactobacillus crispatus LC30 interferes with the morphological transformation ability of Candida albicans

[0112] 1. Test methods

[0113] The Candida albicans culture was centrifuged, the supernatant was removed and washed with PBS, then resuspended with fresh YPD medium, 1 mL was added to a 24-well plate, 0.1% streptomycin solution was added and mixed, and the Lactobacillus crispatus LC30 fermentation broth was centrifuged at 8000rpm for 10 min to collect the Lactobacillus crispatus LC30 fermentation supernatant and add 0.1% streptomycin solution to mix, the negative control did not add 0.1% streptomycin and 1 mL of MRS was used instead of the Lactobacillus crispatus LC30 fermentation supernatant. The above three groups were incubated at 37°C for 6h and 10 μL was taken on a slide. After staining, the growth of Candida albicans hyphae was observed, the number of hyphae phase and yeast phase within the field of view was counted, the proportion of hyphae phase was calculated, and the hyphae length was counted. The results are shown in Table 6.

[0114] 2. Test results

[0115] Table 6 Effect of supernatant of Lactobacillus crispatus LC30 fermentation broth on Candida albicans hyphae

[0116]

[0117]

[0118] Candida albicans Ca is a polymorphic fungus that can grow in the form of unicellular yeast, hyphae or pseudohyphae. The fungus relies on hyphae to invade tissues, and the expression of key virulence factors is coordinated with the formation of hyphae. The data results in Table 6 show that Lactobacillus crispatus LC30 has an inhibitory effect on the formation of Candida albicans hyphae, thereby reducing its adhesion and invasion to vaginal epithelial cells and reducing its toxicity to epithelial cells. Streptomycin can induce the formation of hyphae of Candida albicans. The hyphae accounted for more than 40% in the positive control group, and the hyphae accounted for more than 15% in the negative control group without streptomycin. Compared with the positive control, the fermentation supernatant of Lactobacillus crispatus LC30 with the addition of streptomycin can inhibit the formation of Candida albicans hyphae, and can also inhibit the length of hyphae, and the hyphae accounted for less than 20%.

[0119] Example 6: Adhesion to Hela cells and competitive adhesion with Candida albicans

[0120] 1. Test methods

[0121] (1) Preparation of test strain suspension: The activated test strain was centrifuged at 5000 rpm for 3 min to collect the cells, washed twice with PBS, and the concentration of the suspension was adjusted to 1.5 × 10 8 CFU / mL and place at 4℃ for later use.

[0122] (2) Cell culture: After the HeLa cells are cultured, the culture medium is removed, the cells are washed once with PBS, 1 mL of trypsin is added, and the cells are allowed to stand at room temperature for 3 min. After microscopic examination, the cells are rounded and then aspirated. After the cells are suspended, 2 times the volume of cell culture medium is added to terminate the digestion. The cells are centrifuged at 1200 rpm for 4 min in a 15 mL centrifuge tube, the supernatant is removed, 2 mL of culture medium is added and mixed, and the cells are added to a large culture dish with culture medium to 15 mL and cultured at 37°C for 48 h. The cells are used in experiments after 2-3 generations.

[0123] (3) Cell counting and plating: Adjust the cell concentration to 1.0-2.0×10 5 cell / mL, take 2 mL to a 6-well plate for overnight culture and cell plating.

[0124] (4) Cell adhesion: After overnight culture of HeLa cells in a 6-well plate, the cells were observed under a microscope, the culture medium was removed, the cells were washed once with PBS, 2 mL of the test bacterial suspension with adjusted concentration was added, 10% (v / v) FBS was added, the cells were incubated at 37°C for 3 h, and the cells were washed four times with PBS.

[0125] (5) Competitive occupancy with Candida albicans: preventive adhesion (lactic acid bacteria were first incubated with Hela cells for 3 hours, and then Candida albicans were incubated with Hela cells for another 3 hours), competitive adhesion (lactic acid bacteria and Candida albicans were incubated with Hela cells for 3 hours), therapeutic adhesion (Candida albicans were first incubated with Hela cells for 3 hours, and then lactic acid bacteria were incubated with Hela cells for another 3 hours)

[0126] (6) Cell fixation: 0.5 mL of 4% paraformaldehyde was added to each well of the cell culture plate, fixed at room temperature for 20 min, washed three times with PBS, stained with 0.5 mL of 1 / 1000 crystal violet, washed four times with PBS, dried at room temperature, and observed under a 100× microscope to observe the adhesion of the test strain to the HeLa cells. The results are shown in the table. Figure 2 .

[0127] 2. Test results

[0128] Figure 2 The results showed that Lactobacillus crispatus LC30 had a strong adhesion ability to Hela cells, and its adhesion to Hela cells was stronger than that of Candida albicans Ca. In competitive adhesion and preventive adhesion, Lactobacillus crispatus LC30 had a stronger adhesion to Hela cells, and it could adhere to the surface of Hela cells, thereby reducing the adhesion and occupancy of Candida albicans. In therapeutic adhesion, since Candida albicans adhered first and adhered more to the cells, Lactobacillus crispatus LC30 still had a strong adhesion effect on Hela cells after being added later, occupying the adhesion sites of Candida albicans, reducing the adhesion of Ca, and reducing the damage of Ca to cells.

[0129] Example 7: Study on the chelation of iron ions by Lactobacillus crispatus LC30

[0130] 1. Test methods

[0131] (1) Preparation of lactic acid bacteria samples: Lactobacillus crispatus LC30 was cultured overnight, the fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was collected, the bacteria were washed once with PBS, and resuspended in PBS. The bacterial count was adjusted to 1×10 9 CFU / mL; inactivated bacteria were inactivated by heat inactivation at 80°C water bath for 30 min, and the bacterial count was adjusted to 1×10 9 Cells / mL;

[0132] (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 different components of Lactobacillus crispatus LC30 were 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 the protein. The mixture was centrifuged at 8000 rpm for 5 min. 0.4 mL of the 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 3 .

[0133] Fe 2+ Chelation rate % = (1-A / A0) × 100%

[0134] 2. Test results

[0135] 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 3 The results showed that the fermentation broth (containing bacteria), supernatant (not containing bacteria), live bacteria (not containing fermentation products) and inactivated bacteria (not containing fermentation products) of Lactobacillus crispatus LC30 all have the ability to chelate iron ions. The chelation ability is in the order of live bacteria > supernatant > fermentation broth > inactivated bacteria. By chelating iron ions by Lactobacillus crispatus LC30, the content of free iron ions is reduced, which helps reduce the proliferation of BV pathogenic bacteria in the body.

[0136] Example 8: Study on the inhibitory effect of Lactobacillus crispatus LC30 on pathogenic bacterial biofilm

[0137] 1. Test methods

[0138] (1) 100 μL of Ca was added to a 96-well plate. After 0, 12, and 24 h of culture, 100 μL of Lactobacillus crispatus LC30 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.

[0139] (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;

[0140] (3) Decolorize with 200 μL of anhydrous ethanol for 15 min and measure the OD value at a wavelength of 570 nm. Figure 4 ;

[0141] Inhibition rate of pathogenic bacteria biofilm % = (ODc-ODt) / ODc×100%;

[0142] Among them, ODc: OD value of negative control group; ODt: OD value of experimental group.

[0143] 2. Test results

[0144] 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.

[0145] 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. The fermentation supernatant of Lactobacillus crispatus LC30 was added at these key time points for mediation, and the biofilm formation amount and biofilm reduction rate after mediation at different periods were detected.

[0146] and Figure 4 The data results showed that in the initial adhesion stage of pathogenic bacteria, Lactobacillus crispatus LC30 had a strong inhibitory effect on vaginal Gardnerella and Candida albicans biofilms, with inhibition rates of 51% and 77% respectively. In the early formation stage, when pathogenic bacteria formed preliminary biofilms, Lactobacillus crispatus LC30 had a destructive effect on biofilms, with inhibition rates of 58% and 62% respectively. In the mature biofilm stage of pathogenic bacteria, Lactobacillus crispatus LC30 still had a strong destructive effect on biofilms, with inhibition rates of 77% and 43% respectively. Intervention with Lactobacillus crispatus LC30 in the three stages of pathogenic bacteria's planktonic strain adhesion, initial biofilm formation, and biofilm maturity can effectively reduce the formation of biofilms, thereby reducing reproductive tract infections and recurrences caused by the long-term presence of biofilms.

[0147] Example 9: Study on the antibacterial effect of Lactobacillus crispatus LC30 on the original pathogenic bacteria of vaginitis

[0148] 1. Test methods

[0149] The same as 1.1 of Example 2 was used to verify the antibacterial effect of the fermentation supernatant of Lactobacillus crispatus LC30 on native pathogens isolated from the vagina. The results are shown in Table 7.

[0150] Table 7 Antibacterial effect of Lactobacillus crispatus LC30 fermentation supernatant on vaginal pathogens

[0151]

[0152]

[0153] The six strains of Staphylococcus hominis, Klebsiella pneumoniae, Streptococcus agalactiae, Myxobacterium aureum, Streptococcus angina pectoris, and Staphylococcus haemolyticus are all original pathogens isolated from the secretions of patients with vaginitis. The results in Table 7 show that Lactobacillus crispatus LC30 has an inhibitory effect on these original pathogens of vaginitis, reducing the growth and reproduction of these pathogens, thereby alleviating vaginal inflammation.

[0154] Example 9: Study on sialidase activity of Lactobacillus crispatus LC30

[0155] 1. Test methods

[0156] 1mL BHI medium containing 10% fetal bovine serum was added to the 24-well plate, and vaginal Gardnerella was inoculated at 10% and cultured for 24h. 1mL of Lactobacillus crispatus LC30 fermentation supernatant was added to the above 24-well plate, cultured for 24h, the culture solution was centrifuged at 8000rpm5min, and the supernatant was taken for sialidase activity detection. MRS medium and BHI medium were used as controls instead of Lactobacillus crispatus LC30 fermentation supernatant. The test sample was diluted 5 times according to the instructions for detection, i.e. 10uL sample + 40uL sample diluent, and the sample was operated according to the kit instructions; the sialidase activity was expressed by the concentration of sialidase, and the results are shown in Table 8.

[0157] 2. Test results

[0158] Table 8 Sialidase activity of Lactobacillus crispatus LC30

[0159] Group Sialidase concentration ng / mL MRS control 504.34 BHI control 514.55 Lactobacillus crispatus LC30 fermentation supernatant 321.28

[0160] One of the characteristics of bacterial vaginosis (BV) is that the activity of sialidase in vaginal lavage fluid is higher than that of healthy women. This enzyme is generally believed to play a key role in the occurrence and development of BV. It destroys the mucus layer of the vaginal epithelium, a process that may increase the adhesion of pathogens to the vaginal epithelium and promote the formation of biofilms. Vaginal Gardnerella can secrete sialidase. The results in Table 8 show that LC30 can inhibit the secretion of sialidase by vaginal Gardnerella, thereby protecting the vaginal epithelial mucosa and alleviating vaginal epithelial damage.

[0161] Example 10: Study on the degradation of biogenic amines by Lactobacillus crispatus LC30

[0162] 1. Test methods

[0163] (1) Growth of biogenic amines

[0164] Vaginal simulated fluid (SVF) was prepared by adding cadaverine, putrescine and tyramine respectively, with the concentration of each biogenic amine being 100 mg / L. Lactobacillus crispatus LC30 was added to the SVF containing the three biogenic amines at 5%, 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 10.

[0165] (2) Degradation of biogenic amines

[0166] 1) Preparation of standard curve of biogenic amines

[0167] 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=79.714x+36.65, R2=0.9998; the regression equation of cadaverine is y=55.469x-53.696, R2=0.9999; the regression equation of tyramine is y=57.779x-68.315, R2=0.9998.

[0168] 2) Pre-column derivatization of biogenic amines

[0169] 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.

[0170] 3) HPLC conditions

[0171] Chromatographic separation conditions: chromatographic column: SHIMADZUC18-AQ (5μm, 250mm×4.6mm); mobile phase A: water; mobile phase B: acetonitrile; injection volume: 20μL; column temperature: 35℃; UV detection wavelength: 254nm, gradient elution program: see Table 9.

[0172] Table 9 HPLC gradient elution program

[0173]

[0174]

[0175] 4) Degradation of biogenic amines by Lactobacillus crispatus LC30

[0176] Lactobacillus crispatus LC30 was added to the vaginal simulated fluid (SVF) containing three kinds of biogenic amines (each biogenic amine concentration was 100 mg / L) at 5%, and the one with MRS was used as the control group. The culture was carried out at 37°C for 24 hours, and the bacterial suspension was centrifuged at 8000 rpm for 10 minutes. 1 mL of the supernatant was collected and added with 4 mL of 0.1 mol / L HCl to mix well 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 5 .

[0177] Degradation effect of biogenic amines: Biogenic amine degradation rate % = (W-W1) / W×100%,

[0178] W is the content of biogenic amines in the control group, and W1 is the content of biogenic amines in the experimental group.

[0179] 2. Test results

[0180] 1. Growth of Lactobacillus crispatus LC30 in simulated vaginal fluid (SVF) containing biogenic amines

[0181] Table 10: Growth of Lactobacillus crispatus LC30 in SVF containing biogenic amines

[0182] Cultivation time OD value pH Viable bacteria count CFU / mL 0h 0.218 5.36 6.50E+07 12h 0.384 4.13 2.01E+08 24h 0.501 3.88 2.97E+08

[0183] From the data in Table 10, it can be seen that Lactobacillus crispatus LC30 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.

[0184] 2. Degradation of biogenic amines by Lactobacillus crispatus LC30

[0185] Figure 5The results showed that Lactobacillus crispatus LC30 had different degrees of degradation effects on putrescine, cadaverine and tyramine, three biogenic amines. By reducing the content of these three biogenic amines, it reduced the production of odor, while reducing the pH increase caused by biogenic amines and alleviating the symptoms of vaginitis caused by biogenic amines.

[0186] Example 11: Study on the effect of Lactobacillus crispatus LC30 on cytokine secretion

[0187] 1. Test methods

[0188] (1) Preparation of live bacterial suspension

[0189] 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.

[0190] (2) Preparation of inactivated bacterial suspension

[0191] 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.

[0192] (3) Cell culture

[0193] 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.

[0194] (4) Determination of the effect of strains on the secretion of cellular immune factors

[0195] 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.

[0196] The experimental groups are as follows:

[0197] 1) Negative control group: MEM was added to the wells and incubated for 4 h and 24 h respectively;

[0198] 2) Positive control group (Ca): Candida albicans was added to the wells and incubated for 4 h and 24 h respectively;

[0199] 3) LC30 test group: live bacterial suspension or inactivated bacterial suspension of strain LC30 was added to the wells, and the live bacterial group was incubated for 4 hours, and the inactivated group was incubated for 12 hours;

[0200] 4) LC30 competition group (strain LC30+Ca): Ca and live bacterial suspension or inactivated bacterial suspension of strain LC30 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;

[0201] 5) LC30 treatment group (C.a+ strain LC30): Ca was first added to each well and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria), and then the live bacterial suspension or inactivated bacterial suspension of strain LC30 was added. The live bacteria group was incubated for 4 h, and the inactivated group was incubated for 12 h;

[0202] 6) LC30 prevention group (strain LC30+Ca): strain LC30 bacterial suspension was first added to each well, incubated for 4 h in the live bacteria group and 12 h in the inactivated group, and then Ca was added and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria);

[0203] 7) 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 11-12.

[0204] 2. Test results

[0205] 1. Effect on IL-6:

[0206] Table 11 Effects of live and inactivated bacteria on IL-6 secretion by HeLa

[0207]

[0208]

[0209] IL-6 can regulate the growth and differentiation of a variety of cells, regulate immune responses, acute phase reactions and hematopoiesis, and plays an important role in the body's anti-infection immune response. IL-6 has significant changes in a variety of diseases, and its dysregulated expression can cause many diseases. In patients with vaginitis, the main clinical manifestation is an increase in IL-6 levels during the onset of the disease. From the data in Table 11, it can be seen that the live and inactivated bacteria of LC30 incubated with Hela cells alone can promote the secretion of IL-6, cause physiological inflammatory reactions, and improve the body's immunity. Candida albicans Ca, as a pathogenic bacterium of vaginitis, can stimulate Hela cells to secrete a large amount of IL-6 and increase the level of inflammatory factors. After LC30 intervention, the effect of Ca on Hela cells can be reduced, and the secretion of IL-6 can be reduced. In particular, the effect of the live bacteria groups in inhibiting inflammation is relatively good.

[0210] 2. Effect on IL-8:

[0211] Table 12 Effects of live and inactivated bacteria on IL-8 secretion by HeLa

[0212]

[0213] 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.

[0214] The data in Table 12 show that the live and inactivated bacteria of LC30 incubated with cells alone can promote the secretion of IL-8, induce physiological inflammatory response, and improve the body's immunity. As a pathogenic bacterium of vaginitis, Candida albicans Ca can stimulate the secretion of IL-8 and increase the level of inflammatory factors. After intervention with LC30, the effect of Ca on Hela cells can be reduced, and the secretion of IL-8 can be reduced. In particular, the effect of the live bacteria groups in inhibiting inflammation is relatively good.

[0215] 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 Lactobacillus crispatus LC30, characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the accession number CGMCC NO.31923.

2. A Lactobacillus crispatus LC30 according to claim 1, characterized in that The Lactobacillus crispatus LC30 is derived from the reproductive tract secretions of healthy women.

3. A microbial agent, characterized in that: The method comprises one or more of the live bacteria of Lactobacillus crispatus LC30, the inactivated bacteria of Lactobacillus crispatus LC30, and the fermentation product of Lactobacillus crispatus LC30 as claimed in claim 1.

4. Use of the Lactobacillus crispatus LC30 according to claim 1 or the microbial agent according to claim 2 in preparing a product for preventing and treating female reproductive tract pathogenic bacteria infection, preventing and treating vaginitis, and / or removing genital odor.

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

6. The use according to claim 4, characterized in that: The Lactobacillus crispatus LC30 or microbial agent has one or more of the following effects: 1) Lactobacillus crispatus LC30 or microbial agents can produce hydrogen peroxide; 2) Lactobacillus crispatus LC30 or microbial agents have antibacterial effects on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Staphylococcus hominis, Klebsiella pneumoniae, Streptococcus agalactiae, Corynebacterium aureus, Streptococcus anginae, and Staphylococcus pyogenes; 3) Lactobacillus crispatus LC30 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae; 4) Lactobacillus crispatus LC30 or microbial agents can inhibit the biofilm formation of Gardnerella vaginalis and / or Candida albicans at different stages; 5) Lactobacillus crispatus LC30 or microbial agents can reduce the hydrophobicity of Candida albicans, reduce the adhesion of Candida albicans, and inhibit the formation of biofilm; 6) Lactobacillus crispatus LC30 or microbial agents can chelate iron ions; 7) Lactobacillus crispatus LC30 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage; 8) Lactobacillus crispatus LC30 or microbial agents can grow in an environment containing biogenic amines and can degrade biogenic amines; 9) Lactobacillus crispatus LC30 or microbial agents can reduce the secretion of inflammatory factors IL-6 and IL-8.

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, hygiene products, and family planning products.

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