Lactobacillus plantarum and application thereof

Abstract

According to the present invention, the Lactobacillus plantarum 1-D1 strain and the application thereof are provided, and the Lactobacillus plantarum 1-D1 strain has characteristics of genetic stability, no pathogenic / virulence gene, good safety and strong pathogenic bacterium inhibition ability, can reduce the pH value of the vagina, can maintain the acid environment of the healthy vagina, has the outstanding Hela cell adhesion ability, can adhere to the epithelial cells of the vagina to form the micro-ecological barrier, and can effectively inhibit the growth of the vagina. Pathogens are prevented from colonizing or competing for epithelial cell receptors. The strain can be used for preparing medicines or hygienic products for preventing and treating vaginitis.

Description

Lactobacillus plantarum strain and application thereof Technical Field

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

[0002] In the vagina of healthy women of childbearing age, Lactobacillus is the dominant flora, accounting for over 70% of the vaginal flora. Studies have shown that Lactobacillus in the female reproductive tract primarily inhibits pathogenic microorganisms by producing lactic acid and antibacterial agents.

[0003] Among outpatients in obstetrics and gynecology clinics in my country, reproductive tract infections (RTIs) account for 40.2%-55.6%. At least 200 million patients suffer from RTI-related illnesses, including 100 million with recurrent episodes, resulting in medical costs exceeding 20 billion yuan annually. Surveys show that 99.3% of patients with symptomatic vaginal infections in gynecological clinics experience an imbalance in the vaginal microbiome. In 2016, the Infection Collaboration Group of the Chinese Medical Association's Obstetrics and Gynecology Branch proposed that the root cause of vaginal infection is an imbalance in the vaginal microbiome. Chronic abnormalities in the vaginal microbiome reduce the vagina's resistance to pathogenic microorganisms, often leading to recurrent vaginal infections or the development of new secondary infections.

[0004] Common vaginal infections include bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), Trichomonas vaginitis (TV), and aerobic vaginitis (AV), all of which are accompanied by a decrease or disappearance of Lactobacilli. Conventional vaginal infection treatments focus on antimicrobial therapy. Antibiotics such as clindamycin suppress bacterial vaginosis and the growth of Lactobacilli within the vagina. While metronidazole does not inhibit Lactobacillus growth at given doses, it also fails to promote its recovery. Studies have shown that metronidazole resistance is 63.8%, while clindamycin resistance is 24.1% to 67%.

[0005] Furthermore, the recurrence and resistance rates after antibiotic use are high. Antibiotics can only inhibit planktonic vaginal Gardnerella and temporarily control symptoms, but cannot completely eliminate the biofilm and the vaginal Gardnerella within it. Once treatment is stopped, the bacteria within the biofilm will revive, multiply, and spread again, leading to recurrence of bacterial vaginosis. Biofilms can reduce bacterial sensitivity to antimicrobial drugs and increase resistance to them. Pathogenic strains can acquire resistance through mobile genetic elements, which can weaken or even render antibiotic treatment ineffective.

[0006] Research by Narisu et al. showed that vaginal lactobacilli can interfere with the formation of vaginal Gardnerella biofilm. At present, the infection collaboration group of the Obstetrics and Gynecology Branch of the Chinese Medical Association pointed out that vaginal microecological preparations can be used to restore a weakly acidic environment dominated by functional lactobacilli, promote the balance of vaginal microecology and immune regulation, and reduce the recurrence of vaginal infections (Narisu et al. A preliminary study on the effect of vaginal lactobacilli on vaginal Gardnerella biofilm [J]. Progress in Modern Obstetrics and Gynecology, 2015, 24(09): 641-645.). Currently, there are only two vaginal microecological preparations on the market in China. One is a commercial product named "Yanhua" produced by Xi'an Zhenghao Biopharmaceutical Co., Ltd., which contains a Streptococcus faecalis species. This species is not a dominant vaginal species, and the bacteria under this species are conditionally pathogenic. The other is a commercial product named "Wanze Shuangqi" produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., which contains a lactobacillus species - Lactobacillus delbrueckii.

[0007] Currently available vaginal probiotic preparations fall far short of meeting clinical needs and leave much room for improvement. Therefore, developing Lactobacillus strains with enhanced probiotic potential for use in the prevention and / or treatment of vaginal infections would be of immense value.

[0008] Summary of the Invention

[0009] The present invention aims to provide a Lactobacillus plantarum 1-D1 strain, which can inhibit the growth of pathogenic bacteria in the vagina, regulate the vaginal microecological balance, and has the effect of preventing and treating vaginitis. The strain can be used to prepare medicines, foods, and sanitary products for preventing and treating vaginitis.

[0010] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0011] A Lactobacillus plantarum 1-D1 strain, the preservation number of which is CCTCC NO: M 20221191.

[0012] The present invention provides an application of Lactobacillus plantarum 1-D1 in the preparation of an antibacterial product;

[0013] Furthermore, the antibacterial products include but are not limited to bacterial inhibitors, feed additives and antimicrobial peptides.

[0014] Furthermore, the antibacterial product is a product that inhibits one or more bacteria selected from the group consisting of Gardnerella, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Shigella dysenteriae.

[0015] The present invention provides an application of Lactobacillus plantarum 1-D1 in preparing a product for preventing and / or treating vaginal infectious diseases.

[0016] The term "treatment" (also referred to as "treat" or "treating") refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves a desired effect, i.e., partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., improving vaginal flora structure and function, increasing vaginal flora diversity, reducing the abundance of inflammation-associated flora, treating vaginitis); in some embodiments, the administration of a therapeutic agent according to a therapeutic regimen is associated with the achievement of a desired effect. Such treatment may be directed to subjects who do not exhibit the relevant disease, disorder, and / or condition and / or to subjects who only exhibit early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be directed to subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0017] The products include but are not limited to medicines, foods, health products, sanitary products, etc. When used in the products, the strains are active strains or inactivated strains.

[0018] The term "drug" encompasses drugs for both human and animal use in human and veterinary medicine, as well as drugs for incorporation into animal feed (e.g., livestock feed and / or pet food). In addition, the term "drug" as used herein means any substance that provides a therapeutic, preventive, and / or beneficial effect. The term "drug" as used herein is not necessarily limited to substances that require a marketing authorization, but includes substances that can be used in cosmetics, health products, foods (including, for example, feed and beverages), probiotic cultures, and dietary supplements.

[0019] The Lactobacillus plantarum 1-D1 of the present invention can be added as a beneficial ingredient to fermented foods, immunity-enhancing health foods, female private care solid drinks and other foods, and can enhance human immunity, especially enhance the immunity of the female reproductive tract.

[0020] The vaginal infectious disease is caused by one or more of bacterial vaginosis, vulvovaginal Candida pathogens, Trichomonas vaginitis, and aerobic bacterial vaginitis;

[0021] Furthermore, the aerobic vaginitis pathogens include but are not limited to Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae or Salmonella;

[0022] Furthermore, the bacterial vaginosis pathogen is Gardnerella vaginalis.

[0023] Furthermore, the product is a product for lowering vaginal pH.

[0024] Furthermore, the product is a product that is metabolized in the vaginal environment to produce H2O2, organic acids and / or bacteriocins.

[0025] The invention provides an application of Lactobacillus plantarum 1-D1 in preparing a product for regulating microecological balance of bacterial flora.

[0026] The invention provides an application of Lactobacillus plantarum 1-D1 in the production of sanitary products.

[0027] The present invention also protects the use of Lactobacillus plantarum 1-D1 as a probiotic.

[0028] The present invention also provides a product, the active ingredient of which contains Lactobacillus plantarum; the Lactobacillus plantarum is the aforementioned Lactobacillus plantarum 1-D1.

[0029] Furthermore, the products include but are not limited to food, health products, medicines, daily necessities, etc.;

[0030] Furthermore, the product is a product for preventing and / or treating vaginal infectious diseases.

[0031] The product of the present invention may further include a pharmaceutically acceptable excipient or diluent. Beneficial effects:

[0032] (1) The Lactobacillus plantarum 1-D1 of the present invention has genetic stability, no pathogenicity / virulence genes, good safety, and exhibits good inhibitory ability against Gardnerella vaginalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella Paratyphi B, and Shigella dysenteriae.

[0033] (2) The Lactobacillus plantarum 1-D1 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 flora. At the same time, the antibacterial substances it produces, such as hydrogen peroxide, bacteriocins, and organic acids, can inhibit the growth and reproduction of pathogenic bacteria, improve mucosal immunity, and enhance the host's anti-infection effect.

[0034] (3) The Lactobacillus plantarum 1-D1 provided by the present invention has advantageous cell adhesion, enabling it to adhere to vaginal epithelial cells, forming a microecological barrier to prevent pathogen colonization or competition for epithelial cell receptors.

[0035] (4) The various evaluation indicators of the Lactobacillus plantarum 1-D1 provided by the present invention are significantly better than those of commercially available products, such as Wanze Shuangqi and metronidazole, and have good industrial value and promotion value.

[0036] Strain deposit information:

[0037] Lactobacillus plantarum 1-D1 was deposited in the China Center for Type Culture Collection on July 28, 2022. The strain is deposited with CCTCC NO: M 20221191. The address is Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a frontal photograph of the colony morphology of Lactobacillus plantarum 1-D1;

[0039] Figure 2 is a Gram staining observation picture of Lactobacillus plantarum 1-D1;

[0040] FIG3 is a comparison of the whole genome ANI of Lactobacillus plantarum 1-D1 and the standard strain;

[0041] Figure 4 shows the hemolytic assay results (left: Staphylococcus aureus ATCC 25923; right: Lactobacillus plantarum 1-D);

[0042] Figure 5 is a graph showing the weight change trend of mice in toxicity tests;

[0043] Figure 6 shows the vaginal Lactobacillus colonization scores of SD rats. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific examples. It should be understood by those skilled in the art that this should not be construed as limiting the scope of the claims of the present invention. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. It should also be noted that the reagents or instruments in the present invention, unless otherwise specified, can be obtained commercially. The culture medium preparation methods used in the following examples are all well-known methods.

[0045] The present invention uses the Lactobacillus delbrueckii (hereinafter referred to as DJS-H3) separated from Dingjunsheng as the positive control of each stage, and through low pH culture medium screening, amplification growth ability and calcium dissolution circle screening, quickly isolates the Lactobacillus with low pH tolerance, strong reproductive ability and strong lactic acid production ability. Then, through the ability of adhering to Hela cells and antibacterial test, screens out the dominant Lactobacillus with strong colonization ability and strong ability to inhibit the growth of pathogenic bacteria. Plant lactobacillus 1-D1 provided by the invention is further studied in depth, and its genetic stability and safety are investigated, so as to provide further support for its commercial application. Representative specific embodiments are as follows:

[0046] DMEM medium: Gibco; cat:11995065

[0047] MRS liquid culture medium: Huankai Biotechnology, cat: 1110151

[0048] 2% calcium carbonate-0.8% MRS: Shenggong, H122BA0030

[0049] Agar: Sangon Biotech, A505255-0250

[0050] HeLa cells: Beina Biotechnology, catalog number: BNCC342189

[0051] VK2 / E6E7 cells: Qingqi (Shanghai) Biotechnology

[0052] Example 1 Isolation of Lactobacillus

[0053] Vaginal secretions from healthy female volunteers of childbearing age were examined by Gram staining and Nugent scoring. Twenty-nine healthy female volunteers were recruited. Vaginal swabs were placed in MRS acidic liquid medium and cultured overnight at 37°C. The culture solution was diluted 10-fold in a gradient, and appropriate gradients were plated onto 2% calcium carbonate-0.8% MRS agar medium and incubated at 37°C for 36-48 hours. Single colonies with distinct clear zones on the calcium carbonate-MRS medium were picked and transferred to MRS liquid medium for overnight incubation at 37°C. 1816 strains were amplified by PCR and sent for 16S rRNA sequencing. After eliminating acid-producing non-lactobacilli, 72 Lactobacilli from different samples were selected for subsequent screening.

[0054] Among the isolated strains, the nucleotide sequences (16S rRNA sequences) of the four strains of Lactobacillus plantarum 1-D1, 16-B12, 53-D2, and 51S-H2 are shown in SEQ ID Nos. 1 to 4, respectively.

[0055] Preliminary results showed that the calcium dissolution zone of Lactobacillus plantarum 1-D1 on calcium carbonate-MRS medium was much larger than that of Lactobacillus delbrueckii DJS-H3 isolated from Dingjunsheng, demonstrating its significant advantage in lactic acid production. 16S rRNA sequencing of Lactobacillus plantarum 1-D1 was performed.

[0056] Strain deposit information:

[0057] Lactobacillus plantarum 1-D1 was deposited in China Center for Type Culture Collection on July 28, 2022. The deposit number is CCTCC NO: M 20221191. Address: Wuhan, China.

[0058] Lactobacillus crispatus 51S-H2 was deposited in China Center for Type Culture Collection on July 28, 2022, with the deposit number: CCTCC NO: M 20221194, address: Wuhan, China;

[0059] Lactobacillus paragasseri 16-B12 was deposited in China Center for Type Culture Collection on July 28, 2022, with the deposit number: CCTCC NO: M 20221193, address: Wuhan, China;

[0060] Lactobacillus jensenii 53-D2 was deposited in the China Center for Type Culture Collection on July 28, 2022, with the deposit number: CCTCC NO: M 20221192, address: Wuhan, China.

[0061] Lactobacillus delbrueckii (DJS-H3) was isolated from Dingjunsheng according to the above method for isolating Lactobacillus plantarum.

[0062] Example 2 Growth performance determination of Lactobacillus plantarum 1-D1

[0063] The growth curve data of the lactobacillus isolation stage showed that most lactobacilli entered the late logarithmic phase at 12 hours. Therefore, the viable count of each strain was determined by the agar pouring method 12 hours after inoculation. At the same inoculation time, the strains with high viable counts had a relatively obvious advantage in growth performance. The top 25 strains with growth advantages were selected to enter the next stage of the test. The data showed that Lactobacillus plantarum 1-D1 had excellent growth performance and strong reproductive ability, with a viable count of 6.37×10 in 12 hours. 9 CFU / ml, which is conducive to high-density fermentation during commercial transformation.

[0064] The culture morphology and microscopic characteristics of Lactobacillus plantarum 1-D1 were identified, and the results are shown in Figures 1 and 2.

[0065] Example 3 Adhesion ability of Lactobacillus plantarum 1-D1

[0066] The ability of lactobacilli to adhere to Hela cells and VK2 / E6E7 cells was used to evaluate the colonization ability of lactobacilli. First, activate the lactobacilli: take 50 μL of each lactobacillus and inoculate it into 5 ml of MRS liquid culture medium, culture it at 37°C for 18-24 hours, then take it out for transfer. Lactobacillus transfer: take 50 μL of each lactobacillus activation solution and inoculate it into 5 ml of MRS liquid culture medium, culture it at 37°C for 18-24 hours, centrifuge it at 12000 rpm for 2 minutes, then take it out and resuspend it in DMEM medium and adjust the turbidity to 0.5. Carry out cell plating: take 500 μL of revived cells with a concentration of 10 5 Cells were seeded into 24-well culture plates and placed in a CO2 incubator at 37°C and 5% CO2 overnight. The DMEM stock solution was discarded, and 500 μL of DMEM medium was added after washing three times with PBS. Finally, the bacteria and cells were interacted: 500 μL of a 10 8Lactobacilli cultured overnight at CFU / mL and resuspended in DMEM medium were added to the 24-well culture plate containing the cells and incubated at 37°C, 5% CO2 for 2 and 4 hours, respectively. The plates were then washed four times with PBS to remove any unadhered Lactobacilli. The plates were then digested with 500 μL of 0.25% trypsin for 2 minutes. Digestion was terminated with 600 μL of complete culture medium and mixed thoroughly by pipetting. The cell-bacteria suspension was diluted 10-fold in a sterile PBS gradient. 100 μL of the appropriate dilution was added to a disposable sterile plate. 0.8% MRS agar was then poured over the plate to mix thoroughly. Once solidified, the plates were inverted. The inverted plates were incubated at 37°C for 48 hours, then counted. Three replicates were collected for each group, and the average of the adhered Lactobacilli was used as the final count. A greater number of Lactobacilli indicates a stronger ability to adhere to cells.

[0067] The cell adhesion ability of Lactobacillus plantarum 1-D1 is shown in Tables 1 and 2. Fisher LSD mean comparison analysis revealed that the adhesion ability of Lactobacillus plantarum 1-D1 was far greater than that of DJS-H3 isolated from Dingjunsheng. The differences in adhesion counts between the groups were highly significant. The adhesion count of Lactobacillus plantarum 1-D1 on Hela cells over 4 hours was over 100 times that of Dingjunsheng, and over 10 times that of Dingjunsheng on VK2 / E6E7 cells over 4 hours.

[0068] Table 1 Adhesion ability of Lactobacillus plantarum 1-D1 to Hela cells at different times Note: Different letters indicate significant differences, a: P≤0.05; b: P≤0.01.

[0069] Table 2 Adhesion ability of Lactobacillus plantarum 1-D1 to VK2 / E6E7 cells at different times

[0070] Example 4 Antibacterial ability of Lactobacillus plantarum

[0071] Bacterial inhibition tests were performed to evaluate the ability of Lactobacillus plantarum to inhibit different pathogenic bacteria.

[0072] Lactobacillus is mixed with 0.8% MRS agar medium, and then a 10mm lactobacillus cake is made. Different pathogenic bacteria (Gardnerella vaginalis, Staphylococcus aureus, etc.) are respectively adopted after the culture medium and growth conditions activation transfer suitable for their growth, and then mixed with the corresponding semi-solid culture medium, and pathogenic bacteria plates are respectively made. The lactobacillus cake is gently placed on the surface of the pathogenic bacteria plate, taken out after 37 ℃ of upright cultivation for 18-24h, and the size of the inhibition zone is measured with a vernier caliper, thereby the ability of each lactobacillus to inhibit pathogenic bacteria is assessed. The specific steps are as follows:

[0073] After activating Lactobacillus plantarum and Lactobacillus delbrueckii (DJS-H3) respectively, 0.1 ml of the bacterial solution was mixed with MRS solid medium and poured into a 6 cm plate. After complete solidification, it was cultured at 37°C for 48 h. The plate was removed and holes were punched on the agar medium with a puncher with an inner diameter of 10 mm to obtain lactobacillus cakes for later use.

[0074] Gardnerella vaginalis and Atopobium vaginalis were inoculated into 5% horse serum-anaerobic BHI liquid, activated and transferred, and then diluted 10-fold with 0.9% anaerobic saline. 1 ml of the dilution was mixed with 10 ml of anaerobic BHI agar containing 5% horse serum, poured into a 9 cm plate, and allowed to solidify completely before use.

[0075] Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae were inoculated into nutrient broth culture medium respectively. After activation and transfer, the pathogenic bacteria liquid was diluted to 100 times with 0.9% saline in a 10-fold gradient. 1 ml of the dilution was mixed with 10 ml of nutrient agar, poured into a 9 cm plate, and waited for it to completely solidify for use.

[0076] Gently place a Lactobacillus cake on the surface of each pathogenic bacteria plate, symmetrically placing four cakes per plate, with three replicates for each strain. Place Gardnerella vaginalis and Atopobium vaginalis plates upright in an anaerobic sealed box containing an anaerobic gas-producing bag and incubate at 37°C for 48 hours. Place the other pathogenic bacteria plates upright in an incubator and incubate at 37°C for 24 hours. Finally, measure the size of the inhibition zone on each plate with a vernier caliper. The antibacterial activity of different Lactobacilli is shown in Table 3.

[0077] Table 3 Inhibition zone size of Lactobacillus plantarum against different pathogens (mm)

[0078] The results showed that the comprehensive antibacterial ability of Lactobacillus plantarum 1-D1 was much greater than that of Lactobacillus delbrueckii DJS-H3 isolated from Dingjunsheng and metronidazole.

[0079] Example 5 Whole genome sequence analysis of Lactobacillus plantarum 1-D1

[0080] Whole-genome sequencing of Lactobacillus plantarum 1-D1 was performed. After uploading the whole-genome sequence of Lactobacillus plantarum 1-D1 to EzBiocloud, the average nucleotide identity (ANI) ratio was compared with that of a standard strain, as shown in Figure 3. Whole-genome sequencing confirmed that the strain was Lactiplantibacillus plantarum; its Chinese name is Lactobacillus plantarum.

[0081] Virulence genes in the whole genome sequence were analyzed using the virulence 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)

[0082] And artificial Blast was performed through the NCBI database, and it was found that two hemolysis-related genes (Hemolysin and HemolysinⅢ) had 100% consistency in Lactobacillus plantarum. However, the HemolysinⅢ gene has been proven to be widely present in Lactobacillus plantarum (including commercial Lactobacillus plantarum 299 V, L.plantarum JDM1 and L.plantarum ST-III, etc.) (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)), and this gene can only lyse bacteria or fungi, and has not been found in mammalian cells. Therefore, strains carrying the Hemolysin III gene are not considered to pose a safety concern. Hemolysin requires the simultaneous presence of multiple genes to exhibit hemolytic activity (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). 1-D1 does not contain genes that interact with hemolysin to produce hemolysis. Furthermore, Lactobacillus plantarum 1-D1 lacks plasmids, and the transferable elements in its genome contain no resistance genes, posing no risk of resistance gene transfer. In summary, Lactobacillus plantarum 1-D1 exhibits good genetic safety.

[0083] Example 6 Hemolytic Test of Lactobacillus plantarum 1-D1

[0084] To further confirm the hemolytic activity of Lactobacillus plantarum 1-D1, we streaked the strain on blood agar plates (Chengdu Ruiqi; Sichuan Medical Device Registration No. 20152400001) for 48 hours. A positive control, Staphylococcus aureus ATCC 25923 (Shanghai Baolu Biotechnology), which exhibits beta-hemolysis, was cultured aerobically for 24 hours. The hemolytic activity of Lactobacillus plantarum 1-D1 was determined by observing the formation of a hemolytic ring on the blood agar plate after incubation.

[0085] The results in Figure 4 below show that clear transparent circles were visible around the colonies of ATCC 25923 on the positive control plate, showing typical β-hemolytic characteristics. Lactobacillus plantarum 1-D1 did not produce any hemolytic rings, indicating that Lactobacillus plantarum 1-D1 does not produce hemolysin to dissolve red blood cells, that is, it is not hemolytic.

[0086] Example 7 Toxicity test of Lactobacillus plantarum 1-D1 in mice

[0087] Five mice weighing 18 to 22 g were used, and each mouse was orally gavaged with 0.5 ml of fresh Lactobacillus plantarum 1-D1 solution (no less than 1.0 × 10 9 CFU / 0.5ml), once a day for 3 consecutive days, and continuous observation was performed from the first day of gavage to the seventh day.

[0088] Toxicity tests on mice have shown that the mice have normal body posture, normal limb movements, flexible movements, smooth fur, normal eating and drinking, no abnormal excretions and secretions, no obvious poisoning, no abnormal animal deaths during the experiment, and the mice all increased in weight.

[0089] Example 8 Antibiotic Sensitivity Test of Lactobacillus plantarum 1-D1

[0090] In accordance with the requirements of the antibiotic sensitivity test in the general chapter on microecological live bacteria products in Part III of the 2020 edition of the Pharmacopoeia, the agar diffusion paper method was used to determine the sensitivity of the strain to antibiotics, and the antibiotic sensitivity level of the strain was judged according to the size of the inhibition zone.

[0091] Standard sensitive strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as quality control bacteria. The analysis was carried out in accordance with the international standards of the Clinical and Laboratory Standards Institute (CLSI) of the United States and the health industry standards of the People's Republic of China. A sensitivity determination standard for Lactobacillus plantarum 1-D1 was developed based on the judgment standards for quality control strains. The sensitivity levels were divided into sensitive, intermediate, and resistant, represented by S, I, and R, respectively.

[0092] Antibiotic susceptibility testing showed that Lactobacillus plantarum 1-D1 was resistant to penicillin, oxacillin, azithromycin, gentamicin, kanamycin, vancomycin, enrofloxacin, clindamycin, and metronidazole, but was sensitive to ampicillin, imipenem, ceftriaxone, amoxicillin, piperacillin, and tetracycline, and moderately sensitive to erythromycin. Metronidazole and clindamycin are commonly used clinically to treat bacterial vaginosis, and these results suggest that Lactobacillus plantarum 1-D1 could theoretically be used in combination with these drugs.

[0093] Table 5 Inhibition zone diameter and determination results of drug sensitivity test of Lactobacillus plantarum 1-D1

[0094] Example 9 Colonization test of Lactobacillus plantarum 1-D1 in rat vagina

[0095] SD rats were given Lactobacillus plantarum 1-D1 vaginally once daily for 5 consecutive days and then observed for 10 days. Vaginal secretions were collected and Gram-stained before each administration (D1 secretions were collected before the initial administration) to determine the colonization of Lactobacillus plantarum 1-D1. The experimental scheme is shown in Table 6 below.

[0096] Gram staining microscopy showed that before the initial administration, the vaginal secretions of most rats were free of any microorganisms, while Gram-negative rods and Gram-positive cocci were occasionally found in the vaginal secretions of a very small number of rats. After continued administration, the number of Gram-positive rods in the vaginal secretions of rats given Lactobacillus plantarum 1-D1 was significantly increased compared to the blank control group, indicating that Lactobacillus plantarum 1-D1 can successfully colonize the vagina of SD rats. Five days after stopping administration (D10), Gram-positive rods were still visible in the field of vision. Ten days after stopping administration (D15), the vaginal microenvironment of the rats returned to its pre-dose state.

[0097] Table 6 Experimental plan for colonization of Lactobacillus plantarum 1-D1 in rat vagina

[0098] Table 7 Scoring criteria for vaginal Lactobacillus colonization in SD rats Note: A score of <0 indicates successful Lactobacillus colonization; a score of ≥0 indicates a failure of Lactobacillus colonization.

[0099] Those skilled in the art will appreciate that the embodiments of the present invention described above and shown 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 illustrated in the embodiments. Any variations or modifications may be made to the embodiments of the present invention without departing from the principles described.

Claims

1. A strain of Lactobacillus plantarum 1-D1, characterized in that: The deposit number of this strain is: CCTCC NO: M 20221191.

2. Use of the Lactobacillus plantarum 1-D1 according to claim 1 in the preparation of an antibacterial product; Furthermore, the antibacterial product is a product that inhibits one or more bacteria selected from the group consisting of Gardnerella, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Shigella dysenteriae.

3. Use of the Lactobacillus plantarum 1-D1 according to claim 1 in the preparation of a product for preventing and / or treating vaginal infectious diseases.

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

5. The use according to claim 3, characterized in that The product is a product that lowers vaginal pH.

6. The use according to claim 3, characterized in that The product is metabolized in the vaginal environment to produce H2O2, organic acids and / or bacteriocins.

7. Use of the Lactobacillus plantarum 1-D1 according to claim 1 in preparing a product for regulating the microecological balance of a microflora.

8. Use of the Lactobacillus plantarum 1-D1 according to claim 1 in the production of sanitary products.

9. A product, characterized in that The active ingredient contains Lactobacillus plantarum; the Lactobacillus plantarum is the Lactobacillus plantarum 1-D1 according to claim 1.

10. The product according to claim 9, characterized in that The product is food, health product, medicine or daily necessities; Furthermore, the product is a product for preventing and / or treating vaginal infectious diseases.

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