Lactobacillus salivarius LS360 for preventing and / or improving female genital tract infection and application of lactobacillus salivarius LS360

Abstract

The invention relates to combined lactobacillus salivarius LS360 for preventing and / or improving female genital tract infection and application of the combined lactobacillus salivarius LS360. The combined lactobacillus salivarius LS360 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.32134, and the combined lactobacillus salivarius LS360 has good biological characteristics and endurance capacity and can be used for preventing and / or improving female genital tract infection. The traditional Chinese medicine composition has a relatively strong inhibition effect on main vaginitis pathogenic bacteria, is effective on nutritional pathogenic bacteria, and can also reduce form transformation and biological membrane formation of the nutritional pathogenic bacteria and prevent and treat drug resistance; besides, the bacterial strain and the fermentation product can also reduce the content of pathogenic bacteria metabolites, regulate the secretion of cell factors and play a role in relieving inflammation, so that the bacterial strain and the fermentation product can be used for preparing products for preventing and treating female 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 specifically relates to a saliva-associated lactobacillus LS360 for preventing and / or improving female reproductive tract infection and an application thereof. Background Art

[0002] Vaginitis is the most common reproductive tract disease.

[0003] At present, the treatment for female reproductive tract infection and vaginal inflammation usually adopts oral or topical antibiotics, or washes with antibacterial lotions. The problems are large side effects and easy to cause antibiotic resistance. With the rapid development of microbial application technology, the functions of Lactobacillus are being increasingly developed. As the most important microorganism in the reproductive tract of healthy people, it is almost harmless to the human body. Therefore, it is crucial to screen Lactobacillus strains that can prevent and treat female reproductive tract infection and vaginal inflammation, utilize microbial growth metabolism and competition and synergy between microorganisms, improve the vaginal microenvironment, inhibit vaginal pathogenic bacteria infection, prevent and treat vaginal inflammation, so as to maintain the health of 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 saliva-combined Lactobacillus LS360 for preventing and / or improving female genital tract infections and its application, which can not only effectively prevent and treat genital bacterial infections and vaginitis, but also remove genital odor.

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

[0006] A saliva-associated Lactobacillus LS360, classified and named as saliva-associated Lactobacillus (Ligilactobacillus salivarius), is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCCNO.32134. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is September 30, 2024.

[0007] As a further technical solution, the saliva-lactobacillus LS360 is derived from the reproductive tract secretions of healthy women.

[0008] A microbial agent comprises one or more of the live bacteria of Lactobacillus saliva LS360, the inactivated bacteria of Lactobacillus saliva LS360, and the fermentation product of Lactobacillus saliva LS360.

[0009] The saliva combined with Lactobacillus LS360 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 vaginal odor.

[0010] The vulva pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, and Candida albicans.

[0011] As a further technical solution, the saliva combined with Lactobacillus LS360 or microbial agent has one or more of the following effects:

[0012] 1) Saliva combined with Lactobacillus LS360 or microbial agents have inhibitory effects on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, and Candida albicans;

[0013] 2) Saliva combined with Lactobacillus LS360 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae;

[0014] 3) Saliva combined with Lactobacillus LS360 or microbial agents can inhibit the formation of Candida albicans biofilm at different stages;

[0015] 4) Saliva combined with Lactobacillus LS360 or microbial agents can reduce the hydrophobicity of Candida albicans, reduce the adhesion of Candida albicans, and inhibit the formation of biofilm;

[0016] 5) Saliva combined with Lactobacillus LS360 or microbial agents can tolerate acidic environments and survive in simulated vaginal fluid;

[0017] 6) Saliva combined with Lactobacillus LS360 or microbial agents can chelate iron ions;

[0018] 7) Saliva combined with Lactobacillus LS360 or microbial agents can grow in an environment containing biogenic amines and can degrade biogenic amines;

[0019] 8) Saliva combined with Lactobacillus LS360 or microbial agents can reduce the secretion of inflammatory factor IL-6 and increase the secretion of anti-inflammatory factor IL-10.

[0020] As a further technical solution, the product includes oral products or external products;

[0021] As a further technical solution, the oral product includes food or health care products; such as powders, granules, tablets, yogurt, candy, beverages, etc.

[0022] As a further technical solution, the external-use products include one or more of external-use medicines, sanitary products, and family planning products.

[0023] The sanitary products include sanitary napkins, panty liners, wet wipes, tampons, and antibacterial / antibacterial lotions; family planning products include condoms and lubricants.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The saliva-associated Lactobacillus LS360 isolated and screened by the present invention is derived from the secretions of the reproductive tract of healthy women. Compared with drugs such as antibiotics in traditional technologies, it is safe and healthy and has almost no side effects.

[0026] 2) The saliva-associated Lactobacillus LS360 isolated and screened by the present invention has a strong inhibitory effect on common vaginal pathogens such as Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, and Candida albicans, and can be used to prevent and treat female reproductive tract pathogenic bacteria infection.

[0027] 3) Saliva combined with Lactobacillus LS360 can inhibit the formation of Candida albicans hyphae, reduce the number and length of hyphae, and have a significant effect on female vaginal diseases caused by Candida albicans.

[0028] 4) The vaginal biofilm of healthy women is very thin and sometimes loose, while women with vaginitis often have thick biofilms. Biofilm is a highly heterogeneous, multicellular and multilayered three-dimensional structure. The process of forming this structure is divided into four stages: early (adhesion), intermediate (proliferation, filament and extracellular matrix production), maturity, and diffusion. Biofilms are highly resistant to antimicrobial agents and host defense mechanisms, and all virulence factors of Candida are more or less related to biofilm formation. Therefore, the study of the pathological mechanisms and treatment of various infections must undoubtedly consider the biofilm formation ability of microorganisms. Saliva combined with Lactobacillus LS360 can inhibit the formation of Candida albicans biofilm at different stages, and saliva combined with Lactobacillus LS360 has strong hydrophobicity and adhesion ability, and can reduce the hydrophobicity of Candida albicans, thereby reducing its adhesion effect and reducing the formation of biofilm, thereby preventing the occurrence of Candida albicans resistance.

[0029] 5) Saliva-associated Lactobacillus LS360 can tolerate acidic environments and has good survival ability in vaginal simulated fluid, enabling it to grow and multiply in the female reproductive tract environment and exert a probiotic effect.

[0030] 6) The proliferation of vaginal Gardnerella depends on exogenous iron, and Lactobacillus salivais LS360 can reduce the content of free iron ions by chelating iron ions, thereby limiting the proliferation of vaginal Gardnerella that requires iron.

[0031] 7) Biogenic amines are the main source of vaginal odor. Saliva-combined Lactobacillus LS360 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.

[0032] 8) Saliva combined with live and inactivated Lactobacillus LS360 bacteria can reduce the secretion of inflammatory factor IL-6 and increase the secretion of anti-inflammatory factor IL-10, thereby achieving the purpose of preventing and treating vaginal inflammation.

[0033] 9) The saliva combined with Lactobacillus LS360 of the present invention is non-toxic and harmless, and can be used to prepare a wide range of products, not only can it be prepared into oral products, such as food, medicine, health care products, beverages, candies, etc., but also can be prepared into genital external medicines, and can even be used in sanitary products and family planning products, such as sanitary napkins, female antibacterial / antibacterial lotions, condoms, etc.

[0034] In summary, the saliva-associated Lactobacillus LS360 screened by the present invention has good biological properties and tolerance, has a strong inhibitory effect on major vaginitis pathogens, is not only effective against vegetative pathogens, but also can reduce their morphological transformation and biofilm formation, and prevent the generation of drug resistance; in addition, its strains and fermentation products can also reduce the content of pathogenic bacterial metabolites, regulate the secretion of cytokines, and have the effect of relieving 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

[0035] Figure 1 This is a graph showing the inhibitory effect of fermentation broths of different strains on Candida albicans;

[0036] Figure 2 The graph shows the inhibitory effect of supernatants of different strains on Candida albicans;

[0037] Figure 3 This is a diagram showing the adhesion effects of different strains on Hela cells;

[0038] Figure 4 The following are microscopic images of the adhesion of different strains to Hela cells;

[0039] exist Figure 4 In the figure, A: blank cells; B: strain LS360; C: strain L556; D: strain L557; E: strain L558; F: strain L559; G: strain L661; H: strain L667;

[0040] Figure 5 This is the LS360 hydrophobic performance diagram;

[0041] Figure 6 This is the effect of LS360 on the hydrophobicity of Candida albicans;

[0042] Figure 7 This is a graph showing the growth of LS360 in vaginal simulated fluid;

[0043] Figure 8 This is the graph showing the chelation performance of different components of LS360 on iron ions;

[0044] Fig. 9 This is a diagram showing the degradation effect of LS360 on biogenic amines. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] Example 1: Isolation of saliva-associated Lactobacillus LS360

[0048] 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.

[0049] Example 2, strain screening

[0050] All the isolated lactic acid bacteria 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 effect on Escherichia coli, Staphylococcus aureus, Candida albicans, and Gardnerella vaginalis and strong adhesion was selected and named LS360.

[0051] 1. Test on the inhibitory ability against Escherichia coli and Staphylococcus aureus

[0052] 1.1 Test methods

[0053] (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;

[0054] (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;

[0055] (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.

[0056] (4) Add 1 mL of Escherichia coli (or Staphylococcus aureus) to 100 mL of nutrient agar medium cooled to 50°C and mix thoroughly;

[0057] (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;

[0058] (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.

[0059] 1.2 Test results

[0060] Table 1 Antibacterial results of various lactic acid bacteria on Escherichia coli and Staphylococcus aureus

[0061] Strain number Escherichia coli inhibition zone (mm) Staphylococcus aureus inhibition zone (mm) L557 (Lactobacillus rhamnosus) 14.92±2.62 14.48±0.88 L667 (Lactobacillus rhamnosus) 14.90±0.85 13.38±0.94 LS360 (Salivary Lactobacillus) 19.10±0.85 16.78±0.69 L661 (Lactobacillus salivae) 13.13±0.80 14.04±0.31 L556 (Lactobacillus crispatus) 15.67±1.53 15.13±2.44 L558 (Lactobacillus jensenii) 15.17±1.04 10.90±0.46 L559 (Lactobacillus gasseri) 10.60±0.53 9.08±0.83 MRS 8.00±0.00 8.00±0.00

[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 saliva combined with Lactobacillus LS360 has good inhibitory ability against Escherichia coli and Staphylococcus aureus, and the inhibitory effect is better than other strains. MRS as a control has no inhibitory effect.

[0064] 2. Test of the inhibitory ability to vaginal Gardnerella

[0065] 2.1 Experimental methods

[0066] (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;

[0067] (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;

[0068] (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 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 plates for each group. The results are shown in Table 2.

[0069] 2.2 Experimental Results

[0070] Table 2 Antibacterial effect on vaginal Gardnerella

[0071] strain Inhibition zone diameter L557 (Lactobacillus rhamnosus) 12.05±0.40 L667 (Lactobacillus rhamnosus) 11.22±0.16 LS360 (Salivary Lactobacillus) 17.95±1.2 L661 (Lactobacillus salivae) 12.85±0.97 L556 (Lactobacillus crispatus) 10.30±0.42 L558 (Lactobacillus jensenii) 10.30±0.42 L559 (Lactobacillus gasseri) 10.10±0.14 MRS 8.00±0.00

[0072] Gardnerella vaginalis is the main pathogen of bacterial vaginitis. The results in Table 2 show that LS360 has a strong inhibitory effect on Gardnerella vaginalis, with an inhibitory diameter of 17.95 mm. The inhibitory effect is better than other lactic acid bacteria strains. MRS as a control has no inhibitory effect on Gardnerella vaginalis.

[0073] 3. Test on the inhibitory ability of Candida albicans

[0074] 3.1 Experimental methods

[0075] (1) 5 ml of each lactic acid bacteria fermentation liquid (containing strains) or each lactic acid bacteria fermentation supernatant (without strains) cultured for 16 h and 5 ml of Candida albicans liquid were mixed in equal volumes, and the viable bacteria of Ca were counted at 0 h, 12 h and 24 h, respectively, using selective YPD medium;

[0076] (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.

[0077] (3) Calculate the inhibitory ability of lactic acid bacteria on yeast. The results are shown in Figure 1-2 :

[0078] Candida albicans inhibition rate A=(A0-A t ) / A0×100%, where A t represents the number of Ca viable bacteria in each experimental group after 12 h and 24 h of culture, and A0 represents the number of Ca viable bacteria after 0 h;

[0079] 3.2 Experimental Results

[0080] Candida albicans is the main pathogen of candidal vaginitis. Figure 1-2The results showed that the fermentation broth and fermentation supernatant of LS360 had a strong inhibitory effect on Candida albicans, especially the LS360 fermentation broth, the inhibition rate reached 96% in 24 hours, and the antibacterial effect increased with time; while MRS liquid culture medium with pH 4.0 and pH 6.8 could not inhibit the growth of Candida albicans.

[0081] 4. Adhesion test on Hela cells

[0082] 4.1 Experimental methods

[0083] (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.

[0084] (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.

[0085] (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.

[0086] (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.

[0087] (5) Cell fixation: Add 0.5 mL of 4% paraformaldehyde to each well of the cell culture plate, fix at room temperature for 20 min, wash three times with PBS, add 0.5 mL of 1 / 1000 crystal violet for staining, wash four times with PBS, dry at room temperature, and observe the adhesion of the test strain to the HeLa cells under a 100× microscope.

[0088] (6) Adhesion rate detection: After completing the above step (4), add 2 mL of incomplete culture medium and 4 μL of Triton X-ray distillation, lyse at room temperature for 40 min, mix well and take out 1 mL for the count of live lactic acid bacteria and quantify the adhesion of lactic acid bacteria. The results are shown in the table. Figure 3-4 .

[0089] 4.2 Experimental Results

[0090] Figure 3-4 The results showed that LS360 had adhesion to Hela cells, and the adhesion rate to Hela cells was 10.15%.

[0091] In summary, in this embodiment, the strain LS360 was finally screened out through antibacterial experiments and adhesion experiments, and its antibacterial effect and adhesion effect were strong. LS360 will be further studied in the future.

[0092] Example 3: Identification of strains

[0093] The strain LS360 obtained by screening was identified by 16S rDNA gene, and the nucleotide sequence was shown in SEN ID NO.1. The strain was identified as Ligilactobacillus salivarius and named as Ligilactobacillus salivarius LS360.

[0094] Example 4: Susceptibility of Saliva-Lactobacillus LS360 to Antibiotics

[0095] 1. Test methods:

[0096] The KB method was used to determine the sensitivity of saliva combined with Lactobacillus LS360 to 30 antibiotics, and the quantitative antibiotic paper standard used in the test was produced by the China Food and Drug Inspection Institute. The results are shown in Table 3.

[0097] 2. Test results

[0098] Table 3 The sensitivity of saliva combined with Lactobacillus LS360 to antibiotics

[0099]

[0100] The results in Table 3 show that LS360 is sensitive to 21 antibiotics tested and is insensitive to 6 antibiotics tested.

[0101] Example 5: Study on the inhibitory effect of saliva combined with Lactobacillus LS360 on hyphae production of Candida albicans

[0102] 1. Test methods

[0103] (1) 3 mL of Candida albicans Ca was centrifuged, the supernatant was removed and resuspended in 1 mL of fresh YPD medium, i.e., after concentrating 3 times, 1 mL was added to a 24-well plate;

[0104] (2) 1 mL of saliva combined with Lactobacillus LS360 fermentation broth (containing strains) or fermentation supernatant (without strains) was added to the above 24-well plate and mixed;

[0105] (3) Add 4 μL of penicillin solution (100,000 U / mL) to 2 mL of supernatant + Ca or fermentation broth + Ca mixture to make the final concentration 200 U / mL;

[0106] (4) The positive control was 1 mL of MRS with 4 μL of penicillin added to observe the effect of penicillin on mycelial growth, and MRS without penicillin was used as a negative control;

[0107] (5) Incubate at 37°C for 3 h, take 10 μL of the mixture and place it on a glass slide. After adding a coverslip, observe the growth of Candida albicans hyphae through a 40x microscope eyepiece. Count the number of hyphae and yeast in the field of view, calculate the ratio of hyphae, and count the hyphae length in μm. The results are shown in Table 4.

[0108] 2. Test results

[0109] Table 4 Effect of saliva combined with Lactobacillus LS360 on Candida albicans hyphae

[0110] Grouping Hyphae length μm Mycelial phase percentage Positive control group 23.36±1.48 43.33％±3.32 Negative control group 12.94±2.67 15.69％±2.21 LS360 Fermentation Supernatant Group 15.42±2.41 14.71％±2.18 LS360 Fermentation Broth Group 14.87±1.59 10.99％±3.69

[0111] 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 4 show that saliva combined with Lactobacillus LS360 can inhibit the formation of Candida albicans hyphae, thereby reducing its adhesion and invasion to vaginal epithelial cells and reducing its toxicity to epithelial cells. 200U penicillin can induce the formation of hyphae of Candida albicans. The hyphae accounted for more than 40% in the positive control group and more than 15% in the negative control group without penicillin. Compared with the positive control group, the saliva combined with Lactobacillus LS360 fermentation broth group and LS360 fermentation supernatant group with 200U penicillin can inhibit the formation of Candida albicans hyphae, and can also inhibit the length of hyphae, and the hyphae accounted for less than 15%.

[0112] Example 6: Study on the inhibitory effect of saliva combined with Lactobacillus LS360 on Candida albicans biofilm

[0113] 1. Test methods

[0114] 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. LS360 fermentation supernatant 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.

[0115] (1) 100 μL of Ca was added to a 96-well plate. After 0, 12, and 24 h of culture, 100 μL of saliva combined with the fermentation supernatant of Lactobacillus LS360 (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.

[0116] (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;

[0117] (3) Decolorize with 200 μL of anhydrous ethanol for 15 min, and measure the OD value at a wavelength of 570 nm. The results are shown in Table 5;

[0118] The inhibition rate of albinism biofilm %=(ODc-ODt) / ODc×100%, where ODc represents the OD value of the negative control group, and ODt represents the OD value of the experimental group.

[0119] 2. Test results

[0120] Table 5 Inhibitory effect on biofilm formation of Candida albicans

[0121]

[0122] Biofilm is composed of microbial communities and their coated extracellular polymers and matrix networks. It is a multicellular, multilayered three-dimensional structure that adheres to each other or to the surface of tissues or objects. Biofilm contributes to the formation of microbial resistance. Gene transfer, disease occurrence, and persistent infection are closely related to biofilm. 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 hydrogen peroxide, 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.

[0123] The data results in Table 5 show that in the initial adhesion stage of Candida albicans, saliva combined with Lactobacillus LS360 has a strong inhibitory effect on Candida albicans biofilm, with an inhibition rate of more than 77%. In the initial biofilm formation stage of Candida albicans, LS360 has a destructive effect on Candida albicans biofilm, with an inhibition rate of 60%. In the mature biofilm formation stage of Candida albicans, LS360 still has a strong destructive effect on its biofilm, with an inhibition rate of 69%. Therefore, the intervention of saliva combined with Lactobacillus LS360 in the three stages of Candida albicans planktonic bacteria adhesion stage, biofilm initial formation stage and biofilm maturity can effectively reduce the formation of biofilm, thereby reducing the reproductive tract infection and recurrence problems caused by the long-term existence of biofilm.

[0124] Example 7: Study on the effect of saliva combined with Lactobacillus LS360 on the hydrophobicity of Candida albicans

[0125] 1. Test methods

[0126] (1) Hydrophobicity of saliva-associated Lactobacillus LS360

[0127] The overnight cultured LS360 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 the 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, allowed to stand for 30min, and after the aqueous phase and the organic phase were separated, the OD value of the aqueous phase at 600nm was measured, recorded as A. The results are shown in Figure 5 .

[0128] Cell surface hydrophobicity % = (1-A / A0) × 100%

[0129] Hydrophobicity standard: more than 50% is highly hydrophobic, between 20%-50% is moderately hydrophobic, and less than 20% is non-hydrophobic.

[0130] (2) Effect of saliva combined with Lactobacillus LS360 on the hydrophobicity of Candida albicans

[0131] The overnight culture of Candida albicans was centrifuged at 5000r / min for 10min, the cells were collected, washed with PBS and centrifuged once, the cells were resuspended with PBS, and the concentration of the cell solution was adjusted to an absorbance of about 1.00 at a wavelength of 600nm; 5mL of the above Candida albicans solution was added with 5mL of LS360 fermentation supernatant, mixed and cultured at 37℃ for 24h, and the hydrophobicity was determined. The specific determination method was the same as above, and MRS medium LS360 was used as a control instead of fermentation supernatant. The test results are shown in Figure 6 .

[0132] 2. Test results

[0133] (1) Hydrophobicity of saliva-associated Lactobacillus LS360

[0134] The important premise for the saliva-associated Lactobacillus LS360 to produce beneficial effects on the host is that it 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 saliva-associated Lactobacillus LS360. Figure 5 The results showed that saliva-associated Lactobacillus LS360 had strong hydrophobicity, and the hydrophobicity rates to xylene and chloroform were both greater than 50%, reaching a high hydrophobicity level.

[0135] (2) Effect of saliva combined with Lactobacillus LS360 on the hydrophobicity of Candida albicans

[0136] from Figure 6 It can be seen that the hydrophobicity of Candida albicans is 79.39%. After co-culturing with Candida albicans, the LS360 fermentation supernatant can reduce the hydrophobicity of Candida albicans, thereby reducing the colonization of Candida albicans in the reproductive tract.

[0137] Example 8: Study on the tolerance of saliva combined with Lactobacillus LS360 in a low pH environment

[0138] 1. Test methods

[0139] The activated saliva-lactobacillus LS360 was inoculated at 3% into a liquid MRS medium at pH 4.5 and cultured at 37°C. The number of viable bacteria was measured at 6h, 12h, and 24h to detect the growth of LS360 in a low pH environment. The results are shown in Table 6.

[0140] 2. Test results

[0141] Table 6 Growth status of LS360 under different pH conditions

[0142] Cultivation time Live bacteria count (lgcfu / ml) 6h 7.71±0.02 12h 8.04±0.01 24h 7.83±0.02

[0143] The results in Table 6 show that a low pH environment (pH 4.5) will not inhibit the growth of Lactobacillus salivais LS360. Moreover, as the culture time increases, the number of live bacteria of LS360 increases, indicating that LS360 can adapt to the acidic environment and has tolerance to the acidic environment of the female reproductive tract.

[0144] Example 9: Study on the growth of saliva combined with Lactobacillus LS360 in vaginal simulated fluid

[0145] 1. Test methods

[0146] Simulated Vaginal Fluid (SVF): 3.51 g sodium chloride, 1.4 g potassium hydroxide, 0.222 g calcium hydroxide, 0.018 g bovine serum albumin, 2.0 g lactic acid, 1.0 g acetic acid, 0.16 g glycerol, 0.4 g urea, 5.0 g glucose. Use HCl to adjust the pH to 4.2 and dispense into 10 mL / tube for lactic acid bacteria culture. Bovine serum albumin is added separately to prepare 0.018 g / 10 mL, and 0.1 mL is added to each tube.

[0147] Saliva combined with Lactobacillus LS360 was added to SVF at 5% and the viable count and pH value were determined at 0, 4, 6, 8, 12, and 24 hours. The results are shown in Figure 7 .

[0148] 2. Test results

[0149] Vaginal fluid is a mixture of Bartholin and Skene glands, fallopian tube and endometrial fluid, and cervical mucus. This is a complex fluid containing about 90%-95% water, mucin, carbohydrates, salts, urea, fatty acids, albumin and other macromolecules. The ability of probiotics to adapt to such a complex environment is crucial to vaginal health.

[0150] The results of pH value and viable count showed that saliva-associated Lactobacillus LS360 had a strong survival ability in SVF, was able to use its nutrients to maintain its own growth and reproduction, and produced acidic substances to maintain an acidic environment (see Figure 7 ). It can be seen that saliva-associated Lactobacillus LS360 can tolerate the female reproductive tract environment and can grow and reproduce in it.

[0151] Example 10: Study on the chelation of iron ions by saliva combined with Lactobacillus LS360

[0152] 1. Test methods

[0153] (1) Saliva was combined with Lactobacillus LS360 and cultured overnight. The supernatant was collected by centrifugation at 8000 rpm for 10 min. The cells were washed once with PBS and resuspended in PBS. The cell count was adjusted to 1×10 9 CFU / mL, 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;

[0154] (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 LS360 samples with different components 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 is A0. The test results are shown in Table 1. Figure 8 .

[0155] Fe 2+ Chelation rate % = (1-A / A0) × 100%

[0156] 2. Test results

[0157] 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 8 The results showed that the fermentation broth (containing bacteria), supernatant (without bacteria), live bacteria (without fermentation products) and inactivated bacteria (without fermentation products) of LS360 all have the ability to chelate iron ions. The chelation ability is in the order of live bacteria > supernatant > fermentation broth > inactivated bacteria. The chelation of iron ions by saliva combined with Lactobacillus LS360 reduces the content of free iron ions, helping to reduce the proliferation of BV pathogenic bacteria in the body.

[0158] Example 11: Study on the effect of saliva combined with Lactobacillus LS360 on biogenic amines

[0159] 1. Test methods

[0160] (1) Growth of biogenic amines

[0161] To prepare vaginal simulated fluid SVF, cadaverine, putrescine and tyramine were added respectively, with the concentration of each biogenic amine being 100 mg / L. Saliva combined with Lactobacillus LS360 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 8.

[0162] (2) Degradation of biogenic amines

[0163] 1) Preparation of standard curve of biogenic amines

[0164] 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.

[0165] 2) Pre-column derivatization of biogenic amines

[0166] 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.

[0167] 3) HPLC conditions

[0168] 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 7 below.

[0169] Table 7 HPLC gradient elution program

[0170] 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

[0171] 4) Degradation of biogenic amines by saliva combined with Lactobacillus LS360

[0172] Saliva combined with Lactobacillus LS360 was added to SVF containing three biogenic amines, cadaverine, putrescine and tyramine (each biogenic amine concentration was 100 mg / L) at 5%, and the one with MRS added was used as the control group. The cells were cultured at 37°C for 24 h, and the bacterial suspension was centrifuged at 8000 rpm for 10 min. 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. The sample was 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 Fig. 9 .

[0173] Degradation effect of biogenic amines: biogenic amine degradation rate % = (W-W1) / W×100%, W is the biogenic amine content of the control group, and W1 is the biogenic amine content of the experimental group.

[0174] 2. Test results

[0175] 1. Growth of LS360 in SVF containing biogenic amines

[0176] Table 8: Growth of LS360 in SVF containing biogenic amines

[0177] Cultivation time OD value pH Viable bacteria count CFU / mL 0h 0.138 5.42 5.40E+07 12h 0.302 4.19 1.39E+08 24h 0.336 3.97 1.61E+08

[0178] From the data in Table 8, it can be seen that LS360 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.

[0179] 2. Degradation of biogenic amines by LS360

[0180] Fig. 9 The results showed that LS360 has different degrees of degradation effects on putrescine, cadaverine and tyramine, three biogenic amines. By reducing the content of these three biogenic amines, it reduces the generation of odor, while reducing the pH increase caused by biogenic amines, alleviating the aggravation of vaginal inflammation caused by biogenic amines.

[0181] Example 12: Study on the effect of saliva combined with Lactobacillus LS360 on cytokine secretion

[0182] 1. Experimental Methods

[0183] (1) Preparation of live bacterial suspension

[0184] The test strains were activated for 3 generations in MRS liquid medium, and the bacterial sludge was collected by centrifugation, washed twice with PBS, and resuspended in DMEM culture medium to adjust the bacterial solution concentration to 1.5×10 8 CFU / mL to obtain a live bacterial suspension.

[0185] (2) Preparation of inactivated bacterial suspension

[0186] 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.

[0187] (3) Cell culture

[0188] Macrophage RAW264.7 cells were revived in DMEM complete culture medium containing 10% fetal bovine serum, incubated in a 37°C, 5% CO2 cell culture incubator, and subcultured when the cells grew well and the density reached about 80%. Subcultured three times before conducting subsequent experiments.

[0189] (4) Determination of the effect of strains on the secretion of cellular immune factors

[0190] Macrophage RAW264.7 cells were cultured 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.

[0191] The experimental groups are as follows:

[0192] 1) Negative control group: DMEM was added to the wells and incubated for 4 h and 24 h respectively;

[0193] 2) Positive control group (LPS): lipopolysaccharide (LPS) was added to the wells and incubated for 4 h and 24 h respectively;

[0194] 3) LS360 test group: add live bacterial suspension or inactivated bacterial suspension of the test strain into the wells, incubate the live bacterial group for 4 hours, and incubate the inactivated group for 12 hours;

[0195] 4) LS360 competition group (strain + LPS): LPS and live bacterial suspension or inactivated bacterial suspension of the test strain 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;

[0196] 5) LS360 treatment group (LPS+strain): LPS 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 the test strain was added. The live bacteria group was incubated for 4 h, and the inactivated group was incubated for 12 h;

[0197] 6) LS360 prevention group (strain + LPS): 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 LPS was added and incubated for 4 h (live bacteria) or 12 h (inactivated bacteria);

[0198] 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-10 cytokines in the above different groups were determined according to the instructions of the ELISA kit. The results are shown in Tables 9-10.

[0199] 2. Test results

[0200] 1. Effect on IL-6:

[0201] Table 9 Effects of live and inactivated bacteria on IL-6 secretion by macrophages

[0202]

[0203] 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. The main clinical manifestation is an increase in IL-6 levels during the onset of the disease. From the data in Table 9, it can be seen that the live bacteria and inactivated bacteria of LS360 incubated with cells alone can promote the secretion of IL-6, cause physiological inflammatory reactions, and improve the body's immunity. LPS as a positive control can stimulate the secretion of IL-6 and increase the level of inflammatory factors. After LS360 intervention, the effect of LPS on macrophages can be reduced, and the secretion of IL-6 can be reduced. In particular, the competition group and prevention group of live bacteria and inactivated bacteria have the best effect in inhibiting inflammation.

[0204] 2. Effect on IL-10:

[0205] Table 10 Effects of live and inactivated bacteria on IL-10 secretion by macrophages

[0206]

[0207] IL-10 is a multi-effect cellular anti-inflammatory factor that can play an immunosuppressive or immunostimulatory role in various types of cells. In terms of inflammatory response, IL-10 can downregulate the expression of major histocompatibility antigen II (MHC II) on the surface of monocytes, reduce its antigen presentation function, downregulate T lymphocyte activity, inhibit the activation, migration and adhesion of inflammatory cells, and at the same time, IL-10 can also inhibit the synthesis and release of inflammatory factors. The data in Table 10 show that the live bacteria and inactivated bacteria of LS360 incubated with cells alone can promote the secretion of IL-10 and the secretion of anti-inflammatory factors, thereby reducing the inflammatory response. LPS can stimulate the secretion of IL-10 as a positive control. After LS360 intervention, the effect of LPS on macrophages can be alleviated, and the secretion of the anti-inflammatory factor IL-10 can be promoted. In particular, the competition group and prevention group of live bacteria and inactivated bacteria have the best effect in inhibiting inflammation.

[0208] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A saliva-associated Lactobacillus LS360, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the accession number CGMCC NO.32134.

2. A saliva-associated Lactobacillus LS360 according to claim 1, characterized in that: The saliva-lactobacillus LS360 is derived from the reproductive tract secretions of healthy women.

3. A microbial agent, characterized in that: It comprises one or more of the live bacteria of Lactobacillus saliva LS360, the inactivated bacteria of Lactobacillus saliva LS360, and the fermentation products of Lactobacillus saliva LS360 as claimed in claim 1.

4. Application of the saliva-combined lactobacillus LS360 as claimed in claim 1 or the microbial agent as claimed in claim 2 in preparing a product for preventing and treating female reproductive tract pathogenic bacteria infection, preventing and treating vaginitis, and / or removing pudendal 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, and Candida albicans.

6. The use according to claim 4, characterized in that: The saliva combined with Lactobacillus LS360 or microbial agent has one or more of the following effects: 1) Saliva combined with Lactobacillus LS360 or microbial agents have inhibitory effects on Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, and Candida albicans; 2) Saliva combined with Lactobacillus LS360 or microbial agents can inhibit the formation of Candida albicans hyphae and reduce the number and length of hyphae; 3) Saliva combined with Lactobacillus LS360 or microbial agents can inhibit the formation of Candida albicans biofilm at different stages; 4) Saliva combined with Lactobacillus LS360 or microbial agents can reduce the hydrophobicity of Candida albicans, reduce the adhesion of Candida albicans, and inhibit the formation of biofilm; 5) Saliva combined with Lactobacillus LS360 or microbial agents can tolerate acidic environments and survive in simulated vaginal fluid; 6) Saliva combined with Lactobacillus LS360 or microbial agents can chelate iron ions; 7) Saliva combined with Lactobacillus LS360 or microbial agents can grow in an environment containing biogenic amines and can degrade biogenic amines; 8) Saliva combined with Lactobacillus LS360 or microbial agents can reduce the secretion of inflammatory factor IL-6 and increase the secretion of anti-inflammatory factor IL-10.

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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