A Lactiplantibacillus plantarum strain and its applications

Abstract

The present invention discloses a Lactiplantibacillus plantarum and its application. The Lactiplantibacillus plantarum is named strain LSA-410, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on August 18, 2022, with the deposit number GDMCC No. 62712. The strain LSA-410 has a strong lactic acid production ability, can effectively inhibit the growth of common pathogenic bacteria in the urogenital tract (such as Gardnerella vaginalis, uropathogenic Escherichia coli, etc.), can strongly adhere to cervical epithelium (Hela cells) and human bladder transitional cell carcinoma cells (T24 cells), is non-toxic, non-pathogenic, safe and reliable. Based on these advantages, the strain LSA-410 can be used for the research and development of vaginal microecological preparations for the treatment / prevention of urogenital diseases, and reduce the infection and recurrence rates of urogenital diseases.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a plant lactobacillus strain and application thereof. Background Art

[0002] Lactobacillus is the main dominant bacteria in the vaginal microbiome of healthy women of childbearing age, accounting for 90%-95% of the vaginal microbiome. Lactobacillus can produce lactic acid, hydrogen peroxide and other antibacterial substances, making the vaginal environment acidic, inhibiting the growth of pathogens, and reducing the occurrence of vaginal infections. At the same time, Lactobacillus can occupy the surface of the reproductive tract mucosa and prevent pathogens from colonizing in the vagina through competitive adhesion. In addition, Lactobacillus can participate in regulating the host's immune response, enhance the proliferation and differentiation of immune cells, and improve the host's ability to resist pathogens.

[0003] Bacterial vaginosis (BV) is a common urogenital tract inflammation. BV is a vaginal infection caused by a decrease or disappearance of lactobacilli in the vagina and an increase in facultative anaerobic bacteria such as vaginal Gardnerella. It ranks first in the incidence of reproductive tract infections. If not treated in time, it can further cause a variety of diseases, such as chronic cervicitis, cervical cancer, ovarian cancer, fallopian tube tumors, etc.; at the same time, BV is also the main cause of infertility, premature birth, premature rupture of membranes, recurrent miscarriage, etc.

[0004] Antibiotics are currently the main treatment for urogenital tract inflammation, such as clindamycin and metronidazole. Although they are effective quickly, they are prone to relapse after recovery. More than 50% of BV patients experience symptomatic recurrence within one year after drug treatment. The use of antibiotics not only kills pathogens in the vagina, but also causes the death of normal flora in the vagina, which makes the vaginal microecology unable to recover to a balanced state that can resist the invasion of pathogens. In addition, the widespread and irrational use of antibacterial drugs has caused some pathogens to develop tolerance to drugs, making the treatment of the disease more difficult. Therefore, while killing pathogens, restoring the dominance of Lactobacillus and restoring the acidic environment of the vagina, and maintaining the balance of normal vaginal microecology are the key to treating vaginal inflammation.

[0005] Microecological therapy is a new treatment concept aimed at increasing probiotics and restoring the normal vaginal microecological environment. It includes three steps: killing pathogens, repairing vaginal mucosa, and restoring the balance of vaginal microecology. That is, after using antibiotics to eliminate pathogenic bacteria, microecological preparations are used to supplement lactobacilli isolated from the vagina of healthy women, which adhere and colonize to promote the recovery of the vagina to a normal microenvironment.

[0006] Currently, there are only two types of vaginal probiotic preparations on the market in China, namely, Streptococcus faecalis ) a commercially available drug called "Yanhua" and a drug containing Lactobacillus delbrueckii ( Lactobacillus delbrueckiiCommercially available drugs such as "Ding Junsheng", however, these bacterial strains do not belong to the common bacterial strains in the vaginas of Chinese women and have poor colonization ability. SUMMARY OF THE INVENTION

[0007] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a bacterial strain, which is Lactiplantibacillus plantarum LSA-410, can be used for the research and development of live lactobacillus preparations, has the advantages of safety, reliability, good effect, etc., can be used to treat and / or prevent related diseases such as female lower urinary tract infections and vaginitis, and can also fundamentally restore the vaginal microecological environment, which is beneficial to women's health.

[0008] The present invention also provides a product containing the above-mentioned bacterial strain.

[0009] The present invention also provides the use of the above-mentioned bacterial strain or the product containing the above-mentioned bacterial strain.

[0010] In the first aspect of the present invention, a bacterial strain is provided. The bacterial strain is Lactiplantibacillus plantarum, named LSA-410 strain, which is deposited in the Guangdong Provincial Culture Collection of Microorganisms, with the deposit number of GDMCC No.62712 and the deposit date of August 18, 2022. The LSA-410 strain has a strong lactic acid-producing ability, has good antibacterial effects on urogenital tract pathogenic bacteria such as uropathogenic Escherichia coli and Gardnerella vaginalis, and has a high adhesion ability to human cervical cancer epithelial cells (Hela cells). The LSA-410 strain can be used to develop products such as sanitary products and pharmaceutical preparations, has the advantages of safety, reliability, few adverse reactions, good effect, etc., can be used to prevent and treat related diseases of female lower urinary tract infections and vaginitis, regulate the microecological balance of the female reproductive tract, prevent female reproductive tract diseases, reduce the probability of female reproductive tract diseases, and has very important significance for female reproductive health.

[0011] In some embodiments of the present invention, the LSA-410 strain is isolated from the posterior fornix of the vagina of healthy women.

[0012] In some embodiments of the present invention, the LSA-410 strain has a 16S rDNA gene fragment with a sequence as shown in SEQ ID No:1 and a pheS gene fragment with a sequence as shown in SEQ ID No:2.

[0013] In the second aspect of the present invention, a product is provided, which at least includes one of the following (1) to (6):

[0014] (1) The LSA-410 strain;

[0015] (2) A bacterial agent containing the LSA-410 strain;

[0016] (3) A live bacterial solution containing the LSA-410 strain;

[0017] (4) A dead bacterial solution containing the LSA-410 strain;

[0018] (5) A metabolite obtained by the LSA-410 strain through a metabolic pathway;

[0019] (6) An extract obtained by extracting from the LSA-410 strain.

[0020] In some embodiments of the present invention, the product is a drug, a medical device or a sanitary product.

[0021] In some embodiments of the present invention, the drug further includes a pharmaceutical carrier and / or pharmaceutical excipients.

[0022] In some embodiments of the present invention, the pharmaceutical excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

[0023] In some embodiments of the present invention, the excipient includes water.

[0024] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0025] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, an alginate, gelatin, and polyvinylpyrrolidone.

[0026] In some embodiments of the present invention, the wetting agent includes glycerol.

[0027] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0028] In some embodiments of the present invention, the absorption promoter includes a quaternary ammonium compound.

[0029] In some embodiments of the present invention, the surfactant includes cetyl alcohol.

[0030] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and saponite clay.

[0031] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0032] In some embodiments of the present invention, the medical device is a gel.

[0033] In some embodiments of the present invention, the hygiene product is at least one of a tampon, a sanitary napkin, a menstrual pad, a diaper, a soap, and a condom.

[0034] In some embodiments of the present invention, the sanitary napkin includes a tampon, a menstrual cup, or a pantiliner.

[0035] In some embodiments of the present invention, the form of the product is one of a powder, an ointment, a gel, a drop, a suppository, a lozenge, a granule, a capsule, a spray, a tablet, a pill, or a solution; preferably, the capsule is a soft capsule.

[0036] In the third aspect of the present invention, there is provided the use of the LSA-410 strain or the product in the preparation of an antibacterial product.

[0037] In some embodiments of the present invention, the pathogenic bacteria inhibited by the antibacterial product include any one or more of uropathogenic Escherichia coli, Gardnerella vaginalis, Candida albicans, Candida glabrata, Staphylococcus aureus, Pseudomonas aeruginosa, and Aerococcus christensenii.

[0038] In the fourth aspect of the present invention, there is provided the use of the LSA-410 strain or the product in the preparation of a drug for treating or preventing urogenital tract infections, or a vaginal care product.

[0039] In some embodiments of the present invention, the urogenital tract infections include vaginal infections and lower urinary tract infections.

[0040] In some embodiments of the present invention, the vaginal infections include at least one of bacterial vaginitis, candidal vaginitis, trichomonal vaginitis, infantile vaginitis, menstrual vaginitis, senile vaginitis, mixed infectious vaginitis, and vaginal infections caused by HPV infection, premature birth, or miscarriage.

[0041] In the fifth aspect of the present invention, there is provided the use of the LSA-410 strain or the product in the preparation of a drug delivery carrier. For example, the LSA-410 strain is constructed into a bacterial agent capable of expressing a specific product, and the expression product is delivered into the vagina by using its excellent colonization ability.

[0042] In some embodiments of the present invention, the application is the use in the preparation of a product for adhering to cervical cancer cells.

[0043] In some embodiments of the present invention, the application is the use in the production of lactic acid or the preparation of a product for producing lactic acid.

[0044] In some embodiments of the present invention, the lactic acid is D-lactic acid and L-lactic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Colony morphology diagram of LSA-410 strain in Example 1 of the present invention;

[0046] Figure 2 Morphology diagram of LSA-410 strain after Gram staining in Example 1 of the present invention;

[0047] Figure 3 Growth curve diagram of LSA-410 strain in different pH media in Example 2 of the present invention;

[0048] Figure 4 Detection result diagram of the in vitro antibacterial ability of LSA-410 strain in Example 2 of the present invention. The "MRS control" marked in the figure is the blank control;

[0049] Figure 5 Determination experiment of the hemolytic property of LSA-410 strain in Example 3 of the present invention;

[0050] Figure 6 Drug sensitivity test diagram of LSA-410 strain in Example 3 of the present invention. In the figure, the medium in Figure A contains ampicillin (AM), the medium in Figure B contains clindamycin (CM), the medium in Figure C contains daptomycin (DPC), the medium in Figure D contains erythromycin (ERY), the medium in Figure E contains linezolid (LZ), the medium in Figure F contains meropenem (MRP), and the medium in Figure G contains vancomycin (VA);

[0051] Figure 7 Detection result of the toxicity of LSA-410 strain to Hela cells in Example 3 of the present invention;

[0052] Figure 8 Experimental flow chart for establishing a mouse model of Gardnerella vaginalis infection in Example 4 of the present invention;

[0053] Figure 9 Count histogram of mice before and after vaginal modeling and drug administration in different experimental groups in Example 4 of the present invention. In the figure, Figure A is the counting result of Gardnerella vaginalis, and Figure B is the counting result of Lactobacillus. Detailed implementation manners

[0054] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0055] The components and preparation methods of the bacterial culture media used in the following examples are as follows:

[0056] Preparation of MRS broth / agar: Weigh 24.05 g of the finished MRS broth powder, dissolve it in 500 mL of deionized water, adjust the pH to 6.2 ± 0.2, label the name of the medium and the preparation date, place it in an autoclave and sterilize at 121 °C for 15 min. After cooling, store it in a 4 °C refrigerator for later use; the agar plate is prepared by adding 1.5% agar to the broth medium.

[0057] Preparation of LB broth / agar: Weigh 10.00 g of the finished LB broth powder, dissolve it in 500 mL of deionized water, adjust the pH to 7.0 ± 0.2, label the name of the medium and the preparation date, place it in an autoclave and sterilize at 121 °C for 15 min. After cooling, store it in a 4 °C refrigerator for later use; the agar plate is prepared by adding 1.5% agar to the broth medium.

[0058] Preparation of rabbit blood BHI broth / agar: Weigh 19.25 g of the finished BHI broth powder and 5.0 g of maltose, dissolve them in 500 mL of deionized water, adjust the pH to 7.4 ± 0.2, label the name of the medium and the preparation date, place it in an autoclave and sterilize at 121 °C for 15 min. After the medium cools to 50 - 60 °C, add sterile defibrinated rabbit blood and vitamin mixture. After cooling, store it in a 4 °C refrigerator for later use; the agar plate is prepared by adding 1.5% agar to the broth medium.

[0059] The methods for isolating and obtaining various strains used in each example are as follows:

[0060] 1. Isolation and obtaining of Lactobacillus delbrueckii DM8909

[0061] Purchased the drug "Ding Junsheng" from a pharmacy store. Use an inoculation loop to dip the bacterial powder in the capsule into MRS broth, place it in an anaerobic incubator at 37 °C for 24 h, streak and isolate it on MRS agar, and incubate anaerobically at 37 °C for 48 h. Pick a single colony into MRS broth for expanded culture. Finally, take the cultured bacterial solution for 16S rDNA sequence identification. The 16S rDNA sequence of Lactobacillus delbrueckii DM8909 obtained by identification is shown in SEQ ID No:3. Preserve the bacterial solution and store it frozen in an -80 °C refrigerator to obtain the reference strain Lactobacillus delbrueckii DM8909.

[0062] 2. Obtaining of pathogenic strains

[0063] The vaginal secretion samples of patients with bacterial vaginitis (BV) were diluted and spread on rabbit blood BHI agar, and anaerobically cultured in a 37 °C incubator for 48 h. After picking different morphological single colonies and streaking for purification again, enrichment culture was carried out in rabbit blood BHI broth (37 °C, anaerobic culture for 24 h). Part of the bacterial solution was used for preservation, and the other part was used for strain identification. According to the comparison results, the preserved strains were confirmed to include Gardnerella vaginalis (16S rDNA sequence as shown in SEQ ID No:4), Escherichia coli (16S rDNA sequence as shown in SEQ ID No:5), Pseudomonas aeruginosa (16S rDNA sequence as shown in SEQ ID No:6), and Aerococcus christensenii (16S rDNA sequence as shown in SEQ ID No:7), all of which are common pathogenic bacteria carried by BV patients.

[0064] In addition, the uropathogenic Escherichia coli ATCC700928 strain (isolated from a patient with urinary tract infection) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. in China, and the Gardnerella vaginalis ATCC 14018 type strain (isolated from a BV patient) was purchased from the Guangdong Provincial Culture Collection Center of Microorganisms.

[0065] Example 1 Isolation and identification of LSA-410 strain

[0066] 1. Isolation of Lactobacillus flora

[0067] Vaginal secretion samples of reproductive-age women who passed a physical examination were collected with vaginal swabs. The vaginal swabs were placed in ESwab transport and preservation solution (Copan), shaken and mixed evenly, and the samples were taken and added to sterile PBS solution for serial ten-fold gradient dilution. Different concentration dilutions were spread on MRS agar and anaerobically cultured in a 37 °C incubator for 48 h. Since Lactobacillus has functions such as regulating the human immune function and inhibiting the infection of pathogenic bacteria, and its safety is highly recognized, strains with typical characteristics of Lactobacillus such as white round edges and shiny surfaces in the growth colony morphology were picked and streaked on MRS agar until obvious colonies grew again. Single colonies were picked into MRS broth for enrichment culture. Part of the cultured bacterial solution was used for preservation, and the other part was used for bacterial DNA extraction and gene sequencing identification to confirm the classification information of each strain. One of the isolated Lactobacillus strains had a strong lactic acid-producing ability during the primary screening process and had a strong inhibitory effect on urogenital pathogenic bacteria such as uropathogenic Escherichia coli and Gardnerella vaginalis, so it was named LSA-410 strain and the strain characteristics were further detected.

[0068] 2. Strain culture characteristics

[0069] The LSA-410 strain was streaked on MRS agar and anaerobically cultured for 48 h, and the results are as Figure 1As shown, the colony is white, round, raised, very plump, with a smooth surface, regular edges, and stable size and shape. The colony was picked and smeared for Gram staining, and the microscopic examination results are as Figure 2 shown. This bacterium is Gram-positive, with round-ended short rod shapes, 0.4 - 0.6 μm * 0.9 - 1.8 μm, and can be distributed singly, in pairs, or in short chains.

[0070] 3. Identification of the 16S rDNA gene sequence and pheS gene sequence of the strain

[0071] Identification of the 16S rDNA gene sequence: The DNA of the strain was extracted using a rapid bacterial genomic DNA extraction kit. Primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No: 8) and 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID No: 9) were selected to amplify the conserved region of the 16S rDNA gene. The PCR conditions were: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, for a total of 35 cycles; extension at 72 °C for 10 min.

[0072] Identification of the pheS gene sequence: Primers pheS-21-F: 5’-CAYCCNGCHCGYGAYATGC-3’ (SEQ IDNo: 10) and pheS-23-R: 5’-GGRTGRACCATVCCNGCHCC-3’ (SEQ ID No: 11) were selected. The PCR conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 1 min, annealing at 46 °C for 135 s, extension at 72 °C for 75 s, for a total of 3 cycles; denaturation at 95 °C for 35 s, annealing at 46 °C for 75 s, extension at 72 °C for 75 s, for a total of 30 cycles; extension at 72 °C for 7 min.

[0073] For the above PCR amplifying the 16S rDNA gene and pheS gene, the same amplification system can be used: 12.5 μL TaqPCR Master Mix buffer, 1 μL each of the upstream and downstream primers with a concentration of 10 μmol, 1 - 2 μL DNA template, and the remainder was supplemented with nuclease-free water to 25 μL.

[0074] The obtained PCR products were detected using 1% gel electrophoresis. For positive results, the products were sent to a sequencing company for bidirectional sequencing. Subsequently, Blast sequence analysis of the obtained bacterial sequences was performed on the NCBI database.

[0075] After sequencing and identification, it was determined that the name of strain LSA-410 is Lactiplantibacillus plantarum ( Lactiplantibacillusplantarum ), formerly known as Lactobacillus plantarum ( Lactobacillus plantarum ).

[0076] 4. Determination of physiological and biochemical characteristics of LSA-410 strain

[0077] Centrifuge the bacterial solution of the isolated LSA-410 strain, discard the supernatant, resuspend the cells, and adjust to a 3 McFarland standard bacterial suspension. Use the bioMérieux anaerobic and corynebacterium identification card (VITEK ANC) to determine the physiological and biochemical characteristics. The results are shown in Table 1.

[0078] Table 1 Physiological and biochemical characteristics of Lactobacillus plantarum LSA-410

[0079]

[0080] "+" indicates positive. "w" indicates weakly positive, and "-" indicates negative.

[0081] Example 2 Detection of probiotic characteristics of LSA-410 strain

[0082] 1. Detection of acid tolerance

[0083] Centrifuge the bacterial solution of the LSA-410 strain isolated in Example 1, discard the supernatant, resuspend the cells, and adjust the turbidity of the liquid to an appropriate turbidity (~10 8 CFU / mL). Subsequently, inoculate into MRS broth with pH values of 3.2, 3.7, 4.2, 4.7, 5.2, 5.7, and 6.2 (inoculation amount is 5% v / v), and continuously culture anaerobically at 37 °C for 30 h. According to the change in absorbance of the bacterial solution at OD 600 , determine the acid tolerance of the strain.

[0084] The results are as Figure 3 shown. The LSA-410 strain reaches the stationary phase at 14 - 18 h and grows vigorously under the conditions of pH 4.7 to 6.2; in an environment with pH 4.2, the absorbance value of the LSA-410 strain at OD 600 reaches a maximum of 0.92 and is slightly inhibited; in an environment with pH less than 3.7, the growth of the LSA-410 strain is inhibited. The above results indicate that the LSA-410 strain can well adapt to the acidic environment of the female vagina.

[0085] 2. Determination of lactic acid production

[0086] Since Lactobacillus delbrueckii DM8909 is the only lactobacillus microbial preparation for the treatment of female reproductive health on the current market, it is used as a control strain. Centrifuge the bacterial solutions of Lactobacillus delbrueckii DM8909 and the LSA-410 strain respectively, discard the supernatant, resuspend the cells, and adjust the turbidity to an appropriate turbidity (~108 CFU / mL), inoculate the bacterial suspension into MRS broth (inoculation amount is 5% v / v), and culture anaerobically at 37 °C for 24 h. Centrifuge the obtained bacterial liquid, and take the supernatant for the determination of lactic acid content. Use high-performance liquid chromatography to detect the contents of D-lactic acid and L-lactic acid in the fermentation broth. The specific chromatographic conditions are shown in Table 2, and the determination results of lactic acid content in the fermentation broth are shown in Table 3.

[0087] Table 2 Chromatographic conditions for the determination of lactic acid content in Lactobacillus fermentation broth by high-performance liquid chromatography

[0088]

[0089] Table 3 Lactic acid content in the fermentation broth of LSA-410 strain and control strain

[0090]

[0091] Note: "ND" indicates no production of this type of lactic acid

[0092] As can be seen from Table 3, the LSA-410 strain isolated in Example 1 can produce both D-lactic acid and L-lactic acid, and the lactic acid production is mainly D-lactic acid, accounting for 64.82% of the total lactic acid; the total lactic acid content is significantly higher than that of Lactobacillus delbrueckii DM8909, and it has a strong lactic acid-producing ability. Combining the above results that the LSA-410 strain can continuously grow and produce acid in an environment with a pH value of 3.7 - 6.2, it can be considered that it is of great significance for maintaining the low pH of the vagina. Since pathogenic bacteria such as Candida albicans and Candida glabrata in the genus Candida are more dependent on the pH of the living environment, and the most suitable pH value is about 5.5, it can be inferred that the LSA-410 strain has the effect of inhibiting Candida albicans and Candida glabrata.

[0093] 3. Determination of the adhesion ability to cervical cancer epithelial cells (Hela cells) and human bladder transitional cell carcinoma cells (T24 cells)

[0094] Use Lactobacillus delbrueckii DM8909 as the control strain. Centrifuge the bacterial liquids of Lactobacillus delbrueckii DM8909 and LSA-410 strain respectively, discard the supernatant, resuspend the bacterial cells with PBS buffer and adjust to an appropriate turbidity (~10 8 CFU / mL), and centrifuge to remove the supernatant. When detecting the adhesion to Hela cells, resuspend the bacterial cells with an equal volume of complete medium (90% DMEM high-glucose medium + 10% FBS) to obtain a bacterial suspension; when detecting the adhesion to T24 cells, resuspend the bacterial cells with an equal volume of McCoy's 5a medium to obtain a bacterial suspension. Hela cells (purchased from the Cell Bank of the Chinese Academy of Sciences) or T24 cells (purchased from Beina Biotechnology) cultured in a T75 culture flask at 1×10 6Inoculate 1 mL at a cell density of cells / mL into a 12-well plate. After culturing for 24 h, a single-cell layer is formed. Aspirate the culture medium in the cell culture plate, add 1 mL of PBS solution to each well to wash the cells 3 times, then add 1 mL of bacterial suspension to make the bacteria fully dispersed and uniform. Place the cell culture plate and the remaining bacterial suspension in an incubator at 37 °C and 5% CO2 for adhesion for 3 h, with 3 replicates in each group. After the adhesion is completed, aspirate the culture medium in the cell culture plate, add 1 mL of PBS solution to each well to wash the cells 3 times, add 1 mL of 0.1% TritonX-100 PBS solution to lyse the cells for 5 min, make a bacterial suspension, dilute it in gradient, take 100 μl of the bacterial solution and spread it on MRS agar for anaerobic culture for 48 h, and then count the lactobacilli.

[0095] The calculation formula for the adhesion rate is as follows:

[0096] Adhesion rate = Strain survival rate (%) = N l / N0 × 100%;

[0097] In the formula, N1 - the number of viable bacteria after the strain adhesion treatment (CFU / mL); N0 - the number of viable bacteria before the strain adhesion (CFU / mL).

[0098] Table 4 Results of cell adhesion

[0099]

[0100] The adhesion rate results of Lactiplantibacillus plantarum LSA-410 strain to cervical cancer epithelial cells (Hela cells) and human bladder transitional cell carcinoma cells (T24 cells) are shown in Table 4. The LSA-410 strain has a high adhesion ability to cervical cancer epithelial cells (Hela cells) and human bladder transitional cell carcinoma cells (T24 cells). It can be inferred from the excellent adhesion ability that the colonization ability of the LSA-410 strain is better, which helps to reconstruct a healthy vaginal microecology.

[0101] 4. Determination of the in vitro antibacterial ability of the LSA-410 strain

[0102] Using Lactobacillus delbrueckii DM8909 as the control strain, centrifuge the bacterial suspensions of Lactobacillus delbrueckii DM8909 and LSA-410 strain respectively, discard the supernatant, resuspend the bacterial cells, and adjust to an appropriate turbidity (~10 8 CFU / mL). Take 0.5 mL of the resuspended solution and mix it with 20 mL of MRS agar at 50 - 60 °C. After cooling and solidifying, culture it anaerobically at 37 °C for 24 h, and then punch holes to prepare bacterial cakes. Centrifuge the activated test bacteria, discard the supernatant, resuspend the bacterial cells, and adjust to an appropriate turbidity (~10 8CFU / mL), 100 μL of the test bacterial suspension was inoculated onto the agar medium, evenly spread using glass beads, and the prepared bacterial cake was placed on the agar plate coated with the pathogenic bacterium. Anaerobic culture was carried out at 37 °C until obvious colonies formed, and then the diameter of the inhibition zone was measured using a vernier caliper. The medium for Escherichia coli was LB agar medium, and the media for Gardnerella vaginalis, Aerococcus christensenii, and Pseudomonas aeruginosa were rabbit blood BHI agar medium.

[0103] The experimental results are shown in Figure 4 and Table 5. Figure 4 Uropathogenic Escherichia coli ATCC700928 was cultured in the agar medium of plate A, Figure 4 Escherichia coli isolated from BV patients was cultured in the agar medium of plate B, Figure 4 Gardnerella vaginalis ATCC 14018 was cultured in the agar medium of plate C, Figure 4 Gardnerella vaginalis isolated from BV patients was cultured in the agar medium of plate D, Figure 4 Aerococcus christensenii isolated from BV patients was cultured in the agar medium of plate E, Figure 4 Pseudomonas aeruginosa isolated from BV patients was cultured in the agar medium of plate F. As Figure 4 shown in A, the diameter of the inhibition zone of LSA-410 strain against uropathogenic Escherichia coli was 15.79 mm, which was greater than 14.03 mm of DM8909 strain. As Figure 4 shown in B, the diameter of the inhibition zone of LSA-410 strain against Escherichia coli isolated from BV patients was 16.03 mm, which was about 1 mm higher than the inhibition zone diameter of DM8909 strain. As Figure 4 shown in C and D, the diameter of the inhibition zone of LSA-410 strain against the two strains of Gardnerella vaginalis was as high as 24 - 25 mm, much higher than 17.26 mm and 15.59 mm of DM8909 strain. As Figure 4 shown in E and F, the inhibition zones of LSA-410 strain against Aerococcus christensenii and Pseudomonas aeruginosa isolated from BV patients were also higher than those of DM8909, being 24.11 mm and 17.37 mm respectively. The above results indicate that LSA-410 strain has stronger antibacterial ability than Lactobacillus delbrueckii DM8909 against common pathogenic bacteria in the urogenital tract.

[0104] Table 5 Inhibition zone diameters (mm) of Lactiplantibacillus plantarum LSA-410 against different test bacteria

[0105]

[0106] Example 3 Safety detection of LSA-410 strain

[0107] 1. Hemolytic assay of LSA-410 strain

[0108] Using Listeria innocua ( Listeria innocua ), CICC 10417 as the negative control and Staphylococcus aureus ( Staphylococcus aureus ), CICC 10473 as the positive control, the LSA-410 strain was inoculated on a blood agar plate to determine its hemolytic activity. The results are as shown in Figure 5 . Staphylococcus aureus CICC 10473 produced an obvious hemolytic ring, while neither Listeria innocua CICC 10417 nor the LSA-410 strain showed a hemolytic ring, indicating that the strain is non-pathogenic.

[0109] 2. Antimicrobial susceptibility assay of LSA-410 strain

[0110] According to the reference method for antimicrobial susceptibility testing of fastidious and rare bacteria by broth dilution method CLSI M45-A3, the susceptibilities of the LSA-410 strain to ampicillin (AM), clindamycin (CM), daptomycin (DPC), erythromycin (ERY), linezolid (LZ), meropenem (MRP), and vancomycin (VA) were determined. The results are as shown in Figure 6 and Table 6. The LSA-410 strain was sensitive to ampicillin, daptomycin, erythromycin, linezolid, and meropenem; intermediate to clindamycin; and resistant to vancomycin.

[0111] Table 6 MIC values and drug susceptibilities of Lactiplantibacillus plantarum LSA-410 to antimicrobial agents

[0112]

[0113] 3. Toxicity assay on cervical cancer epithelial cells (Hela cells)

[0114] Using Lactobacillus delbrueckii subsp. bulgaricus DM8909 as the control strain, the bacterial suspensions of Lactobacillus delbrueckii subsp. bulgaricus DM8909 and the LSA-410 strain were centrifuged respectively, the supernatants were discarded, and the bacterial cells were resuspended in PBS buffer and adjusted to an appropriate turbidity (~10 8 CFU / mL). After centrifugation to remove the supernatant, the bacterial cells were resuspended with an equal volume of complete medium (90% DMEM high-glucose medium + 10% FBS) to obtain bacterial suspensions. Hela cells cultured in a T75 cell culture flask were seeded at a density of 1×10 6Inoculate 1 mL at a cell density of cells / mL into a 12-well plate. After culturing for 24 h, a single-cell layer is formed. Aspirate the culture medium in the cell culture plate, add 1 mL of PBS solution to each well to wash the cells 3 times, and then add 1 mL of bacterial suspension to evenly disperse the bacteria. Place the cell culture plate in a 37 °C, 5% CO2 incubator for co-culture. There are 2 parallels in each group, and the cell density viability is detected at 0, 2, 4, 5, 6, 7, 8, 9, and 10 h respectively.

[0115] The results are as Figure 7 shown. During the experiment, the viable cell density of different experimental groups was approximately 10 6 Cells / mL. Although the viable cell density of the two Lactobacillus experimental groups showed different downward trends at 7 h, the number of viable cells of the LSA-410 strain was higher than or similar to that of the DM8909 group during the adhesion process. See Figure 7 A. The cell survival rate of the LSA-410 strain group remained above 90% in the first 7 h of the experiment, showed a downward trend after 8 h, and decreased to 68.46% at 10 h. The cell survival rate of the DM8909 group began to decrease after 7 h, decreased to 66.31% at 10 h, and was significantly lower than that of the Lactobacillus plantarum LSA-410 group at the three time points of 7, 8, and 9 h. See Figure 7 B. The results indicate that the adhesion of the LSA-410 strain has no significant effect on cervical cancer epithelial cells (Hela cells) and no obvious toxic effect.

[0116] Example 4 Determination of the Therapeutic Ability of Lactobacillus plantarum LSA-410 on a Mouse Model Infected with Gardnerella vaginalis

[0117] Select 24 female C57BL / 6 mice weighing 19 - 22 g and 5 weeks old, and randomly divide them into a healthy group, an infection group, a metronidazole group, and a Lactobacillus (LSA-410 strain) group, with 6 mice in each group. The modeling and treatment protocols are set as Figure 8 shown. Define the first day of starting to establish the Gardnerella vaginalis model in mice as Day1. From Day1 to Day3 of establishing the Gardnerella vaginalis model in mice, each mouse is injected with 0.5 mg of estradiol daily for estrogenization. Subsequently, 20 μL of a suitable concentration of Gardnerella vaginalis solution (~10 8 CFU / mL) is injected into the vagina of the mice, and inoculation is continued for 4 days (Day4 - Day8). The mice in the healthy group are injected with 20 μL of normal saline instead of the bacterial solution. On Day8, a small amount of mucus is dipped from the vagina of the mice with a sterile swab, and it is observed under a microscope after staining to check for the presence of Gardnerella vaginalis, so as to ensure the colonization of Gardnerella vaginalis in the vagina of the mice and the establishment of the Gardnerella vaginalis model in mice.

[0118] Centrifuge the activated LSA-410 strain, discard the supernatant, and resuspend the bacterial pellet with PBS solution to an appropriate turbidity (~10 9 CFU / mL). From the first day to the seventh day after modeling (Day8~Day15), for 7 consecutive days, once a day, intravaginally instill 20 μL of freshly prepared LSA-410 strain bacterial solution into the mice in the Lactobacillus group; inject 20 μL of metronidazole solution into the vagina of the mice in the metronidazole group; give the mice in the healthy group and the infection group the same volume of normal saline intravaginally every day.

[0119] Before drug administration, on the 1st day after drug administration, and on the 7th day after drug administration, use a micropipette to take 50 μL of normal saline and repeatedly rinse the vagina of the mice 5-6 times. Take 30 μL of the above lavage fluid, dilute it to an appropriate gradient, and then spread it on an agar medium for counting Lactobacillus and Gardnerella vaginalis. The counting of Lactobacillus uses MRS agar, and the counting of Gardnerella vaginalis uses Columbia blood agar plates supplemented with gentamicin sulfate (4 mg / L), nalidixic acid (30 mg / L), and amphotericin B (2 mg / L).

[0120] The results are as Figure 9 shown. It can be seen that on the 1st day after drug administration, compared with the infection group, the number of Gardnerella vaginalis in the vagina of the mice instilled with LSA-410 strain bacterial solution decreased significantly by about one order of magnitude. Although the number of viable Gardnerella vaginalis in the vagina of the Lactobacillus group was higher than that in the metronidazole group, the number of Lactobacillus in its sample was about 8 times that of the metronidazole group and about 5 times that of the infection group. On the 7th day after drug administration, the number of Gardnerella vaginalis in the vagina of the mice in the Lactobacillus group was similar to that in the metronidazole group, but the number of Lactobacillus was about 3 orders of magnitude higher than that in the metronidazole group and about 6 times that of the healthy group. The above results indicate that the LSA-410 strain has an inhibitory ability similar to that of metronidazole against Gardnerella vaginalis, and at the same time can successfully colonize in the vagina of mice and restore the number of Lactobacillus in the vagina to normal levels and above, indicating that the LSA-410 strain has a therapeutic effect on bacterial vaginitis caused by Gardnerella vaginalis infection.

[0121] Based on the above detection results, the LSA-410 strain has a strong lactic acid-producing ability, has good antibacterial effects against urogenital pathogenic bacteria such as uropathogenic Escherichia coli and Gardnerella vaginalis, and has a high adhesion ability to human cervical cancer epithelial cells (Hela cells). The LSA-410 strain can be used to develop products such as hygiene products and pharmaceutical preparations, and has the advantages of safety, reliability, few adverse reactions, and good effects. It can be used to treat and / or prevent female lower urinary tract infections and vaginitis-related diseases, regulate the microecological balance of the female reproductive tract, prevent female reproductive tract diseases, reduce the probability of female reproductive tract diseases, and is of great significance to female reproductive health. Therefore, on August 18, 2022, the LSA-410 strain was deposited with the Guangdong Provincial Culture Collection Center of Microorganisms (abbreviation: GDMCC, address: 5th Floor, Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province), and the deposit number is GDMCC No. 62712.

[0122] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A strain, characterized in that, The strain is Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), which is deposited in the Guangdong Microbial Culture Collection Center with the deposit number of GDMCC No. 62712 and the deposit date of August 18, 2022.

2. A product, characterized in that, comprises at least one of the following (1) to (6): (1) the strain according to claim 1; (2) a microbial agent containing the strain according to claim 1; (3) a viable bacteria solution containing the strain according to claim 1.

3. The product according to claim 2, wherein The product is a drug, a medical device or a sanitary product.

4. The product according to claim 3, wherein The sanitary product is at least one of a tampon, a sanitary napkin, a menstrual pad, a diaper, a soap and a condom.

5. The product according to claim 3, wherein The drug further comprises a pharmaceutical carrier and / or pharmaceutical excipients, and the pharmaceutical excipients comprise at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, a sweetening agent and a flavoring agent.

6. The product according to claim 2, wherein The dosage form of the product is one of a powder, an ointment, a gel, a drop, a suppository, a lozenge, a granule, a capsule, a spray, a tablet, a pill or a solution.

7. The product according to claim 6, characterized in that, The capsule is a soft capsule.

8. Use of the strain according to claim 1, or the product according to any one of claims 2 to 7, in the preparation of an antibacterial product, wherein the pathogenic bacteria inhibited by the antibacterial product include any one or more of uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa and Aerococcus christensenii.

9. Use of the strain according to claim 1, or the product according to any one of claims 2 to 7, in the preparation of a drug for treating or preventing urogenital tract infections, or a vaginal care product.

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