A probiotic composition comprising lactobacillus gasseri ln32 and lactobacillus crispatus ln70 and use in reducing the risk of vaginal infections

Abstract

The present application provides a kind of lactobacillus gasseri LN32 and lactobacillus crispatus LN70 containing probiotic composition and application in reducing the risk of vaginal infection, belong to microbial application technical field.Containing 4 strains of probiotics, including lactobacillus plantarum LN66, lactobacillus rhamnosus LN56, lactobacillus gasseri LN32 and lactobacillus crispatus LN70.The probiotic composition is realized by oral route, vaginal flora reconstruction, inhibit the reproduction of gardnerella vaginalis and candida albicans, increase lactobacillus colonization, eliminate abnormal leucorrhea, vaginal burning and itching, frequent urination, urgency and pain.The probiotic composition is combined with conventional carrier, applied to prepare food, dietary supplement or drug for preventing and / or treating vaginal and urethral microecological disorder, bacterial vaginitis, mycotic vaginitis and urethral infection, and then the present application realizes inhibiting harmful bacteria in vagina, reconstructing vaginal flora to reduce the risk of vaginal infection and reduce the risk of urethral infection.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a probiotic composition containing Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 and its application in reducing the risk of vaginal infections. Background Technology

[0002] Female reproductive tract infections, including bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), have a high incidence rate. The main symptoms include abnormal vaginal discharge, vaginal burning and itching, frequent urination, painful urination, and urgency. If left untreated, they can lead to adverse pregnancy reactions, increased risk of sexually transmitted infections, and pelvic inflammatory disease. Current treatments primarily involve antibiotics or antifungal drugs, including oral and topical medications. However, antibiotic treatment often fails, has a high recurrence rate, and significant side effects.

[0003] Studies have found that the human vaginal microbiota is crucial for maintaining vaginal health. Lactobacilli are the main component of healthy vaginal microbiota, accounting for more than 70% of the total vaginal flora. There are relatively few types of lactobacilli in a healthy vagina, mainly including Lactobacillus gasseri, Lactobacillus curvaturei, Lactobacillus janniae, and Lactobacillus indolentus. Lactobacilli can produce lactic acid, acidifying the vaginal environment and inhibiting the proliferation of various pathogens. They can also inhibit the transformation of Candida albicans from the yeast stage to the hyphal stage, reducing its pathogenicity, preventing Candida albicans from adhering to and invading vaginal epithelial cells, and reducing the risk of infection.

[0004] Although the pathogenesis of vaginitis is not fully understood, studies have shown that its occurrence is closely related to vaginal microbiome dysbiosis and a reduction in vaginal lactobacilli. In recent years, increasing research has demonstrated that the human body is an integrated system, with the vagina communicating bidirectionally with distal organs (including the intestines) through immune, neural, endocrine, and metabolic pathways. While the gut-vaginal axis is characterized by distal interactions, the rectum is close to the vagina, and the migration of microorganisms between the rectum and vagina is a unique proximal axis feature; microorganisms can migrate directly from the anus to the vagina and urethra. Furthermore, gut microbiota and their metabolites may affect vaginal microbiota through immune mechanisms. Therefore, there is an interaction between the gut and vaginal microbiota, and most vaginal microbiome dysbiosis is related to gut microbiome dysbiosis; women with BV also suffer from chronic intestinal diseases.

[0005] Currently, there are topical medications available both domestically and internationally that administer lactobacilli directly into the vagina, including suppositories and gels. However, topical medications increase the risk of infection due to direct contact. Furthermore, even if topical medications are effective in the short term, they cannot resolve the problem of intestinal microbial imbalance, resulting in a high recurrence rate.

[0006] In addition, *Lactobacillus plantarum* LN66 and *Lactobacillus rhamnosus* LN56 are published probiotics, as can be found in invention patents ZL 201911218939.4 and ZL 202011412221.1. These two probiotics have significant inhibitory properties against harmful bacteria and play an important role in flora reconstruction, reducing the risk of halitosis-causing bacteria and *Helicobacter pylori*. However, their application in inhibiting harmful vaginal bacteria and reducing the risk of vaginal infections has not yet been developed.

[0007] Therefore, to address the aforementioned issues of increased infection risk and high recurrence rate, researching oral probiotic compositions that synergistically complement gut-vaginal axis strains to reduce antibiotic use, balance gut microbiota, lower the risk and recurrence rate of vaginal infections, and improve women's overall health has significant social and economic value. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies, the present invention aims to provide a probiotic composition containing *Lactobacillus gasseri* LN32 and *Lactobacillus curvatureii* LN70, and its application in reducing the risk of vaginal infections. Through oral administration, it achieves vaginal flora reconstruction, inhibits the proliferation of *Gardnerella vaginalis* and *Candida albicans*, increases lactobacillus colonization, and eliminates abnormal vaginal discharge, vaginal burning and itching, frequent urination, urgency, and painful urination. This probiotic composition, combined with conventional carriers, can be used in the preparation of foods, dietary supplements, or pharmaceuticals for the prevention and / or treatment of vaginal and urethral microecological imbalances, bacterial vaginosis, fungal vaginitis, and urethral infections. Thus, the present invention achieves the goals of inhibiting harmful vaginal bacteria, rebuilding the vaginal flora to reduce the risk of vaginal and urethral infections.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] A probiotic composition containing four strains of probiotics, including Lactobacillus plantarum LN66 and Lactobacillus rhamnosus LN56, and the probiotic composition further includes Lactobacillus gasseri LN32 and Lactobacillus curvature LN70.

[0011] Preferably, in the probiotic composition, the viable counts of *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70 are all greater than 1.0 × 10⁻⁶. 9 CFU / g(ml).

[0012] The present invention also provides the application of the above-mentioned probiotic composition combined with a conventional carrier in the preparation of drugs for preventing and treating vaginal infections.

[0013] The present invention also provides the application of the above-mentioned probiotic composition combined with a conventional carrier in the preparation of drugs for the prevention and treatment of urinary tract infections.

[0014] The present invention also provides an application of the above-mentioned probiotic composition in vaginal flora reconstruction.

[0015] The present invention also provides a strain combination of probiotic compositions, the strain combination comprising Lactobacillus gasseri LN32 and Lactobacillus curvature LN70;

[0016] The strain of *Lactobacillus gasseri* is LN32, the deposit date is November 18, 2019, the deposit location is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No. 18958;

[0017] The strain of *Lactobacillus curvature* is LN70, the deposit date is April 22, 2020, the deposit location is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No. 19702.

[0018] The present invention also provides the application of the above-mentioned probiotic composition strain combination in the preparation of a product for generating hydrogen peroxide, specifically the application of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 in the generation of hydrogen peroxide.

[0019] The present invention also provides the use of the above-mentioned probiotic composition strain combination in the preparation of products for adhesion to intestinal epithelial cells and vaginal epithelial cells, specifically the use of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 in adhesion to intestinal epithelial cells and vaginal epithelial cells.

[0020] The present invention also provides the use of the above-mentioned probiotic composition strain combination in the preparation of a product for inhibiting the proliferation of Gardnerella vaginalis, that is, the use of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 in inhibiting the proliferation of Gardnerella vaginalis.

[0021] The present invention also provides the application of the strain combination of the above-mentioned probiotic composition in the preparation of a product for inhibiting the proliferation of Candida albicans, that is, the application of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 in inhibiting the proliferation of Candida albicans.

[0022] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0023] This invention provides a probiotic composition and its application in reducing the risk of vaginal infections. The probiotic composition contains *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70. In addition to the existing health functions of *Lactobacillus plantarum* LN66 and *Lactobacillus rhamnosus* LN56, it also includes the ability to inhibit harmful bacteria, improve oral health, and reduce the risk of *Helicobacter pylori* infection.

[0024] The Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 provided by this invention have probiotic functions such as the ability to produce hydrogen peroxide, the ability to adhere to intestinal epithelial cells and vaginal epithelial cells, and the ability to inhibit the proliferation of Gardnerella vaginalis and Candida albicans.

[0025] The probiotic composition provided by this invention reduces the risk of vaginal infections by inhibiting Gardnerella vaginalis and Candida albicans and rebuilding the vaginal flora. It also relates to the combination of the probiotic composition with a conventional carrier, which can be used in the preparation of foods, dietary supplements, or pharmaceuticals for the prevention and / or treatment of vaginal and urethral microecological imbalances, bacterial vaginosis, fungal vaginitis, and urethral infections. Through the combination of *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70, it achieves the inhibition of harmful vaginal bacteria, rebuilds the vaginal flora to reduce the risk of vaginal infections, and also reduces the risk of urethral infections.

[0026] Preservation Instructions

[0027] Lactobacillus gasseri ( Lactobacillus gasseri The strain number is LN32, the deposit date is November 18, 2019, the deposit location is China General Microbiological Culture Collection Center, the specific address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the biological preservation number is CGMCC No. 18958.

[0028] Lactobacillus curvaturei ( Lactobacillus crispatus The strain number is LN70, the deposit date is April 22, 2020, the deposit location is China General Microbiological Culture Collection Center, the specific address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the biological preservation number is CGMCC No. 19702. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments provided by the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] In this invention, the *Lactobacillus gasseri* LN32 is directly isolated from the feces of healthy infants. It is rod-shaped, Gram-positive, does not form spores, produces lactic acid, is catalase-negative, and is facultatively anaerobic. A partial 16S rDNA sequence (1313 bp) of the *Lactobacillus gasseri* LN32 is as follows:

[0031]

[0032] The *Lactobacillus curvature* LN70 of this invention was directly isolated from the feces of healthy infants. It is morphologically a slender rod, slightly bent into chains, Gram-positive, does not form spores, produces lactic acid, is catalase-negative, and is facultatively anaerobic. A partial 16S rDNA sequence (1416 bp) of the *Lactobacillus curvature* LN70 is as follows:

[0033]

[0034] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0035] Example 1

[0036] In this embodiment, *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 were subjected to microscopic examination, MRS plate purification and isolation, and fermentation to verify their ability to produce hydrogen peroxide, thereby confirming the characteristics and probiotic functions of the strains. The specific process includes:

[0037] First, under aseptic conditions, Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 were picked up with an inoculation loop, smeared on a glass slide, prepared, and Gram stained. The morphology of the bacteria was then observed under a microscope.

[0038] Next, under aseptic conditions, Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 were picked up with an inoculation loop and streaked on MRS plates. The plates were then placed in an incubator and incubated at 37°C for 72 h. Single colonies were selected for microscopic examination to achieve pure culture isolation of the strains.

[0039] Finally, using an inoculation loop, two loops of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 were picked from the slant and placed into two 50 ml MRS liquid culture media. They were cultured at 37°C for 24 h, and then inoculated into 50 ml MRS liquid culture media at a 5% inoculation rate and cultured at 37°C for 72 h, thus obtaining the fermentation broths of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70, respectively.

[0040] After completing the above process, the H2O2 production capacity of the two lactobacilli was determined, specifically including:

[0041] Freshly cultured lactobacilli (greater than 10) 8 The cells were serially diluted to a certain concentration (CFU / ml), and 100 μL was inoculated onto an H2O2 identification plate, i.e., spread on an MRS agar plate containing 0.25 mg / mL TMB (3,3',5,5'-tetramethylbenzidine) and 0.01 mg / mL horseradish peroxidase. The plates were then anaerobically incubated at 37°C for 48 to 72 hours. After removing the MRS agar plates, they were exposed to air. Colonies producing H2O2 turned blue, while colonies not producing H2O2 remained unchanged. The H2O2 production was semi-quantitatively determined based on the color change time, and the results are shown in Table 1.

[0042] Table 1. Semi-quantitative determination criteria for H2O2

[0043]

[0044] In Table 1, strain scores are assigned as weak (1 point, time ≥ 20 minutes), medium (2 points, time 10-20 minutes), strong (3 points, time ≤ 10 minutes), and 0 points, indicating no blue production. According to the results in Table 2, the ability of *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 to produce H2O2 is the same as that of *Lactobacillus plantarum* LN66, superior to other *Lactobacillus gasseri* LN20 and *Lactobacillus curvature* LN26, and also superior to *Lactobacillus rhamnosus* LN56. The colonies of viable *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 begin to show a slight blue tinge after 2-3 minutes, and a large amount of blue tinge appears within 9 minutes. Based on the criteria shown in Table 1, the semi-quantitative score for the ability of *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 to produce hydrogen peroxide is 3 points.

[0045] Table 2. Ability to produce H2O2

[0046]

[0047] Table 2 shows that Lactobacillus gasseri LN32, Lactobacillus curvature LN70, and Lactobacillus plantarum LN66 have a significant advantage over other strains in terms of H2O2 production capacity.

[0048] Example 2

[0049] In this embodiment, the inhibitory activity of Lactobacillus gasseri LN32 and Lactobacillus curvature LN70 against harmful vaginal bacteria (Gardnerella vaginalis and Candida albicans) and their adhesion to intestinal epithelial cells and vaginal epithelial cells were verified, elucidating their mechanism of action in vaginal flora reconstruction.

[0050] First, the inhibitory activity of the two types of lactobacilli provided in this invention against Gardnerella vaginalis (ATCC14018) was verified. The specific procedure was as follows: 10 μL of freshly cultured lactobacilli suspension (greater than 10 μL) was taken. 8 The CFU / ml samples were spotted onto the surface of MRS agar and anaerobically cultured at 37°C for 48 hours. 100µL of freshly cultured Candida albicans suspension was mixed thoroughly with 5mL of soft BHI agar (0.7% agar) in a 50°C water bath and poured onto the solid plates containing the spotted lactobacilli. After solidification, the plates were anaerobically cultured at 37°C for 12–48 hours. The inhibition of Gardnerella vaginalis activity was evaluated by observing the growth of Gardnerella vaginalis. The results are shown in Table 3.

[0051] Table 3. Activity of Gardnerella vaginalis in inhibiting vaginal bacteria

[0052]

[0053] The inhibitory activity was assessed as follows: +++, high inhibitory activity against Gardnerella vaginalis, i.e., complete inhibition of Gardnerella vaginalis growth; ++, moderate inhibitory activity against Gardnerella vaginalis; +, low inhibitory activity against Gardnerella vaginalis; 0, no inhibitory activity, i.e., Gardnerella vaginalis grows normally. According to the results in Table 3, *Lactobacillus gasseri* LN32 and *Lactobacillus curvatureii* LN70 showed superior inhibitory activity against vaginal Gardnerella vaginalis compared to other strains such as *Lactobacillus gasseri* LN20 and *Lactobacillus curvatureii* LN26, and also superior to *Lactobacillus plantarum* LN66 and *Lactobacillus rhamnosus* LN56. Therefore, *Lactobacillus gasseri* LN32 and *Lactobacillus curvatureii* LN70 have a significant advantage over other strains in inhibiting vaginal Gardnerella vaginalis activity.

[0054] Secondly, the inhibitory activity of the two lactobacilli provided in this invention against Candida albicans (ATCC90028) was verified. The specific process was as follows: 10 μL of freshly cultured lactobacillus suspension (greater than 10 μL) was taken. 8 The CFU / ml samples were spot-inoculated onto the surface of MRS agar and anaerobically cultured at 37°C for 48 hours. 100 μL of freshly cultured Candida albicans was added to 5 mL of soft YM agar (0.7% agar), mixed thoroughly in a 50°C water bath, and poured onto the Lactobacillus MRS agar plates that had been cultured for 48 hours. After solidification, the plates were aerobically cultured at 37°C for 12-48 hours. The inhibitory activity against Candida albicans was evaluated by the growth of yeast cells. The results are shown in Table 4.

[0055] Table 4. Activity against Candida albicans

[0056]

[0057] The inhibitory activity was assessed as follows: +++, high inhibitory activity against Candida albicans, i.e., complete inhibition of Candida albicans growth; ++, moderate inhibitory activity against Candida albicans; +, low inhibitory activity against Candida albicans; 0, no inhibitory activity, i.e., normal growth of Candida albicans. According to the results in Table 4, *Lactobacillus gasseri* LN32 and *Lactobacillus curvatureii* LN70 showed superior inhibitory activity against Candida albicans compared to other *Lactobacillus gasseri* strains, including LN20 and LN26, as well as Lactobacillus plantarum LN66 and *Lactobacillus rhamnosus* LN56. Therefore, *Lactobacillus gasseri* LN32 and Lactobacillus curvatureii LN70 have a significant advantage over other strains in inhibiting Candida albicans activity.

[0058] Next, the adhesion ability of the two lactobacilli provided in this invention to intestinal epithelial cells was verified. The adhesion performance of different lactobacilli was determined based on the number of lactobacilli adhering to a single cell layer of intestinal epithelial cells. The specific process was as follows: Four strains of lactobacilli were cultured in MRS liquid medium for 48 hours (greater than 10). 8(CFU / ml), centrifuged, washed three times with PBS, resuspended in DMEM complete culture medium, and set aside for use; simultaneously, Caco-2 cells were adjusted to 600,000 cells / mL and seeded into 6-well plates. After culturing for 48 hours to form a monolayer, the cells were washed twice with sterile PBS, and the cells in the first well were digested with trypsin and counted using a hemocytometer. Additionally, 1 mL each of the previously prepared Lactobacillus and blank control PBS were added to 5 wells of the plate, and after co-incubation for 2 hours, the unattached Lactobacillus was washed away, and the cells were lysed with 1 mL of 0.05% Triton X-100 to prepare a bacterial suspension. This suspension was diluted, and 100 μL of the suspension was evenly spread onto MRS plates. After anaerobic incubation for 48 hours, the number of Lactobacillus on each plate was counted, and the number of attached Lactobacillus was calculated. The results are shown in Table 5.

[0059] Table 5 Adhesion ability to intestinal epithelial cells

[0060]

[0061] The adhesion rate to intestinal epithelial cells is calculated using the following formula:

[0062] Adhesion rate (%) = Number of adhering lactobacilli / Total number of cells × 100;

[0063] Based on the results in Table 5, *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 exhibited superior adhesion to intestinal epithelial cells compared to other *Lactobacillus gasseri* strains, such as LN20 and LN26. *Lactobacillus plantarum* LN66 and *Lactobacillus rhamnosus* LN56 also demonstrated excellent adhesion to intestinal epithelial cells. Therefore, *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 showed a significant advantage over other strains of the same species in terms of intestinal epithelial cell adhesion.

[0064] Finally, the adhesion ability of the two lactobacilli provided in this invention to vaginal epithelial cells was verified. The adhesion performance of different lactobacilli was determined based on the number of lactobacilli adhering to a single cell layer of vaginal epithelial cells. The specific process was as follows: Four strains of lactobacilli were cultured in MRS liquid medium for 48 hours (greater than 10). 8(CFU / ml) were prepared for use; simultaneously, human vaginal epithelial cells Vk2 / E6E7 were seeded at a density of 5 million per well in 6-well culture plates and cultured at 37°C and 5% CO2 for 48 hours to form a monolayer. The cells were washed twice with sterile PBS, and the first well was digested with trypsin. The number of cells was counted using a hemocytometer. In addition, 1 mL of the previously prepared lactobacillus and 1 mL of blank control PBS were added to each of the other 5 wells. After co-incubation for 2 hours, the unattached lactobacillus was washed off, and the cells were lysed with 1 mL of 0.05% Triton X-100 to prepare a bacterial suspension. The suspension was diluted, and 100 μL of the suspension was evenly spread on MRS plates. After anaerobic culture for 48 hours, the number of lactobacillus on each plate was counted, and the number of attached lactobacillus was calculated. The results are shown in Table 6.

[0065] Table 6 Adhesion ability to vaginal epithelial cells

[0066]

[0067] The adhesion rate of vaginal epithelial cells is calculated using the following formula:

[0068] Adhesion rate (%) = Number of adhering lactobacilli / Total number of cells × 100;

[0069] Based on the results in Table 6, *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 exhibited superior adhesion to vaginal epithelial cells compared to other *Lactobacillus gasseri* strains such as LN20 and LN26, as well as *Lactobacillus plantarum* LN66 and *Lactobacillus rhamnosus* LN56. Therefore, *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70 demonstrate a significant advantage over other strains in terms of vaginal epithelial cell adhesion.

[0070] Example 3

[0071] In this embodiment, the effectiveness of a probiotic composition containing Lactobacillus gasseri LN32, Lactobacillus curvature LN70, Lactobacillus plantarum LN66, and Lactobacillus rhamnosus LN56 in reducing the risk of vaginal infections was verified through animal models and clinical trials.

[0072] First, animal experiments were conducted to investigate the reduction of the risk of Gardnerella vaginalis infection using a probiotic combination containing Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32, and Lactobacillus curvature LN70. The reduction in the risk of bacterial vaginosis was determined by inhibiting the number of Gardnerella vaginalis and increasing the number of lactobacilli in the animals. The specific methods are as follows:

[0073] Fifty female BALB / c mice were randomly divided into five groups of ten each: a blank control group, a model group, a combination of two publicly available bacterial strains (Lactobacillus plantarum LN66 + Lactobacillus rhamnosus LN56, i.e., the LN66+LN56 group), a combination of two novel bacterial strains (Lactobacillus gasseri LN32 + Lactobacillus curvature LN70, i.e., the LN32+LN70 group), and a combination of four probiotic strains (Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32, and Lactobacillus curvature LN70, i.e., the LN66+LN56+LN32+LN70 group).

[0074] An animal model of bacterial vaginosis was established based on the model described by Sabbatini et al. (Sabbatini S, Monari C, Ballet N, et al. Saccharomyces cerevisiae–based probiotic as novel antimicrobial agent for therapy of bacterial vaginosis, Virulence, 2018, 9(1): 954-966). Three days before infection (i.e., day -3) and on the day of infection (i.e., day 0), all mice except the control group were subcutaneously injected with 100 µL of estradiol valerate (0.5 mg estradiol valerate dissolved in 100 µL of medium-chain triglycerides). On day 0, the vaginal inoculation count was 10... 8 A mouse model of bacterial vaginosis was established by administering 20 µL of CFU / mL Gardnerella vaginalis suspension for 5 consecutive days, ending on day 4. Post-infection (day 6), except for the model group, the control group received 200 µL of PBS buffer by gavage once daily, while the other three groups received 200 µL of PBS buffer at a concentration of 10 CFU / mL by gavage once daily. 10 A probiotic suspension of CFU / mL was administered by gavage once daily until the end of the experiment, for a total of 14 days.

[0075] Then, the vaginal flora of mice was tested. Using a micropipette containing 50µL of physiological saline, the mice vagina were repeatedly flushed 5-6 times during the following time periods: after modeling (day 5), day 1 after gavage (day 20), and day 6 after gavage (day 25). 30µL of the above vaginal irrigation fluid was collected to count Gardnerella vaginalis and Lactobacillus colonies, and compared with the model group and the blank control group. The percentages of Gardnerella vaginalis and Lactobacillus in each group were calculated. That is, if the Lactobacillus in the blank control group was 100%, then the Lactobacillus (%) in the model group = Lactobacillus count in the model group × 100 / Lactobacillus count in the blank control group. The Lactobacillus (%) in each experimental group was calculated accordingly. Similarly, if the Gardnerella vaginalis in the model control group was 100%, then the Gardnerella vaginalis (%) in the blank control group = Gardnerella vaginalis count in the blank control group × 100 / Gardnerella vaginalis count in the model group. The results are shown in Table 7.

[0076] The results in Table 7 indicate that on day 20 (the first day after gavage), the number of Gardnerella vaginalis colonizations in the three probiotic groups was significantly lower than that in the model group: 25.48% in the LN66+LN56 group, 11.72% in the LN32+LN70 group, and 3.65% in the LN66+LN56+LN32+LN70 group. Conversely, the number of Lactobacillus colonizations in the vagina was significantly higher than that in the control group: 1346.56%, 1428.23%, and 1834.15%, respectively. On day 6 after gavage, the number of Gardnerella vaginalis colonizations in the three probiotic groups was less than 1% of that in the model group, while the number of Lactobacillus colonizations continued to increase. This demonstrates that oral administration of individual strains of LN66+LN56, LN32+LN70, and LN66+LN56+LN32+LN70 can inhibit Gardnerella vaginalis infection while increasing the number of Lactobacillus. The results of comparing the three groups of probiotics showed that the combination of LN66+LN56+LN32+LN70 was superior to the other two combinations, namely LN66+LN56 and LN32+LN70, in inhibiting Gardnerella vaginalis infection.

[0077] Table 7. Percentages of Gardnerella vaginalis and Lactobacillus in vaginal irrigation fluid of each experimental group

[0078]

[0079] Secondly, animal experiments were conducted to investigate the reduction of the risk of Candida albicans infection by oral administration of a probiotic combination of Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32, and Lactobacillus curvature LN70. The reduction of the risk of vaginal Candida albicans infection by probiotics was determined based on the inhibition of the number of Candida albicans in the vagina of animals and the increase of the number of lactobacilli.

[0080] The specific method is as follows: Fifty SPF-grade C57BL / 6 female mice were randomly divided into 5 groups of 10 mice each: a blank control group, a model group, a combination group of 2 publicly available bacterial strains (Lactobacillus plantarum LN66 + Lactobacillus rhamnosus LN56, i.e., LN66 + LN56 group), a combination group of 2 new bacterial strains (Lactobacillus gasseri LN32 + Lactobacillus curvature LN70, i.e., LN32 + LN70 group), and a combination group of 4 probiotic strains (Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32, and Lactobacillus curvature LN70, i.e., LN66 + LN56 + LN32 + LN70 group). Five days before infection (i.e., day -5), except for the control group, each group used a micropipette to rinse the vagina of mice with 50 μL of a certain concentration of lincomycin hydrochloride solution once a day for 5 consecutive days. The number of live bacteria inoculated vaginally on day 0 was 2.0 × 10⁻⁶. 7 A mouse model of vaginal Candida albicans infection was established by vaginal inoculation with 20 µL of CFU / mL Candida albicans suspension once daily for 6 consecutive days, ending on day 5. Post-infection (day 7), except for the model group, the blank control group received 200 µL of PBS buffer by gavage once daily, while the other three groups received 200 µL of PBS buffer at a concentration of 10 CFU / mL by gavage once daily. 10 A probiotic suspension of CFU / mL was administered by gavage once daily until the end of the experiment, for a total of 14 days.

[0081] The vaginal flora of mice was then tested. Using a micropipette containing 50µL of physiological saline, the mice vaginas were repeatedly flushed 5-6 times during the following time periods: after modeling (day 6), day 1 after gavage (day 21), and day 6 after gavage (day 26). 30µL of the vaginal irrigation fluid was collected for colony counting of Candida albicans and Lactobacillus, and compared with the model group and the blank control group. The percentages of Candida albicans and Lactobacillus in each group were calculated. That is, if the Lactobacillus in the blank control group was 100%, then the Lactobacillus (%) in the model group = Lactobacillus count in the model group × 100 / Lactobacillus count in the blank control group, and so on. Similarly, if the Candida albicans in the model control group was 100%, then the Candida albicans (%) in the blank control group = Candida albicans count in the blank control group × 100 / Candida albicans count in the model group, and so on. The results are shown in Table 8.

[0082] Table 8. Percentages of Candida albicans and Lactobacillus in vaginal douches of each experimental group

[0083]

[0084] As shown in Table 8, on day 21 after gavage, the number of Candida albicans colonized in the vagina of mice in the three probiotic groups was significantly lower than that in the model group: 15.49% in the LN66+LN56 group, 10.36% in the LN32+LN70 group, and 5.78% in the LN66+LN56+LN32+LN70 group. Meanwhile, the number of Lactobacillus colonized in the vagina was significantly higher than that in the control group: 1143.27%, 1317.45%, and 1816.34%, respectively. On day 6 after gavage, the number of Candida albicans colonized in the three probiotic groups was less than 10% of that in the model group, while the number of Lactobacillus colonized continued to increase. This indicates that oral administration of individual LN66+LN56, LN32+LN70, and LN66+LN56+LN32+LN70 strains can inhibit Candida albicans infection while increasing the number of Lactobacillus. The results of comparing the three groups of probiotics showed that the combination of LN66+LN56+LN32+LN70 was superior to the other two combinations, namely LN66+LN56 and LN32+LN70, in inhibiting Candida albicans infection.

[0085] Example 4

[0086] This embodiment mainly focuses on the clinical trial of the present invention. Firstly, two probiotic composition products prepared according to the present invention are applied. One is a probiotic composition (containing *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70 at concentrations of 1.0 × 10⁻⁶). 9 CFU / day, total viable bacteria count is 4.0 × 10⁻⁶ 9 A product combining 4 billion CFU / day with a conventional carrier (referred to as 4 billion CFU / day) is available. Another option is a low-dose combination of this probiotic composition (containing 1.0 × 10⁻⁶ CFU / day each of *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70). 8 CFU / day, total viable bacteria count is 4.0 × 10⁻⁶ 8 The product (400 million CFU / day) is a combination of CFU / day and a conventional carrier. The above two probiotic compositions are used to treat bacterial vaginosis. The Amsel "gold standard" and Gram staining Nugent score for the clinical diagnosis of bacterial vaginosis (BV) were used to evaluate the changes in BV-related indicators before and after oral administration of the probiotic composition of the present invention. The specific results are shown in Table 9.

[0087] Table 9. Therapeutic effects of two probiotic compositions on BV

[0088]

[0089]

[0090] The number of lactobacilli was scored based on the average number of lactobacilli morphological bacteria observed per 10 oil immersion fields: 0+, no lactobacilli observed; 1+, <1 lactobacillus; 2+, 1~4 lactobacilli; 3+, 5~30 lactobacilli; 4+, >30 lactobacilli.

[0091] According to the results shown in Table 9, based on the Amsel "gold standard" for clinical diagnosis and the Gram staining Nugent score, patients with bacterial vaginosis showed improvement in both clinical symptoms and lactobacillus counts after taking the 4 billion CFU / day probiotic composition of this invention for 4 weeks. A control was established using a lower dose of the probiotic composition than specified in this invention, i.e., 4.0 × 10⁻⁶ CFU / day. 8 The probiotic composition with CFU / day also showed significantly lower efficacy in improving bacterial vaginosis after 4 weeks of use compared to the probiotic composition described in this invention. The results indicate that the live bacteria content in the probiotic composition of this invention, namely *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70, is not less than 1.0 × 10⁻⁶ CFU / day. 9 CFU / day, total viable count not less than 4.0 × 10⁻⁶ 9 CFU / day.

[0092] Secondly, two probiotic composition products prepared using this invention were applied. One was a probiotic composition containing 1.0 × 10⁻⁶ Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32, and Lactobacillus curvature LN70. 9 CFU / day, total viable bacteria count is 4.0 × 10⁻⁶ 9 A product combining 4 billion CFU / day with a conventional carrier (referred to as 4 billion CFU / day) is available. Another option is a low-dose combination of this probiotic composition (containing 1.0 × 10⁻⁶ CFU / day each of *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70). 8 CFU / day, total viable bacteria count is 4.0 × 10⁻⁶ 8 The product (400 million CFU / day) is a combination of CFU / day and a conventional carrier. The above two probiotic compositions are used to treat vulvovaginal candidiasis. According to the diagnostic criteria of the "Chinese Guidelines for the Diagnosis and Treatment of Vulvovaginal Candidiasis (2024 Edition)," the changes in vulvovaginal candidiasis-related indicators before and after oral administration of the probiotic composition of this invention were evaluated. The specific results are shown in Table 10.

[0093] Table 10. The therapeutic effects of two probiotic compositions on fungal vaginitis.

[0094]

[0095]

[0096] As shown in Table 10, based on clinical symptoms and vaginal Candida albicans testing, patients with vulvovaginal candidiasis experienced improvement in their clinical symptoms and a change from positive to negative Candida albicans test results after taking the 4 billion CFU / day probiotic composition of this invention for 4 weeks. A control was established using a lower dose of the probiotic composition than specified in this invention, i.e., 4.0 × 10⁻⁶ CFU / day. 8 The probiotic composition with CFU / day, after 4 weeks of use, also showed a significantly lower effect on improving fungal vaginitis than the probiotic composition described in this invention. The results indicate that the live bacteria content in the probiotic composition of this invention, namely *Lactobacillus plantarum* LN66, *Lactobacillus rhamnosus* LN56, *Lactobacillus gasseri* LN32, and *Lactobacillus curvature* LN70, is not less than 1.0 × 10⁻⁶ CFU / day. 9 CFU / day, total viable count not less than 4.0 × 10⁻⁶ 9 CFU / day.

[0097] Therefore, by employing the aforementioned probiotic composition containing *Lactobacillus gasseri* LN32 and *Lactobacillus curvature* LN70, and its application in reducing the risk of vaginal infections, vaginal flora reconstruction is achieved through oral administration. This inhibits the proliferation of *Gardnerella vaginalis* and *Candida albicans*, increases lactobacillus colonization, and eliminates abnormal vaginal discharge, vaginal burning and itching, urinary frequency, urgency, and dysuria. This probiotic composition, combined with conventional carriers, can be used in the preparation of foods, dietary supplements, or pharmaceuticals for the prevention and / or treatment of vaginal and urethral microecological imbalances, bacterial vaginosis, fungal vaginitis, and urethral infections. Thus, this invention achieves the goals of inhibiting harmful vaginal bacteria, rebuilding the vaginal flora, and reducing the risk of vaginal and urethral infections.

[0098] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0099] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A probiotic composition containing four strains of probiotics, including *Lactobacillus plantarum* (…). Lactobacillus plantarum LN66, Lactobacillus rhamnosus ( Lactobacillus rhamnosus LN56, characterized in that, The probiotic composition further comprises Lactobacillus gasseri (Lactobacillus gasseri) Lactobacillus gasseri ) LN32 and Lactobacillus crispatus (Lactobacillus crispatus) Lactobacillus crispatus ) LN70; the strain of Lactobacillus gasseri is LN32, the preservation time is November 18, 2019, the preservation place is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No. 18958. The strain of the Lactobacillus crispatus is LN70, the preservation time is April 22, 2020, the preservation place is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.19702; The strain of the Lactobacillus plantarum is LN66, the classification name is Lactobacillus plantarum, the preservation time is March 20, 2019, the preservation place is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.17369. The strain of the Lactobacillus rhamnosus is LN56, the classification name is Lactobacillus rhamnosus, the preservation time is March 20, 2019, the preservation place is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.17370.

2. The probiotic composition according to claim 1, characterized in that, In the probiotic composition, the viable cell number of the Lactiplantibacillus halli LN66, Lactosaccharo-carbohydrate LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 is each greater than 1.0 x 10 9 CFU / g or CFU / ml.

3. A combination of strains of a probiotic composition, characterized in that, The strain combination comprises Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70; The strain of the Lactobacillus gasseri is LN32, the preservation time is November 18, 2019, the preservation place is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.18958. The strain of the Lactobacillus crispatus is LN70, the preservation time is April 22, 2020, the preservation place is China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.19702.

4. Use of a strain combination of the probiotic composition according to claim 3 in the preparation of a product for producing hydrogen peroxide.

5. Use of a strain combination of the probiotic composition according to claim 3 in the preparation of a product for inhibiting the proliferation of Gardnerella.

6. Use of a strain combination of the probiotic composition according to claim 3 in the preparation of a product for inhibiting the proliferation of Candida albicans.

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