Application of lactobacillus gasseri LG20 in female private part health maintenance

Abstract

The invention relates to an application of lactobacillus gasseri LG20 in health maintenance of female private parts, the lactobacillus gasseri LG20 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.32035. The lactobacillus gasseri LG20 has good hydrogen peroxide production capability and adhesion capability, has a relatively strong inhibition effect on main vaginitis pathogenic bacteria, and can be used for preparing a health care product for female private parts. The bactericide is effective to nutritional pathogenic bacteria, and can also reduce the form transformation and the formation of biological membranes and prevent the generation of 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 pudendum 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 the application of Lactobacillus gasseri LG20 in maintaining the health of female private parts. Background Art

[0002] Female private health is an important area related to women's overall health and quality of life. Due to the particularity of women's physiological structure, the private parts are easily affected by various factors, such as bacterial and fungal infections, microecological imbalance and other problems. Common diseases include vaginitis, vulvitis, cervicitis, etc. These problems not only bring physical discomfort to women, such as itching, odor, abnormal secretions, etc., but may also have a negative impact on their psychology and life. There are many kinds of female private care products on the market, including lotions, suppositories, etc. However, most of these products have certain limitations. Chemical lotions may destroy the normal balance of flora in the private parts, and long-term use may lead to dysbiosis and increase the risk of infection. Some suppositories are effective in treating specific diseases, but have limited effects on preventing and maintaining the overall healthy environment of the private parts, and some products may have side effects or cause allergic reactions.

[0003] The relationship between microorganisms and human health has received increasing attention, especially in the microecology of the female private parts, where beneficial microorganisms are essential for maintaining a healthy and balanced environment. Preventing and improving private health problems by regulating the microbial community has gradually become a research hotspot. Lactic acid bacteria can interact with the epithelial cells of the private mucosa, and prevent the adhesion and colonization of harmful bacteria on the mucosal surface through mechanisms such as space protection and competition for nutrients. Moreover, lactic acid bacteria can also stimulate the mucosal immune system and induce the production of immune factors, thereby enhancing the immune function of the private mucosa, further improving the defense ability of the private parts, and providing a new, natural and safe way to maintain the health of the female private parts, which has broad application prospects. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide an application of Lactobacillus gasseri LG20 in maintaining the health of female private parts, which can not only effectively prevent and treat private part pathogenic bacteria infection and vaginitis, but also remove private part odor and maintain the health of female private parts.

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

[0006] A Lactobacillus gasseri LG20, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.32035, the deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the deposit time is September 23, 2024.

[0007] As a further technical solution, the Lactobacillus gasseri LG20 is derived from the reproductive tract secretions of healthy women.

[0008] A microbial agent comprises one or more of the live bacteria of Lactobacillus gasseri LG20, the inactivated bacteria of Lactobacillus gasseri LG20, and the fermentation product of Lactobacillus gasseri LG20.

[0009] The Lactobacillus gasseri LG20 or the microbial agent is used in preparing products for preventing and treating genital tract pathogenic bacteria infection, preventing and treating vaginitis and / or removing genital odor and maintaining the health of female private parts.

[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, and Streptococcus angina pectoris.

[0011] As a further technical solution, the Lactobacillus gasseri LG20 or microbial agent has one or more of the following effects;

[0012] 1) Lactobacillus gasseri LG20 or microbial agents can produce hydrogen peroxide;

[0013] 2) Lactobacillus gasseri LG20 or microbial agents have antibacterial effects on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, Staphylococcus hemolyticus, and Streptococcus angina pectoris;

[0014] 3) Lactobacillus gasseri LG20 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae;

[0015] 4) Lactobacillus gasseri LG20 or microbial agents can inhibit the biofilm formation of Gardnerella vaginalis and / or Candida albicans at different stages;

[0016] 5) Lactobacillus gasseri LG20 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 gasseri LG20 or microbial agents have strong adhesion to Hela cells and can compete with Candida albicans for adhesion to Hela cells;

[0018] 7) Lactobacillus gasseri LG20 or microbial agents can chelate iron ions;

[0019] 8) Lactobacillus gasseri LG20 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage;

[0020] 9) Lactobacillus gasseri LG20 or microbial agents can grow in an environment containing biogenic amines and can degrade biogenic amines;

[0021] 10) Lactobacillus gasseri LG20 or microbial agents can reduce the secretion of inflammatory factors IL-6 and IL-8.

[0022] As a further technical solution, the product includes an oral product or an external product;

[0023] As a further technical solution, the oral product includes food or health care products; such as powders, granules, tablets, yogurt, candy, beverages, etc.

[0024] As a further technical solution, the external-use products include one or more of external-use medicines, sanitary products, and family planning products.

[0025] The sanitary products include sanitary napkins, panty liners, wet wipes, tampons, and antibacterial / antibacterial lotions; family planning products include condoms, lubricants, etc.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1) The Lactobacillus gasseri LG20 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.

[0028] 2) The Lactobacillus gasseri LG20 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 Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, hemolytic Staphylococcus, and Streptococcus angina pectoris, and can be used to prevent and treat female vaginal pathogen infection.

[0029] 3) Lactobacillus gasseri LG20 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.

[0030] 4) Lactobacillus gasseri LG20 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.

[0031] 5) Lactobacillus gasseri LG20 has a strong adhesion ability to Hela cells and can compete with Candida albicans for adhesion to Hela cells;

[0032] 6) Iron ions can promote the growth of vaginal Gardnerella, while Lactobacillus gasseri LG20 can chelate iron ions and reduce the iron ion content, thereby reducing the growth and reproduction rate of vaginal Gardnerella.

[0033] 7) Lactobacillus gasseri LG20 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage;

[0034] 8) Lactobacillus gasseri LG20 can grow in an environment containing biogenic amines and can use biogenic amines as a nitrogen source for growth and reproduction, thereby achieving the effect of degrading biogenic amines and removing vaginal odor.

[0035] 9) The fermentation products of Lactobacillus gasseri LG20 can reduce the secretion of inflammatory factors IL-6 and IL-8, thereby achieving the purpose of preventing and treating vaginal inflammation.

[0036] 10) The Lactobacillus gasseri LG20 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 genital topical medicines, and can even be used in sanitary products and family planning products, such as sanitary napkins, female antibacterial / antibacterial lotions, condoms, etc.

[0037] In summary, the Lactobacillus gasseri LG20 screened by the present invention has good hydrogen peroxide production ability and adhesion ability, has a strong inhibitory effect on major 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 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 vulvar pathogenic bacteria infection, preventing and treating vaginitis and / or removing vulvar odor, and maintaining the health of female private parts. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a diagram showing the effect of Lactobacillus gasseri LG20 producing hydrogen peroxide;

[0039] Figure 2 This is a diagram showing the adhesion effect of Lactobacillus gasseri LG20 and Candida albicans on Hela cells;

[0040] exist Figure 2 Middle, a: blank cell control group, b: LG20 adhesion; c: Ca adhesion;

[0041] Figure 3 This is a diagram showing the competitive adhesion effect of Lactobacillus gasseri LG20 and Candida albicans on Hela cells;

[0042] exist Figure 3 In the figure, a: competitive adhesion of LG20 to Candida albicans; b: preventive adhesion of LG20 to Candida albicans; c: therapeutic adhesion of LG20 to Candida albicans;

[0043] Figure 4 This is a graph showing the effect of Lactobacillus gasseri LG20 on the hyphae formation ability of Candida albicans;

[0044] exist Figure 4 In, a: MRS, 3h; b: LG20, 3h; c: MRS, 6h; d: LG20, 6h;

[0045] Figure 5 This is a diagram showing the chelation effect of Lactobacillus gasseri LG20 on iron ions;

[0046] Figure 6 This is a diagram showing the degradation effect of Lactobacillus gasseri LG20 on biogenic amines. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] Example 1: Isolation of Lactobacillus gasseri LG20

[0050] Take a sample of secretions from a healthy woman and place it in an MRS medium for anaerobically culture. Take 1 mL of the culture solution and dilute it with 9 mL of sterile saline solution, 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 strain with consistent colony and bacterial morphology for preservation.

[0051] Example 2, strain screening

[0052] The strain LG20 with milky white colonies, round protrusions, neat edges, smooth surface, rod-shaped, Gram-positive, and catalase-negative under a microscope was taken for 16S rRNA gene identification, and its nucleotide sequence is shown in SEN ID NO.1; the identification results showed that the strain LG20 was Lactobacillus gasseri, so it was named Lactobacillus gasseri LG20, and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.32035, and 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 23, 2024.

[0053] Example 3: Hydrogen peroxide production capacity and antibacterial test of Lactobacillus gasseri LG20

[0054] 1. Test on the inhibitory ability of Escherichia coli

[0055] 1.1 Test methods

[0056] (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;

[0057] (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.

[0058] (3) Lactobacillus gasseri LG20 was inoculated into MRS liquid medium at 3% and cultured at 37°C for 24 h. The fermentation liquid 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 1.

[0059] 1.2 Test results

[0060] Table 1 The antibacterial results of Lactobacillus gasseri LG20 on Escherichia coli

[0061]

[0062] Note: Since the hole of the Oxford cup has a diameter of 8mm, only the antibacterial zone diameter ≥ 8mm indicates an antibacterial effect.

[0063] The data results in Table 1 show that the antibacterial ability of the fermentation supernatant of Lactobacillus gasseri LG20 on Escherichia coli and Staphylococcus aureus was studied by double-layer agar diffusion method. The results in Table 1 show that Lactobacillus gasseri LG20 has good antibacterial effects on both Escherichia coli and Staphylococcus aureus, and MRS as a control has no inhibitory effect.

[0064] 2. Hydrogen peroxide production capacity of Lactobacillus gasseri LG20

[0065] 2.1 Experimental methods

[0066] (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.

[0067] (2) Streak the bacterial liquid of Lactobacillus gasseri LG20 on a TMB-HRP-MRS plate and culture it anaerobically at 37°C for 48 hours. Then expose the 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 (-).

[0068] 2.2 Experimental Results

[0069] The hydrogen peroxide produced by lactic acid bacteria has a strong antibacterial effect and can inhibit Gram-negative bacteria and catalase-positive bacteria such as Pseudomonas, Escherichia coli and Salmonella. Since 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized by hydrogen peroxide and horseradish peroxidase (HRP), a blue product is formed. The higher the hydrogen peroxide content, the darker the blue color. Figure 1 The results showed that the ability of Lactobacillus gasseri LG20 to produce hydrogen peroxide was positive.

[0070] 3. Experiment on inhibition of Candida albicans by Lactobacillus gasseri LG20

[0071] 3.1 Experimental methods

[0072] (1) The fermentation broth of Lactobacillus gasseri LG20 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 a selective YPD medium;

[0073] (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.

[0074] (3) Calculate the inhibitory ability of lactic acid bacteria on Candida albicans. The results are shown in Table 3:

[0075] Candida albicans inhibition rate A=(A 0 -A t ) / A 0 × 100%, where A t represents the number of Ca viable bacteria in each experimental group after 12h and 24h of culture, A 0 Represents the number of Ca viable bacteria after 0h;

[0076] 3.2 Experimental Results

[0077] Table 2 Antibacterial effect of Lactobacillus gasseri LG20 on Candida albicans

[0078] Species 12h inhibition rate 24h inhibition rate Lactobacillus gasseri LG20 71.68％ 63.80％ pH4.0MRS 0.00±0.00 0.00±0.00 pH6.8MRS 0.00±0.00 0.00±0.00

[0079] Candida albicans is the main pathogen of candidal vaginitis. The results in Table 2 show that Lactobacillus gasseri LG20 has a good inhibitory effect on Candida albicans, while MRS at pH 4.0 and pH 6.8 cannot inhibit the growth of Candida albicans.

[0080] 4. Test on the inhibitory ability of Lactobacillus gasseri LG20 on vaginal Gardnerella

[0081] 4.1 Experimental methods

[0082] (1) Lactobacillus gasseri LG20 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 / min for 10 min.

[0083] (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;

[0084] (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 3.

[0085] 4.2 Experimental Results

[0086] Table 3 Antibacterial effect of Lactobacillus gasseri LG20 on vaginal Gardnerella

[0087] Species Diameter of inhibition zone (unit: mm) Lactobacillus gasseri LG20 21.57 MRS 8.00

[0088] Gardnerella vaginalis is the main pathogenic bacteria of vaginitis. The results in Table 3 show that Lactobacillus gasseri LG20 has a good inhibitory effect on Gardnerella vaginalis, while MRS has no inhibitory effect on Gardnerella vaginalis.

[0089] Example 4: Study on the hydrophobicity of Lactobacillus gasseri LG20

[0090] 1. Experimental Methods

[0091] The overnight cultured Lactobacillus gasseri LG20 was centrifuged at 5000 rpm for 3 min, the bacterial sludge was collected and washed once with PBS, then resuspended in PBS, and the OD value was measured at a wavelength of 600 nm to be 0.7-0.8, recorded as A0. The LG20 bacterial suspension was mixed with xylene, chloroform, and ethyl acetate in a ratio of 3:1, respectively, and fully shaken and mixed for 30 s, allowed to stand for 10 s, shaken and mixed for 30 s, allowed to stand for 30 min, and after the aqueous phase and the organic phase were separated, the OD value of the aqueous phase at 600 nm was measured, recorded as A, and the hydrophobicity of the Lactobacillus gasseri LG20 cell surface was calculated. The results are shown in Table 4.

[0092] Cell surface hydrophobicity % = (1-A / A0) × 100%;

[0093] Hydrophobicity standard: more than 50% is highly hydrophobic, between 20%-50% is moderately hydrophobic, and less than 20% is non-hydrophobic.

[0094] 2. Experimental Results

[0095] Table 4 Hydrophobicity of Lactobacillus gasseri LG20

[0096] Organic phase Hydrophobicity Xylene 77.29％ Chloroform 85.02％ Ethyl acetate 68.77％

[0097] An important prerequisite for lactic acid bacteria to produce beneficial 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 4 show that Lactobacillus gasseri LG20 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.

[0098] Example 5: Hydrophobicity of Candida albicans mediated by Lactobacillus gasseri LG20

[0099] 1. Experimental Methods

[0100] (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.

[0101] (2) Take 4.5 ml of the Candida albicans solution, add 4.5 ml of Lactobacillus gasseri LG20 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.

[0102] (3) The above bacterial suspension was adjusted to a concentration where the absorbance at 560 nm was about 1.000, which was recorded as A 0 3.0 mL of bacterial suspension was mixed with 0.6 mL of xylene, ethyl acetate and chloroform respectively, 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 water 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 was taken. The results are shown in Table 5.

[0103] Hydrophobicity % = (1-A / A 0 )×100%

[0104] The specific standards for the hydrophobicity are as follows: >50% is highly hydrophobic, between 20% and 50% is moderately hydrophobic, and <20% is non-hydrophobic.

[0105] 2. Experimental Results and Analysis

[0106] Table 5 Effect on the hydrophobicity of Candida albicans

[0107] Xylene Ethyl acetate Chloroform Lactobacillus gasseri LG20 55.43％ 10.71％ 22.86％ MRS 79.39％ 32.57％ 38.00％

[0108] Hydrophobicity is a nonspecific adhesion mechanism, which is related to the ability of strains to adhere and colonize in the reproductive tract. By comparing the data in Table 5, it can be seen that the fermentation supernatant of Lactobacillus gasseri LG20 can reduce the hydrophobicity of Candida albicans, indicating that its fermentation supernatant product can reduce the ability of Candida albicans to nonspecifically adhere to the reproductive tract, and can significantly affect the colonization of Candida albicans in the reproductive tract.

[0109] Example 6: Ability of Lactobacillus gasseri LG20 to inhibit biofilm formation of pathogenic bacteria

[0110] 1. Experimental Methods

[0111] (1) 100 μL of vaginal Gardnerella or Candida albicans was added to a 96-well plate. After culturing for 0, 12, and 24 h, 100 μL of Lactobacillus gasseri LG20 fermentation supernatant was added (8000 rpm for 10 min) 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.

[0112] (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;

[0113] (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 biofilm. The results are shown in Table 6-7;

[0114] Biofilm inhibition rate (%) = (ODc-ODt) / ODc×100%.

[0115] Among them, ODc: OD value of negative control group; ODt: OD value of experimental group.

[0116] 2. Experimental Results and Analysis

[0117] The biofilm in the vagina of healthy women is thin and loose, while women with vaginitis tend to have thick biofilm. Biofilm is a multicellular, multi-layered three-dimensional structure that is highly resistant to antimicrobial agents and host defenses. The physical structure of the biofilm helps pathogens evade many negative stimuli and increases the ability to resist antimicrobial substances (such as H 2 O 2 During conventional antibiotic treatment, the patient's clinical symptoms disappear in a short period of time, but after stopping the drug, a dense and active biofilm quickly appears on the vaginal mucosa, leading to persistent infection or recurrence of vaginitis.

[0118] 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 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.

[0119] (1) Experimental results on inhibition of vaginal Gardnerella biofilm

[0120] Table 6 Inhibitory effect on vaginal Gardnerella biofilm

[0121] strain 0h 12h 24h Lactobacillus gasseri LG20 57.17％ 62.61％ 69.84％ Chlorhexidine acetate 48.03％ 67.87％ 65.25％

[0122] Table 6 shows that Lactobacillus gasseri LG20 can significantly inhibit the formation of vaginal Gardnerella biofilm at different stages from initial adhesion to biofilm maturity, and destroy the mature pathogenic bacteria biofilm.

[0123] (2) Experimental results on inhibition of Candida albicans biofilm

[0124] Table 7 Inhibitory effect on Candida albicans biofilm

[0125] strain 0h 12h 24h Lactobacillus gasseri LG20 76.41％ 71.57％ 69.25％ Chlorhexidine acetate 82.34％ 64.65％ 62.22％

[0126] The results in Table 7 show that Lactobacillus gasseri LG20 can significantly inhibit the formation of Candida albicans biofilm and destroy the mature pathogenic bacteria biofilm in the initial adhesion stage, formation stage and maturity stage of Candida albicans.

[0127] In summary, the present invention can utilize the destructive effect of Lactobacillus gasseri LG20 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.

[0128] Example 7: Competitive Adhesion Effect of Lactobacillus gasseri LG20 and Candida albicans

[0129] 1. Experimental Methods

[0130] (1) Preparation of test strain suspension: The activated Lactobacillus gasseri LG20 or Candida albicans was centrifuged at 5000 rpm for 3 min to collect the cells, washed twice with PBS, and the bacterial suspension concentration was adjusted to 1.5×10 8 CFU / mL and place at 4℃ for later use.

[0131] (2) Cell counting and plating: After the HeLa cells were cultured, the culture medium was removed and the cells were washed once with PBS. 2 mL of trypsin was added and the cells were allowed to stand at room temperature for 3 min. After microscopic examination, the cells became round and then aspirated. After the cells were suspended, 2 times the volume of cell culture medium was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 4 min in a 15 mL centrifuge tube. The supernatant was removed and the culture medium was added to mix well for cell counting. The cell concentration was adjusted to 1.5 × 10 5 cell / mL, take 2 mL to a 6-well plate for overnight culture and cell plating.

[0132] (3) Cell adhesion: After the HeLa cells were cultured overnight in a 6-well plate, they were observed under a microscope, the culture medium was removed, the cells were washed once with PBS, 2 mL of the adjusted concentration of Lactobacillus gasseri LG20 or Candida albicans suspension was added, 10% (v / v) FBS was added, the cells were incubated at 37°C for 3 h, and the cells were washed 4 times with PBS. In the competitive adhesion experiment, 1 mL of the adjusted concentration of Lactobacillus gasseri LG20 suspension and 1 mL of the Candida albicans suspension were added to the 6-well plate at the same time, 10% (v / v) FBS was added, the cells were incubated at 37°C for 3 h, and the cells were washed 4 times with PBS. In the preventive adhesion experiment, 1 mL of the adjusted concentration of Lactobacillus gasseri LG20 suspension was added to the 6-well plate, the cells were incubated at 37°C for 3 h, and then 1 mL of the Candida albicans suspension was added, the cells were incubated at 37°C for 3 h, and the cells were washed 4 times with PBS. In the therapeutic adhesion experiment, 1 mL of Candida albicans suspension with adjusted concentration was added to the 6-well plate and incubated at 37°C for 3 h, then 1 mL of Lactobacillus gasseri LG20 suspension was added, incubated at 37°C for 3 h, and washed 4 times with PBS.

[0133] (4) 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 1-2 .

[0134] 2. Experimental Results

[0135] 1. Adhesion of Lactobacillus gasseri LG20 and Candida albicans to Hela cells

[0136] Figure 2 The results showed that Lactobacillus gasseri LG20 had a stronger adhesion to Hela cells, and Candida albicans had a certain adhesion to Hela cells.

[0137] 2. Competitive adhesion of Lactobacillus gasseri LG20 and Candida albicans to Hela cells

[0138] Lactobacillus gasseri LG20 and Candida albicans Ca have competitive adhesion, preventive adhesion and therapeutic adhesion. Figure 3 The results showed that the adhesion of Lactobacillus gasseri LG20 to Hela cells was stronger than that of Candida albicans Ca. In competitive adhesion and preventive adhesion, Lactobacillus gasseri LG20 had a stronger adhesion to Hela cells, and it could adhere to the surface of Hela cells, thereby reducing the adhesion and occupation of Candida albicans. In therapeutic adhesion, since Candida albicans adhered first and adhered more to the cells, the subsequent addition of Lactobacillus gasseri LG20 still had an adhesion effect on Hela cells, occupying the adhesion sites of Candida albicans, reducing the adhesion of Candida albicans, and thus reducing the damage of Candida albicans to cells.

[0139] Example 8: Antibacterial effect of Lactobacillus gasseri LG20 on native pathogenic bacteria

[0140] 1. Experimental Methods

[0141] (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;

[0142] (2) The activated clinical pathogenic bacteria are added to a nutrient agar medium (containing 0.8% agar concentration) and mixed evenly. 10 mL is added to a culture dish. After the culture medium solidifies, the Oxford cup is taken out to form an Oxford cup hole.

[0143] (3) Lactobacillus gasseri LG20 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.

[0144] 2. Experimental Results and Analysis

[0145] Table 8 Antibacterial results of Lactobacillus gasseri LG20 on native pathogenic bacteria

[0146]

[0147] The results in Table 8 show that Lactobacillus gasseri LG20 has an inhibitory effect on six strains of native pathogenic bacteria, including Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, Staphylococcus hemolyticus, and Streptococcus angina pectoris.

[0148] Example 9: Effect of Lactobacillus gasseri LG20 on sialidase activity

[0149] 1. Experimental Methods

[0150] (1) Vaginal Gardnerella culture

[0151] 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.

[0152] (2) Lactobacillus gasseri LG20 intervention

[0153] 1 mL of Lactobacillus gasseri LG20 fermentation supernatant was added to the above 24-well plate, cultured for 24 h, the culture solution was centrifuged at 8000 rpm for 5 min, and the supernatant was taken for sialidase activity detection. MRS medium and BHI medium were used as controls instead of lactic acid bacteria fermentation supernatant.

[0154] (3) Sialidase detection

[0155] The samples were operated according to the instructions of the kit, and the results are shown in Table 9;

[0156] 2. Experimental Results and Analysis

[0157] Table 9: Effect of Lactobacillus gasseri LG20 on sialidase activity

[0158] Group Sialidase concentration ng / mL MRS control 504.34 BHI control 514.55 Lactobacillus gasseri LG20 423.80 blank ------

[0159] 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.

[0160] The results in Table 9 show that vaginal Gardnerella has the ability to secrete sialidase, and its sialidase content reaches 500 ng / mL after 48 h of culture. Compared with the control group, the fermentation supernatant of Lactobacillus gasseri LG20 can inhibit the secretion of sialidase by vaginal Gardnerella, and the content of sialidase in the sample is only 423.80 ng / mL, which can reduce its activity, thereby playing a role in slowing down the damage to the vaginal epithelial mucosa.

[0161] Example 10: Lactobacillus gasseri LG20 interferes with the morphological transformation ability of Candida albicans

[0162] 1. Experimental Methods

[0163] (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;

[0164] (2) centrifuging Lactobacillus gasseri LG20 at 8000 rpm for 10 min, collecting the supernatant, taking 1 mL and adding it to the above 24-well plate and mixing;

[0165] (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 Lactobacillus gasseri LG20;

[0166] (4) After incubation at 37°C for 3 h and 6 h, 10 μL was taken on a glass slide and the growth of Candida albicans hyphae was observed under a microscope. The number of hyphae and yeast in the field of view was counted, the proportion of hyphae was calculated, and the hyphae length in μm was counted. The results are shown in Tables 10 and Figure 4 ;

[0167] 2. Experimental Results and Analysis

[0168] Table 10 Effect of Lactobacillus gasseri LG20 on the hyphae formation ability of Candida albicans

[0169]

[0170] Table 10 and Figure 4 The results showed that when Lactobacillus gasseri LG20 was co-cultured with Candida albicans for 3h and 6h, Lactobacillus gasseri LG20 could inhibit the formation of Candida albicans hyphae, reduce the hyphae ratio, and reduce the hyphae length of Candida albicans, thereby reducing the toxicity of Candida albicans and the damage to the body.

[0171] Example 11: Regulation of Hela cell inflammatory factors by Lactobacillus gasseri LG20

[0172] 1. Experimental methods:

[0173] 1. Strain activation, enrichment and cell culture

[0174] (1) Preparation of live bacterial suspension

[0175] 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.

[0176] (2) Preparation of inactivated bacterial suspension

[0177] 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.

[0178] (3) Cell culture

[0179] Hela cells were revived in MEM complete culture medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO 2 Incubate in a cell culture incubator, and subculture when the cells are in good growth condition and the density reaches about 80%. Follow-up experiments are performed after three subcultures.

[0180] 2. Determination of the effect of strains on the secretion of cellular immune factors

[0181] 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.

[0182] The experimental groups are as follows:

[0183] (1) Negative control group (MEM): MEM was added to the wells and incubated for 4 h and 24 h respectively;

[0184] (2) Positive control group (Ca): Candida albicans was added to the wells and incubated for 4 h and 24 h, respectively;

[0185] (3) Test strain group (LG20): LG20 live bacterial suspension or inactivated bacterial suspension was added to the wells. The live bacterial group was incubated for 4 h, and the inactivated group was incubated for 12 h.

[0186] (4) Competition group (LG20+Ca): Candida albicans and LG20 live bacterial suspension or inactivated bacterial suspension were added to each well at the same time. The live bacterial group was incubated for 4 h, and the inactivated group was incubated for 12 h.

[0187] (5) Treatment group (Ca+LG20): Candida albicans was first added to each well and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria), and then LG20 live bacteria suspension or inactivated bacteria suspension was added. The live bacteria group was incubated for 4 h, and the inactivated group was incubated for 12 h;

[0188] (6) Prevention group (LG20+Ca): LG20 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 Candida albicans was added and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria);

[0189] The cell culture supernatant was collected, centrifuged at 8000 r / min for 10 min, and then dispensed into sterile centrifuge tubes and stored at -80°C for later use. The secretion of IL-6 and IL-8 cytokines in the above different groups was determined according to the instructions of the ELISA kit. The results are shown in Tables 11-12.

[0190] 2. Experimental Results and Analysis

[0191] 1. Experimental results of IL-8

[0192] Table 11 Results of IL-8 secretion by live and dead Lactobacillus gasseri LG20 on Hela cells

[0193]

[0194] IL-8 is a common inflammatory chemokine that can activate and aggregate inflammatory cells, thereby promoting inflammatory responses. 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.

[0195] From the results in Table 11, 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 IL-8; compared with the negative control group, the co-incubation of the Lactobacillus gasseri LG20 live bacteria group with cells has little effect on the secretion of IL-8. In the competition group, Lactobacillus gasseri LG20 live bacteria and Candida albicans Ca were co-incubated with cells at the same time, which significantly reduced the secretion of IL-8 compared with the positive control. In the treatment group, Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then LG20 live bacteria were added for incubation. The secretion of IL-8 was significantly lower than that of Candida albicans Ca alone. In the prevention group, Lactobacillus gasseri LG20 live bacteria were first co-incubated with cells, and then Candida albicans Ca was added for incubation, which significantly reduced the secretion of IL-8 compared with the positive control.

[0196] The results of the inactivated bacteria group test showed that compared with the negative control group, inactivated Lactobacillus gasseri LG20 bacteria co-incubated with cells could promote the secretion of cytokine IL-8 and cause physiological inflammatory response. In the competition group, inactivated bacteria and Candida albicans Ca were co-incubated with cells at the same time. Compared with the positive control, Lactobacillus gasseri LG20 could significantly reduce the secretion of IL-8. In the treatment group, Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then inactivated Lactobacillus gasseri LG20 was added for incubation. The IL-8 secretion was lower than that of Candida albicans Ca alone. In the prevention group, inactivated Lactobacillus gasseri LG20 bacteria were first co-incubated with cells, and then Candida albicans Ca was added for incubation. Compared with the positive control, the IL-8 secretion was lower than that of Candida albicans Ca alone.

[0197] 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 Lactobacillus gasseri LG20 can reduce the secretion of IL-8 and improve the inflammatory state of cells.

[0198] 2. Experimental results of IL-6

[0199] Table 12 Results of IL-6 secretion by live and dead Lactobacillus gasseri LG20 on Hela cells

[0200]

[0201] 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.

[0202] From the results in Table 12, it can be seen that the test results of the live bacteria group showed that in the positive control group, Candida albicans Ca could stimulate Hela cells to secrete a large amount of IL-6; compared with the negative control group, the co-incubation of the Lactobacillus gasseri LG20 live bacteria group with cells had little effect on the secretion of IL-6. In the competition group, LG20 live bacteria and Candida albicans Ca were co-incubated with cells at the same time, which significantly reduced the secretion of IL-6 compared with the positive control. In the treatment group, Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then LG20 live bacteria were added for incubation. The IL-6 secretion was lower than the secretion of Candida albicans Ca alone. In the prevention group, LG20 live bacteria were first co-incubated with cells, and then Candida albicans Ca was added for incubation, which could reduce the secretion of IL-6 compared with the positive control.

[0203] The results of the inactivated bacteria group test showed that compared with the negative control group, the inactivated bacteria LG20 co-incubated with cells could stimulate the secretion of cytokine IL-6 and cause a physiological inflammatory response. In the competition group, the inactivated bacteria and Candida albicans Ca were co-incubated with cells at the same time. Compared with the positive control, LG20 could reduce the secretion of IL-6. In the treatment group, Candida albicans Ca was first co-incubated with cells to create a cell inflammation model, and then inactivated bacteria LG20 was added for incubation. The IL-6 secretion was lower than that of Candida albicans Ca alone. In the prevention group, inactivated bacteria LG20 was first co-incubated with cells, and then Candida albicans Ca was added for incubation. The IL-6 secretion was lower than that of Candida albicans Ca alone.

[0204] 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 LG20 bacteria can reduce the secretion of IL-6 and improve the inflammatory state of cells.

[0205] Example 12: Chelation of iron ions by Lactobacillus gasseri LG20

[0206] 1. Test methods

[0207] (1) Lactobacillus gasseri LG20 was cultured overnight and centrifuged at 8000 rpm for 10 min to collect the supernatant. The cells were washed once with PBS and resuspended in PBS. The cell count was adjusted to 1×10 9 CFU / mL, dead bacteria were heat-inactivated in a 80°C water bath for 30 min, and the bacterial count was adjusted to 1×10 9 Cells / mL;

[0208] (2) 0.1 mL of 1% ascorbic acid, 0.1 mL of 2% FeSO 4, 1mL 0.2mol / L NaOH, mix thoroughly, add 0.5mL Lactobacillus gasseri LG20 sample, let stand at 37℃ for 120min to chelate iron ions, add 0.5mL 10% TCA to precipitate protein, centrifuge at 8000rpm for 5min, take 0.4mL supernatant and mix with 4mL 0.1% o-phenanthroline, let stand at 37℃ for 10min, measure the absorbance at 510nm, the blank group is replaced by MRS medium or PBS, which is A0. The test results are shown in Figure 5 .

[0209] Fe 2+ Chelation rate % = (1-A / A0) × 100%

[0210] 2. Test results and analysis

[0211] 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.

[0212] Figure 5 The results showed that the fermentation broth (containing bacteria), supernatant (without bacteria), live bacteria (without fermentation products) and inactivated bacteria (without fermentation products) of LG20 all have the ability to chelate iron ions. The chelation ability is in the order of live bacteria > supernatant > fermentation broth > inactivated bacteria. Through the chelation of iron ions by Lactobacillus gasseri LG20, the content of free iron ions is reduced, which helps reduce the proliferation of BV pathogenic bacteria in the body.

[0213] Example 13: Effect of Lactobacillus gasseri LG20 on biogenic amines

[0214] 1. Test methods

[0215] 1. Growth in biogenic amines

[0216] To prepare vaginal simulated fluid SVF, cadaverine, putrescine and tyramine were added respectively, with the concentration of each biogenic amine being 100 mg / L. 5% of Lactobacillus gasseri LG20 was added 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 14.

[0217] 2. Degradation of biogenic amines

[0218] (1) Preparation of standard curve of biogenic amines

[0219] 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.

[0220] (2) Pre-column derivatization of biogenic amines

[0221] 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.

[0222] (3) HPLC conditions

[0223] 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 13 below.

[0224] Table 13 HPLC gradient elution program

[0225] 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

[0226] (4) Degradation of biogenic amines by Lactobacillus gasseri LG20

[0227] Lactobacillus gasseri LG20 was added to SVF containing three 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 dissolve the biogenic amines as the sample treatment solution. After derivatization, the sample was 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 6 .

[0228] Degradation effect of biogenic amines: Biogenic amine degradation rate % = (WW 1 ) / W×100%, W is the biogenic amine content of the control group, W 1 is the content of biogenic amines in the experimental group.

[0229] 2. Test results and analysis

[0230] 1. Growth of Lactobacillus gasseri LG20 in SVF containing biogenic amines

[0231] Table 14: Growth of Lactobacillus gasseri LG20 in SVF containing biogenic amines

[0232] Cultivation time OD value pH Viable bacteria count CFU / mL 0h 0.149 5.42 4.85E+07 12h 0.357 4.18 2.26E+08 24h 0.425 3.89 2.83E+08

[0233] From the data in Table 14, it can be seen that Lactobacillus gasseri LG20 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.

[0234] 2. Degradation of biogenic amines by Lactobacillus gasseri LG20

[0235] Figure 6 The results showed that Lactobacillus gasseri LG20 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.

[0236] 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 gasseri LG20, characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the accession number CGMCC NO.32035.

2. A Lactobacillus gasseri LG20 according to claim 1, characterized in that The Lactobacillus gasseri LG20 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 gasseri LG20, the inactivated bacteria of Lactobacillus gasseri LG20, and the fermentation product of Lactobacillus gasseri LG20 as claimed in claim 1.

4. Use of the Lactobacillus gasseri LG20 according to claim 1 or the microbial agent according to claim 2 in preparing products for preventing and treating genital tract pathogenic bacteria infection, preventing and treating vaginitis and / or removing genital odor and maintaining the health of female private parts.

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, and Streptococcus angina pectoris.

6. The use according to claim 4, characterized in that: The Lactobacillus gasseri LG20 or microbial agent has one or more of the following effects; 1) Lactobacillus gasseri LG20 or microbial agents can produce hydrogen peroxide; 2) Lactobacillus gasseri LG20 or microbial agents have antibacterial effects on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Klebsiella pneumoniae, Corynebacterium aureus, Staphylococcus hominis, Streptococcus agalactiae, Staphylococcus hemolyticus, and Streptococcus angina pectoris; 3) Lactobacillus gasseri LG20 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae; 4) Lactobacillus gasseri LG20 or microbial agents can inhibit the biofilm formation of Gardnerella vaginalis and / or Candida albicans at different stages; 5) Lactobacillus gasseri LG20 or microbial agents can reduce the hydrophobicity of Candida albicans, reduce the adhesion of Candida albicans, and inhibit the formation of biofilm; 6) Lactobacillus gasseri LG20 or microbial agents have strong adhesion to Hela cells and can compete with Candida albicans for adhesion to Hela cells; 7) Lactobacillus gasseri LG20 or microbial agents can chelate iron ions; 8) Lactobacillus gasseri LG20 or microbial agents can inhibit the secretion of sialidase by Gardnerella vaginalis, protect the vaginal epithelial mucosa, and alleviate vaginal epithelial damage; 9) Lactobacillus gasseri LG20 or microbial agents can grow in an environment containing biogenic amines and can degrade biogenic amines; 10) Lactobacillus gasseri LG20 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, sanitary products, and family planning products.

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