Lactipranthi Bacillus plantarum strains and their uses

Abstract

The present invention provides Lactiplantibacillus plantarum 1-D1 strain and uses thereof. The Lactiplantibacillus plantarum 1-D1 strain provided by the present invention is genetically stable, lacks pathogenicity / virulence genes, is highly safe, has strong antibacterial activity against pathogenic bacteria, lowers vaginal pH, and maintains a healthy vaginal acidic environment. At the same time, it has excellent adhesion to HeLa cells, adhering to vaginal epithelial cells to form a microecological barrier, preventing pathogens from adhering to or competing with epithelial cell receptors. This strain can be used in the preparation of medicines, foods, and hygiene products for the prevention and treatment of vaginitis.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a lactipranthi Bacillus plantarum strain and its uses. [Background technology]

[0002] Lactobacillus is the predominant bacterial flora in the vagina of healthy women of childbearing age, accounting for more than 70% of the vaginal flora. Studies have shown that Lactobacillus in the female genital tract suppresses pathogenic microorganisms primarily through the production of lactic acid and bacteriocins.

[0003] Genital tract infections account for 40.2% to 55.6% of outpatient visits to obstetrics and gynecology departments in China. There are at least 200 million cases of genital tract infection-related diseases, 100 million of which are recurrent, resulting in annual medical costs exceeding 20 billion yuan. Research has shown that 99.3% of gynecological outpatients with vaginal infection symptoms have an imbalance in the vaginal microecology. In 2016, the Infectious Diseases Collaboration Group of the Obstetrics and Gynecology Branch of the Chinese Medical Society proposed that the essence of vaginal infection is "vaginal microecological imbalance." Prolonged abnormalities in the vaginal microecology weaken the vagina's resistance to pathogenic microorganisms, which is often a major cause of recurrent vaginal infections and secondary infections.

[0004] Common vaginal infections include bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), trichomonas vaginitis (TV), and aerobic vaginitis (AV), all of which are associated with a reduction or disappearance of Lactobacillus bacteria. Conventional treatment strategies for vaginal infections are primarily antibacterial, with treatment with antibiotics such as clindamycin suppressing the growth of vaginal Lactobacillus bacteria while inhibiting the growth of bacterial vaginosis pathogens. Metronidazole, while not inhibiting Lactobacillus growth at the appropriate dosage, does not promote Lactobacillus recovery. Furthermore, research has shown that the resistance rate to metronidazole is 63.8% and to clindamycin is 24.1%-67%.

[0005] Furthermore, the recurrence and resistance rates after antibiotic use are very high. Antibiotics can only suppress floating Gardnerella vaginalis and temporarily alleviate symptoms, but they cannot completely eliminate the biofilm and the Gardnerella vaginalis contained within it. Once treatment is discontinued, the bacteria in the biofilm can recover, multiply, and spread again, leading to recurrence of bacterial vaginosis. Biofilms can reduce bacterial sensitivity to antibiotics and increase their resistance, and resistance in pathogenic strains can be acquired through mobile genetic elements, making antibiotic treatment less effective or even ineffective.

[0006] Research by Narisu et al. has shown that vaginal Lactobacillus bacteria inhibit biofilm formation by Gardnerella vaginalis. The Infectious Disease Collaborative Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association has currently suggested that the application of vaginal microecological preparations can restore a mildly acidic environment dominated by functional Lactobacillus bacteria, promote vaginal microecological balance and immunomodulation, and reduce the recurrence of vaginal infections (Narisu, et al., "Preliminary Study on the Effects of Vaginal Lactobacillus on Gardnerella vaginalis Biofilms [J]." Progress in Obstetrics and Gynecology, 2015, 24(09): 641-645). Currently, there are only two vaginal microecological preparations commercially available in China. One, called "Yanhua" manufactured by Xi'an Zhenghao Biopharmaceutical Co., Ltd., contains a Streptococcus faecalis strain, which is not the dominant vaginal bacterial species and is conditionally pathogenic. The other is a commercially available product called "Wanze Shuangqi" manufactured by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., which contains Lactobacillus delbrueckii, a type of lactobacillus bacteria.

[0007] Currently available vaginal microbiological preparations are far from meeting clinical needs and have significant room for optimization. Therefore, developing Lactobacillus strains with stronger probiotic potential for the prevention and / or treatment of vaginal infections would be of great application value. Summary of the Invention

[0008] The objective of the present invention is to provide a Lactiplantibacillus plantarum 1-D1 strain that can inhibit the growth of pathogenic bacteria in the vagina and regulate the balance of the vaginal microecology, has the effects of preventing and treating vaginitis, and can be used in the preparation of medicines, foods, and hygiene products for preventing and treating vaginitis.

[0009] In order to achieve the objectives of the present invention, the present invention provides the following technical solutions:

[0010] The strain is Lactiplantibillus plantarum 1-D1, which has the accession number of CCTCC NO: M 20221191.

[0011] Use of the Lactiplantibacillus plantarum 1-D1 strain provided by the present invention in the preparation of an antibacterial product.

[0012] Furthermore, the antibacterial products include, but are not limited to, bacteria inhibitors, feed additives, antibacterial peptides, etc.

[0013] Furthermore, the antibacterial product is a product that inhibits one or more of Gardnerella, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Shigella dysenteriae.

[0014] Use of the Lactiplantibacillus plantarum 1-D1 strain provided by the present invention in the preparation of a product for preventing and / or treating vaginal infectious diseases.

[0015] The term "treatment" (also referred to as "treat" or "treating") refers to the optional administration of a therapeutic agent according to a treatment regimen that achieves a desired effect of partially or completely alleviating, ameliorating, alleviating, suppressing, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., improving the structure and function of the vaginal flora, increasing the diversity of the vaginal flora, reducing the amount of flora associated with inflammation, treating vaginitis). In some embodiments, the administration of a therapeutic agent according to a treatment regimen is associated with achieving a desired effect. Such treatment may be directed to subjects who do not exhibit the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be directed to subjects who exhibit one or more confirmed signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0016] Such products include, but are not limited to, medicines, foods, health products, hygiene products, etc., and when applied to products, the strains may be active or inactivated.

[0017] The term "drug" includes drugs used in both human and veterinary medicine for both humans and animals, as well as drugs for incorporation into animal feed (e.g., livestock feed and / or pet food). Furthermore, as used herein, the term "drug" refers to any substance that provides a therapeutic, preventative, and / or beneficial effect. As used herein, the term "drug" is not necessarily limited to substances that require marketing approval, but includes substances that can be used in cosmetics, health products, foods (including, e.g., feed and beverages), probiotic cultures, and dietary supplements.

[0018] The Lactiplantibacillus plantarum 1-D1 strain of the present invention can be added as a beneficial ingredient to fermented foods, immune-boosting health foods, solid beverages for personal care for women and other foods, and can enhance human immunity, especially immunity in the female reproductive tract.

[0019] The vaginal infectious disease is caused by one or more of the pathogens of bacterial vaginosis, pathogens of vulvovaginal candidiasis, Trichomonas vaginitis, and aerobic vaginitis.

[0020] Furthermore, the pathogenic bacteria of aerobic vaginitis include, but are not limited to, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella, and the like.

[0021] Furthermore, the causative bacterium of bacterial vaginosis is Gardnerella vaginalis.

[0022] Additionally, the product is a product that lowers vaginal pH.

[0023] Furthermore, the product is one that is metabolized in the vaginal environment to produce H2O2, organic acids and / or bacteriocins.

[0024] Use of the Lactiplantibacillus plantarum 1-D1 strain provided by the present invention in the preparation of a product for regulating the balance of the microecology of bacterial flora.

[0025] Use of the Lactiplantibacillus plantarum 1-D1 strain provided by the present invention in the production of hygiene products.

[0026] The present invention also covers the use of the Lactiplantibacillus plantarum strain 1-D1 as a probiotic.

[0027] The present invention also provides a product containing Lactiplantibacillus plantarum as an active ingredient, wherein the Lactiplantibacillus plantarum is the aforementioned Lactiplantibacillus plantarum 1-D1 strain.

[0028] Furthermore, the products include, but are not limited to, foods, health products, medicines, daily necessities, etc.

[0029] Furthermore, the product is for preventing and / or treating infectious vaginal diseases.

[0030] The products of the present invention may also include a pharmaceutically acceptable excipient or diluent.

[0031] Beneficial Effects: (1) The Lactiplantibacillus plantarum 1-D1 strain of the present invention is genetically stable, does not contain any pathogenic / virulence genes, is highly safe, and exhibits good inhibitory ability against Gardnerella vaginalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B, and Shigella dysenteriae.

[0032] (2) The Lactiplantibacillus plantarum 1-D1 strain provided by the present invention has a strong ability to produce lactic acid, which can lower the vaginal pH, maintain a healthy vaginal acidic environment, and improve the balance of vaginal bacterial flora. At the same time, the antibacterial substances it produces, such as hydrogen peroxide, bacteriocins, and organic acids, can inhibit the growth and proliferation of pathogenic bacteria, improve mucosal immunity, and enhance the host's anti-infection effect.

[0033] (3) The Lactiplantibacillus plantarum 1-D1 strain provided by the present invention has advantageous cell adhesion properties and can adhere to vaginal epithelial cells to form a microecological barrier, preventing pathogens from settling on or competing with epithelial cell receptors.

[0034] (4) The various evaluation indexes of the Lactiplantibacillus plantarum 1-D1 strain provided by the present invention are significantly superior to those of commercially available products such as Wanze Shuangqi and Metronidazole, and have good industrial and promotional value.

[0035] Strain storage information: Lactiplantibacillus plantarum 1-D1 strain was developed on July 2, 2022. 7The strain was deposited at the China Center for Type Culture Collection on the date of this writing. The strain has the accession number CCTCC NO: M 20221191 and its address is Wuhan, China. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 is a front photograph of the colony morphology of Lactiplantibacillus plantarum strain 1-D1. [Figure 2] FIG. 2 is a photograph of the Gram staining observation of Lactiplantibacillus plantarum 1-D1 strain. [Figure 3] Figure 3 shows the ANI comparison of the whole genomes of Lactiplantibacillus plantarum strain 1-D1 and the standard strain. [Figure 4] FIG. 4 shows the results of the hemolytic test (left: Staphylococcus aureus ATCC 25923 strain, right: Lactiplantibacillus plantarum 1-D1 strain). [Figure 5] FIG. 5 shows the trend of body weight change in a toxicity test in mice. [Figure 6] FIG. 6 shows the colonization score of Lactobacillus vaginalis in SD rats. DETAILED DESCRIPTION OF THE INVENTION

[0037] In order to allow those skilled in the art to better understand the present invention, the present invention will be described in more detail below with reference to specific embodiments. Those skilled in the art should understand that this should not be interpreted as limiting the scope of the claims of the present invention. Unless defined otherwise, technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It should also be noted that the reagents or instruments used in the present invention are commercially available unless otherwise specified. The methods for preparing the culture media used in the following embodiments are all known methods.

[0038] In this invention, Lactobacillus delbrueckii (hereinafter collectively referred to as DJS-H3) isolated from the Dingjunsheng dynasty was used as a positive control at each stage. Through screening in low pH medium, screening for amplifying growth ability, and screening for calcium carbonate clear zones, lactobacillus bacteria with low pH tolerance, strong reproductive ability, and strong lactic acid production ability were rapidly isolated. Next, through adhesion ability to HeLa cells and antibacterial tests, dominant lactobacillus bacteria with strong colonization ability and strong ability to inhibit the growth of pathogenic bacteria were selected. Further detailed research was conducted on the Lactiplantibacillus plantarum 1-D1 strain provided by this invention to examine its genetic stability and safety, providing further support for its commercial application. Representative specific embodiments are as follows:

[0039] DMEM medium: Gibco, cat:11995065 MRS liquid medium: HuanKai Biology, cat:1110151 2% Calcium Carbonate-0.8% MRS: Sangon, H122BA0030 Agar: Sangon Biotech, A505255-0250 HeLa cells: BeiNa Biotech, product number: BNCC342189 VK2 / E6E7 cells:Bluef(Shanghai)Biotechnology

[0040] Example 1 Isolation of Lactobacillus Vaginal secretions from healthy, reproductive-age female volunteers were examined by Gram stain microscopy and Nugent scoring, and 29 healthy, reproductive-age female volunteers were selected. Vaginal swabs from healthy volunteers were placed in MRS acidic liquid medium and incubated overnight at 37°C. The culture was diluted 10-fold, and appropriate gradients were applied to 2% calcium carbonate-0.8% MRS agar plates and incubated at 37°C for 36–48 hours. Single colonies with clear, transparent circles were selected from the cultured calcium carbonate-MRS plates, transferred to MRS liquid medium, and incubated overnight at 37°C. 1,816 cultured strains were amplified by PCR and sequenced for 16S rRNA sequencing. After removing remaining acid-producing non-lactobacilli, a total of 72 Lactobacillus strains were selected from the different samples and subjected to subsequent screening tests.

[0041] Of the isolated strains, the nucleotide sequences (16S rRNA sequences) of four strains, Lactiplantibacillus plantarum 1-D1, 16-B12, 53-D2, and 51S-H2, are shown in SEQ ID Nos. 1 to 4, respectively.

[0042] Preliminary results showed that the calcium carbonate clear zone formed by Lactiplantibacillus plantarum 1-D1 on calcium carbonate-MRS medium was much larger than that of Lactobacillus delbrueckii DJS-H3 isolated from Dingjunsheng, demonstrating that Lactiplantibacillus plantarum 1-D1 has a significant advantage in lactic acid production. The 16S rRNA sequence of Lactiplantibacillus plantarum 1-D1 was determined.

[0043] Strain storage information: Lactiplantibacillus plantarum 1-D1 strain was identified as a microbial strain of Lactiplantibacillus plantarum.7 The strain was deposited at the China Center for Typical Culture Collection on the date of its release. The accession number is CCTCC NO: M 20221191, and the address is Wuhan, China.

[0044] Lactobacillus crispatus strain 51S-H2 was approved on July 2, 2022. 7 The strain was deposited at the China Center for Typical Culture Collection on the date of its release. The accession number is CCTCC NO: M 20221194, and the address is Wuhan, China.

[0045] Lactobacillus paragasseri strain 16-B12 was approved on July 2, 2022. 7 The strain was deposited at the China Center for Typical Culture Collection on the date of its release. The strain has the accession number CCTCC NO: M 20221193 and its address is Wuhan, China.

[0046] Lactobacillus jensenii strain 53-D2 was identified as a genotype of lactic acid bacteria. 7 The strain was deposited at the China Center for Typical Culture Collection on the date of its release. The accession number is CCTCC NO: M 20221192, and the address is Wuhan, China.

[0047] Following the method for isolating Lactobacillus plantarum described above, Lactobacillus delbrueckii (DJS-H3) was isolated from the jellyfish.

[0048] Example 2 Measurement of growth performance of Lactiplantibacillus plantarum 1-D1 strain Growth curve data from the lactobacillus isolation stage showed that most lactobacilli entered the late logarithmic phase at 12 hours. Therefore, the viable cell count of each strain was measured using the agar injection method 12 hours after inoculation. For the same inoculation time, strains with higher viable cell counts had a clear advantage in growth performance. The top 25 strains with favorable growth were selected for the next test stage. The data showed that Lactiplantibacillus plantarum 1-D1 strain had excellent growth performance and strong reproductive ability, with a viable cell count of 6.37 x 10 after 12 hours. 9 CFU / mL, indicating that the cells are suitable for high-density fermentation in commercial transformation processes.

[0049] The results of determining the culture morphology and microscopic characteristics of Lactiplantibacillus plantarum strain 1-D1 are shown in Figures 1 and 2.

[0050] Example 3 Adhesion ability of Lactiplantibacillus plantarum strain 1-D1 The colonization ability of lactobacilli was evaluated using their adhesion ability to HeLa cells and VK2 / E6E7 cells. First, the lactobacilli were activated: 50 μL of each lactobacillus was taken, placed in 5 mL of MRS liquid medium, and cultured at 37°C for 18-24 hours, then removed and prepared for transfection. The lactobacilli were transfected: 50 μL of the activation solution of each lactobacillus was taken, placed in 5 mL of MRS liquid medium, and cultured at 37°C for 18-24 hours, then centrifuged at 12,000 rpm for 2 minutes to remove the cells, resuspended in DMEM medium, and adjusted to a McFarland turbidity of 0.5. Cell plating: Resuscitated cells were plated at 10 5 500 μL of the cells / mL were inoculated into a 24-well culture plate and placed in a carbon dioxide incubator at 37°C and 5% CO2 for overnight incubation. The DMEM stock solution was discarded, washed three times with PBS, and then 500 μL of DMEM medium was added. Finally, the bacteria and cells were allowed to interact: overnight activated cells suspended in DMEM medium at a concentration of 10 8500 μL of CFU / mL lactobacilli were added to the 24-well culture plate containing the cells and placed in a carbon dioxide incubator. The plates were incubated at 37°C and 5% CO2 for 2 and 4 hours, respectively, and then washed four times with PBS to remove unattached lactobacilli. 500 μL of 0.25% trypsin was added for 2 minutes, followed by the addition of 600 μL of complete medium to terminate the digestion and mix with a pipette. A 10-fold gradient dilution of the cell-bacteria suspension was performed with sterile PBS. 100 μL of the appropriate gradient dilution was added to a disposable sterile plate, over which 0.8% MRS agar medium was added, mixed, and allowed to solidify. The inverted plates were incubated at 37°C for 48 hours, removed, and counted. Each group contained three parallel plates, and the average was used as the final adherent count. A higher number of lactobacilli demonstrated a stronger ability to adhere to cells.

[0051] The cell adhesion ability of Lactiplantibacillus plantarum strain 1-D1 is shown in Tables 1 and 2 below. Fisher LSD average comparison analysis showed that the adhesion ability of Lactiplantibacillus plantarum strain 1-D1 was much higher than that of strain DJS-H3 isolated from Dingjunsheng, with significant differences in adhesion between the groups. The 4-hour adhesion of Lactiplantibacillus plantarum strain 1-D1 to HeLa cells was more than 100 times that of Dingjunsheng, and the 4-hour adhesion of Lactiplantibacillus plantarum strain 1-D1 to VK2 / E6E7 cells was more than 10 times that of Dingjunsheng.

[0052] [Table 1]

[0053] [Table 2]

[0054] Example 4 Antibacterial activity of Lactiplantibacillus plantarum Antibacterial tests were used to evaluate the ability of Lactiplantibacillus plantarum to inhibit various pathogens.

[0055] Lactobacillus bacteria were mixed with 0.8% MRS agar medium to prepare 10 mm lactobacillus cakes. Different pathogenic bacteria (e.g., Gardnerella vaginalis, Staphylococcus aureus) were activated and transfected using the appropriate medium and growth conditions, and then mixed with the corresponding semi-solid medium to prepare separate pathogen plates. The lactobacillus cakes were gently placed on the surface of the pathogen plates and incubated at 37°C for 18-24 hours. The size of the inhibition zone was measured with a vernier caliper to evaluate the ability of each lactobacillus to inhibit pathogens. The specific procedure was as follows:

[0056] After activating Lactiplantibacillus plantarum and Lactobacillus delbrueckii (DJS-H3), 0.1 mL of the bacterial solution was mixed with MRS solid medium and poured into a 6 cm flat dish. After solidification, the mixture was incubated at 37°C for 48 hours. The dish was removed, and a hole was made in the agar medium using a 10 mm inner diameter punch to obtain a Lactobacillus cake for later use.

[0057] Gardnerella vaginalis and Atopobium vaginalis were inoculated into 5% horse serum-anaerobic BHI solution, respectively, and after activation and transfer, they were diluted 10-fold with 0.9% anaerobic saline. 1 mL of the diluted solution was mixed with 10 mL of anaerobic BHI agar medium containing 5% horse serum, poured into a 9 cm flat dish, and left to solidify completely for later use.

[0058] Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella Paratyphi B, and Shigella dysenteriae were inoculated into nutrient broth medium, respectively, and after activation and transfer, the pathogen solution was diluted 100-fold with 0.9% saline in a 10-fold gradient. 1 mL of the diluted solution was mixed with 10 mL of nutrient agar, poured into a 9 cm flat dish, and allowed to solidify completely for later use.

[0059] A Lactobacillus cake was lightly placed on the surface of each pathogen plate, with four bacterial cakes placed symmetrically on each plate, with three parallel plates for each strain. The Gardnerella vaginalis and Atopobium vaginalis plates were placed face up in an anaerobic sealed box equipped with an anaerobic gas-generating bag and incubated at 37°C for 48 hours, while the other pathogen plates were placed face up in an incubator and incubated at 37°C for 24 hours. Finally, the size of the inhibition zone on each plate was measured with a vernier caliper. The antibacterial activity of different Lactobacillus bacteria is shown in Table 3 below.

[0060] [Table 3]

[0061] The results showed that the overall antibacterial activity of Lactiplantibacillus plantarum 1-D1 strain was far superior to that of Lactobacillus delbrueckii DJS-H3 strain isolated from Teijinsho and metronidazole.

[0062] Example 5 Whole genome sequence analysis of Lactiplantibacillus plantarum strain 1-D1 We performed whole-genome sequencing of the Lactiplantibacillus plantarum 1-D1 strain. The whole-genome sequence of the Lactiplantibacillus plantarum 1-D1 strain was uploaded to EzBiocloud and compared with the standard strain. The average nucleotide identity (ANI) ratio was obtained as shown in Figure 3. The whole-genome sequencing results confirmed that the strain was Lactiplantibacillus plantarum (Chinese name: plant lactobacillus).

[0063] The virulence genes in the whole genome sequence were analyzed using the pathogenicity gene database VFDB (identity > 70%, coverage length > 70%) (A defined commensal consortium elicits CD8 T cells and anti-cancer immunity [J]. Nature, 2019, 565 (7741): 1). Artificial Blast analysis using the NCBI database revealed that two hemolysis-related genes (hemolysin and hemolysin III) were 100% identical to those in Lactiplantibacillus plantarum. However, the hemolysin III gene has been confirmed to be widely present in Lactiplantibacillus plantarum (including commercially available Lactiplantibacillus plantarum strains such as L. plantarum 299 V, L. plantarum JDM1, and L. plantarum ST-III) (Chokesajjawatee N, Santiyanont P, Chantarasakha K, et al. Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis [J]. Scientific Reports, 2020, 10(1)). This gene can only lyse bacteria or fungi, but has not been found to lyse mammalian cells. Therefore, strains containing the hemolysin III gene are considered to be safe.For hemolysin to exhibit hemolytic activity, multiple genes must be present simultaneously (Karina, Arellano, Jorge, et al. Safety Evaluation and Whole-Genome Annotation of Lactobacillus plantarum Strains from Different Sources with Special Focus on Isolates from Green Tea. [J]. Probiotics and antimicrobial proteins, 2019). The 1-D1 strain does not contain any genes that interact with hemolysin to cause hemolysis. At the same time, the Lactiplantibacillus plantarum 1-D1 strain does not possess a plasmid and does not contain resistance genes in mobile elements within its genome, eliminating the risk of resistance gene transfer. In summary, the Lactiplantibacillus plantarum 1-D1 strain has good safety performance at the genetic level.

[0064] Example 6 Hemolytic test of Lactiplantibacillus plantarum 1-D1 strain To further confirm the hemolytic potential of Lactiplantibacillus plantarum 1-D1, the strain was streaked onto a blood agar plate (Chengdu Ruiqi, Sichuan Medical Device No. 20152400001) and cultured anaerobically for 48 hours. As a positive control, the β-hemolytic Staphylococcus aureus ATCC 25923 strain (Shanghai Bioplus Biotech Co., Ltd.) was cultured aerobically for 24 hours. The hemolytic potential of Lactiplantibacillus plantarum 1-D1 was determined by observing whether hemolytic rings were formed on the blood agar plate after culture.

[0065] The results in Figure 4 below show that a clear transparent circle, which is typical of β-hemolysis, was observed around the colonies of ATCC 25923 on the positive control plate, but the Lactiplantibacillus plantarum 1-D1 strain did not form a hemolytic ring, indicating that the Lactiplantibacillus plantarum 1-D1 strain does not produce hemolysin that lyses red blood cells, i.e., does not have hemolytic activity.

[0066] Example 7 Toxicity test of Lactiplantibacillus plantarum strain 1-D1 in mice Five mice weighing 18–22 g were used, and each mouse was inoculated with a fresh bacterial solution of Lactiplantibacillus plantarum 1-D1 strain (1.0 × 10 9 CFU / 0.5 mL or more) was administered by oral gavage once daily for three consecutive days, and the animals were continuously observed from the first day to the seventh day of oral gavage.

[0067] The mouse toxicity test showed that the mice had normal body posture, normal limb activity, flexible movement, smooth fur, normal eating and drinking, no abnormalities in excretion or secretion, no obvious symptoms of poisoning, no abnormal animal deaths during the experiment period, and the mice gained weight.

[0068] Example 8 Antibiotic susceptibility test of Lactiplantibacillus plantarum 1-D1 strain In accordance with the requirements for antibiotic susceptibility testing in the 2020 Pharmacopoeia, Part 3, General Description of Microbiological Probiotic Preparations, the agar diffusion disk method was used to measure the antibiotic susceptibility of the strains, and the size of the inhibition zone was used to determine the antibiotic susceptibility level of the strains.

[0069] Standard susceptible strains of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as quality control bacteria. Analysis was performed according to the international standards of the Clinical Laboratory Standards Institute (CLSI) and the People's Republic of China's Standard for Health Industry. Based on the criteria for quality control strains, the susceptibility criteria for Lactiplantibacillus plantarum 1-D1 were established. The susceptibility levels were classified as susceptible, intermediate, and resistant, and represented by S, I, and R, respectively.

[0070] Antibiotic susceptibility testing showed that Lactiplantibacillus plantarum 1-D1 strain was resistant to penicillin, oxacillin, azithromycin, gentamicin, kanamycin, vancomycin, enrofloxacin, clindamycin, and metronidazole, and was susceptible to ampicillin, imipenem, ceftriaxone, amoxicillin, piperacillin, and tetracycline, and intermediately susceptible to erythromycin. On the other hand, metronidazole and clindamycin are commonly used clinically in the treatment of bacterial vaginosis, and the results of this experiment suggest that Lactiplantibacillus plantarum 1-D1 strain could theoretically be used in combination with metronidazole and clindamycin.

[0071] [Table 4]

[0072] [Table 5]

[0073] Example 9 Colonization test of Lactiplantibacillus plantarum 1-D1 in the rat vagina Lactiplantibacillus plantarum 1-D1 strain was administered intravaginally to SD rats once daily for five consecutive days, followed by 10 days of observation. Vaginal secretions were collected before each administration and examined under a Gram stain microscope (D1 secretions were collected before the first administration) to determine colonization with Lactiplantibacillus plantarum 1-D1 strain. The experimental design is shown in Table 6 below.

[0074] Gram staining microscopy revealed that most rats had no microorganisms in their vaginal secretions before the first administration, while a small number of rats occasionally had gram-negative bacilli and gram-positive cocci. After successive administrations, rats administered Lactiplantibacillus plantarum 1-D1 had significantly increased numbers of gram-positive bacilli in their vaginal secretions compared with the blank control group and the pre-administration and blank control groups, demonstrating that Lactiplantibacillus plantarum 1-D1 can successfully colonize the vagina of SD rats. Five days after discontinuation of administration (D10), gram-positive bacilli were still visible within the visual field. Ten days after discontinuation of administration (D15), the vaginal microbial environment of the rats returned to its pre-administration state.

[0075] [Table 6]

[0076] [Table 7]

[0077] It should be understood by those skilled in the art that the embodiments of the present invention described above and illustrated in the accompanying drawings are intended to be illustrative only and are not intended to limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments, but the embodiments of the present invention can be varied or modified in any way without departing from the principles. [Translation of the Certificate of Trust] TIFF2025526081000026.tif221170TIFF2025526081000027.tif220170TIFF2025526081000028.tif223170TIFF2025526081000029.tif222170

Claims

1. Lactiplantibacillus plantarum 1-D1 strain, characterized in that it is a Lactiplantibillus plantarum 1-D1 strain, having the accession number of CCTCC NO: M 20221191.

2. 10. Use of the Lactiplantibacillus plantarum 1-D1 strain according to claim 1 in the manufacture of an antibacterial product, wherein the antibacterial product inhibits one or more of Gardnerella, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Shigella dysenteriae.

3. 10. Use of the Lactiplantibacillus plantarum strain 1-D1 according to claim 1 in the preparation of a product for the prevention and / or treatment of vaginal infectious diseases.

4. 4. The use according to claim 3, wherein the vaginal infectious disease is caused by one or more of pathogens of bacterial vaginosis, pathogens of vulvovaginal candidiasis, Trichomonas vaginitis, and aerobic vaginitis, wherein the pathogens of aerobic vaginitis are Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, or Salmonella, and the pathogen of bacterial vaginosis is Gardnerella vaginalis.

5. 4. Use according to claim 3, characterized in that the product is a product for lowering vaginal pH.

6. The product is metabolized in the vaginal environment to produce H ２ O ２ 4. The use according to claim 3, characterized in that it is a product that produces organic acids and / or bacteriocins.

7. 10. Use of the Lactiplantibacillus plantarum 1-D1 strain according to claim 1 in the preparation of a product for regulating the balance of the microecology of bacterial flora.

8. 10. Use of the Lactiplantibacillus plantarum 1-D1 strain according to claim 1 in the production of hygiene products.

9. A product containing Lactiplantibacillus plantarum as an active ingredient, wherein the Lactiplantibacillus plantarum is the Lactiplantibacillus plantarum 1-D1 strain according to claim 1.

10. 10. The product according to claim 9, wherein the product is a food product, a health product, a medicine or a daily necessities product, and further wherein the product is a product for preventing and / or treating infectious vaginal diseases.

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