A probiotic composition of lab4 and lactobacillus gasseri cul09 and use in reducing the risk of vaginal infections

Abstract

This invention provides a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 and its application in reducing the risk of vaginal infections, belonging to the field of microbial application technology. Containing the Lab4 probiotic group and the novel strain Lactobacillus gasseri CUL09, this probiotic composition achieves vaginal flora reconstruction via oral administration, effectively improving vaginal pH, eliminating odor, and increasing lactobacillus colonization. This invention also relates to the combination of the probiotic composition with vitamin B6, cranberry extract, and conventional carriers, 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, vaginitis, vaginal candidiasis, and urethral infections. Furthermore, this invention, through the combination of Lab4 and Lactobacillus gasseri CUL09, achieves the inhibition of harmful vaginal bacteria, reconstruction of the vaginal flora to reduce the risk of vaginal infections, and can also prevent and treat urethral infections.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 and its application in reducing the risk of vaginal infections. Background Technology

[0002] Lab4, a known probiotic blend, contains four strains: Lactobacillus acidophilus CUL60, CUL21, Bifidobacterium animalis subsp. lactis CUL34, and Bifidobacterium bifidum CUL20. Since its development in 1999, over 20 studies have confirmed its functions in the following areas: Gut health: inhibiting harmful gut bacteria, rebuilding gut microbiota (e.g., gut microbiota recovery after antibiotic treatment), reducing antibiotic-related side effects such as Clostridium difficile infection, and improving symptoms of irritable bowel syndrome. Immune regulation: enhancing immunity and regulating cytokine production by peripheral blood mononuclear cells. However, research on Lab4 has primarily focused on gut health, and its application in female reproductive health (e.g., vaginal microecological regulation) has not yet been fully explored.

[0003] Furthermore, bacterial vaginosis (BV) is a common disease among women of reproductive age, with a global incidence rate of 23%–29%. Main symptoms include abnormal discharge, odor, and itching. Untreated, it can lead to pregnancy complications, increased risk of sexually transmitted infections, and pelvic inflammatory disease. Its pathogenesis is mainly due to vaginal microecological imbalance, with a decrease or disappearance of hydrogen peroxide-producing lactobacilli and an overgrowth of facultative anaerobes such as Gardnerella vaginalis and anaerobic bacteria, resulting in an elevated vaginal pH. However, current treatments mostly involve local medications (such as lactobacillus suppositories and gels) that need to be applied directly to the vagina, which is inconvenient and may disrupt the natural flora balance.

[0004] In addition, there are already suppositories or gels for direct vaginal administration of lactobacillus both domestically and internationally. However, there are still few novel oral probiotic compositions based on the "gut-vaginal axis" interaction. Therefore, based on the previous Lab4 research, developing an oral probiotic composition to reduce the risk of vaginal infection and improve women's health is of great value. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 and its application in reducing the risk of vaginal infections. The composition comprises Lab4 probiotics and the novel strain Lactobacillus gasseri CUL09. This probiotic composition, administered orally, achieves vaginal flora reconstruction, effectively improving vaginal pH, eliminating odor, and increasing lactobacillus colonization. The present invention also relates to the combination of the probiotic composition with vitamin B6, cranberry extract, and conventional carriers, 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, vaginitis, vaginal candidiasis, and urethral infections. Furthermore, the present invention, through the combination of Lab4 and Lactobacillus gasseri CUL09, achieves the inhibition of harmful vaginal bacteria, reconstruction of vaginal flora to reduce the risk of vaginal infections, and can also prevent and treat urethral infections.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] A probiotic composition comprising Lab4 and Lactobacillus gasseri CUL09, wherein the probiotic composition contains Lab4 and Lactobacillus gasseri CUL09, wherein Lab4 contains four probiotic strains, namely Lactobacillus acidophilus CUL60, Lactobacillus acidophilus CUL21, Bifidobacterium animalis subsp. lactis CUL34 and Bifidobacterium bifidum CUL20.

[0008] Preferably, in the probiotic composition, the viable counts of Lab4 and Lactobacillus gasseri CUL09 are both greater than 1.0 × 10⁻⁶. 9 CFU / ml(g).

[0009] The present invention also provides the application of the above-mentioned probiotic composition of Lab4 and Lactobacillus gasseri CUL09 in the reconstruction of vaginal flora.

[0010] The present invention also provides the application of the above-mentioned probiotic composition of Lab4 and Lactobacillus gasseri CUL09 combined with vitamin B6, cranberry extract and conventional carrier in the preparation of food, supplement or medicine for the prevention and treatment of vaginal infections.

[0011] The present invention also provides the application of the above-mentioned probiotic composition of Lab4 and Lactobacillus gasseri CUL09 combined with vitamin B6, cranberry extract and conventional carrier in the preparation of food, supplements or medicines for the prevention and treatment of urinary tract infections.

[0012] The present invention also provides a strain of Lactobacillus gasseri CUL09, wherein the strain number of Lactobacillus gasseri CUL09 is CUL09, the deposit date is March 14, 2003, the deposit location is the British Microbiological Culture Collection (NCIMB), and the biological accession number is NCIMB 30155.

[0013] The present invention also provides the application of Lactobacillus gasseri CUL09 in the production of hydrogen peroxide.

[0014] The present invention also provides the application of Lactobacillus gasseri CUL09 in adherent intestinal epithelial cells and vaginal epithelial cells.

[0015] The present invention also provides the application of Lactobacillus gasseri CUL09 in inhibiting the proliferation of Gardnerella vaginalis.

[0016] This invention also provides the application of Lactobacillus gasseri CUL09 in inhibiting the proliferation of Candida albicans.

[0017] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0018] This invention provides a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 and its application in reducing the risk of vaginal infections. The composition contains Lab4 and a novel strain of Lactobacillus gasseri, CUL09. Lab4 contains four probiotic strains: Lactobacillus acidophilus CUL60, Lactobacillus acidophilus CUL21, Bifidobacterium animalis subsp. lactis CUL34, and Bifidobacterium bifidum CUL20. In addition to the existing clinically proven health functions of Lab4, including inhibiting harmful intestinal bacteria, rebuilding the intestinal flora, reducing antibiotic-related side effects, improving irritable bowel syndrome, and enhancing immunity, the probiotic composition provided by this invention, in its embodiments, includes Lactobacillus gasseri CUL09 with the ability to produce hydrogen peroxide, dominant intestinal and vaginal epithelial cell adhesion, and inhibition of Gardnerella vaginalis and Candida albicans proliferation. The Lab4 and Lactobacillus gasseri CUL09 composition reduces the risk of vaginal infections by inhibiting Gardnerella vaginalis and rebuilding the vaginal flora. This invention also relates to the combination of the probiotic composition with vitamin B6, cranberry extract, and conventional carriers, which can be used in the preparation of foods, dietary supplements, or medicines for the prevention and / or treatment of vaginal and urethral microecological disorders, vaginitis, vaginal candidiasis, and urethral infections. By combining Lab4 with Lactobacillus gasseri CUL09, it can inhibit harmful vaginal bacteria, rebuild the vaginal flora to reduce the risk of vaginal infections, and also prevent and treat urethral infections. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the 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.

[0020] The present invention provides a probiotic composition of Lab4 and Lactobacillus gasseri CUL09, wherein the probiotic composition contains Lab4 and Lactobacillus gasseri CUL09, wherein Lab4 contains four probiotic strains, namely Lactobacillus acidophilus CUL60, Lactobacillus acidophilus CUL21, Bifidobacterium animalis subsp. lactis CUL34 and Bifidobacterium bifidum CUL20.

[0021] Specifically, in the probiotic composition, the viable counts of Lab4 and Lactobacillus gasseri CUL09 are both greater than 1.0 × 10⁻⁶. 9 CFU / ml(g).

[0022] As another embodiment, the present invention also provides the application of the above-mentioned probiotic composition of Lab4 and Lactobacillus gasseri CUL09 in vaginal flora reconstruction.

[0023] As another embodiment, the present invention also provides the application of the above-mentioned probiotic composition of Lab4 and Lactobacillus gasseri CUL09 combined with vitamin B6, cranberry extract and conventional carrier in the preparation of food, supplements or medicines for the prevention and treatment of vaginal infections.

[0024] As another embodiment, the present invention also provides the application of the above-mentioned probiotic composition of Lab4 and Lactobacillus gasseri CUL09 combined with vitamin B6, cranberry extract and conventional carrier in the preparation of food, supplements or medicines for the prevention and treatment of urinary tract infections.

[0025] As another embodiment, the present invention also provides a Lactobacillus gasseri CUL09, wherein the Lactobacillus gasseri CUL09 is classified as Lactobacillus gasseri and its Latin name is [missing information]. Lactobacillus gasseri The strain number is CUL09, the viability status is live, the deposit date is March 14, 2003, the deposit location is the British Microbial Culture Collection (NCIMB), and the biological accession number is NCIMB 30155.

[0026] As another embodiment, the present invention also provides the application of Lactobacillus gasseri CUL09 in the production of hydrogen peroxide.

[0027] As another embodiment, the present invention also provides the application of Lactobacillus gasseri CUL09 in adherent intestinal epithelial cells and vaginal epithelial cells.

[0028] As another embodiment, the present invention also provides the application of Lactobacillus gasseri CUL09 in inhibiting the proliferation of Gardnerella vaginalis.

[0029] As another embodiment, the present invention also provides the application of Lactobacillus gasseri CUL09 in inhibiting the proliferation of Candida albicans.

[0030] It should be noted that the Lactobacillus gasseri CUL09 of the present invention was 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.

[0031] The partial 16S rDNA sequence (1466 bp) of Lactobacillus gasseri CUL09 described in this invention is as follows:

[0032]

[0033] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to specific embodiments, mainly covering specific embodiments such as strain preparation, characteristic determination and composition application.

[0034] Example 1

[0035] In this embodiment, microscopic examination of the bacterial strain is required. Specifically, under aseptic conditions, Lactobacillus gasseri CUL09 is picked up with an inoculation loop, smeared onto a glass slide, prepared, and Gram-stained. The bacterial morphology is then observed under a microscope. The results show that the bacterial cells are rod-shaped, Gram-positive, and non-spore-forming, consistent with the characteristics of the Lactobacillus genus.

[0036] Example 2

[0037] In this embodiment, MRS plate purification and separation are required. Specifically, under aseptic conditions, Lactobacillus gasseri CUL09 is picked up with an inoculation loop, streaked on an MRS plate, and the plate is placed in an incubator and incubated at 37°C for 72 hours. Single colonies are selected for microscopic examination to achieve pure culture isolation of the strain, thereby obtaining single-morphological colonies and confirming the purity of the strain.

[0038] Example 3

[0039] In this embodiment, two loops of Lactobacillus gasseri CUL09 were picked up from the slant and placed into a 50 ml MRS liquid medium. The mixture was cultured at 37°C for 24 h and then transferred to a new MRS liquid medium at a 5% inoculation rate. The mixture was cultured at 37°C for 72 h to obtain the Lactobacillus gasseri CUL09 fermentation broth.

[0040] Subsequently, the H2O2 production capacity was determined, specifically by introducing freshly cultured lactobacilli (i.e., greater than 10...). 8 CFU / ml serially diluted to a certain concentration, 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, and anaerobically incubated at 37°C for 48 to 72 hours. After removing the plate, it was 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. The judgment criteria are shown in Table 1.

[0041] Table 1. Semi-quantitative determination criteria for H2O2

[0042] Colony discoloration time <![CDATA[Generate semi - quantitative score for H2O2]]> ≤10 minutes 3 points 10-20 minutes 2 points ≥20 minutes 1 point >30 minutes or no color change 0 points

[0043] According to the test results, Lactobacillus gasseri CUL09 has a better ability to produce H2O2 than other Lactobacillus gasseri CUL36, and also better than Lactobacillus acidophilus CUL60 and Lactobacillus acidophilus CUL21, as detailed in Table 2.

[0044] Table 2. Ability to produce H2O2

[0045] strain <![CDATA[Generate semi - quantitative score of H2O2]]> Lactobacillus gasseri CUL09 3 points Lactobacillus gasseri CUL36 2 points Lactobacillus acidophilus CUL60 1 point Lactobacillus acidophilus CUL21 0 points

[0046] The colonies of live *Lactobacillus gasseri* CUL60 began to show a slight blue tinge after 2-3 minutes, the blue color deepened further after 5 minutes, and a large amount of blue appeared after 6-9 minutes. According to the judgment criteria shown in Table 1, the ability of *Lactobacillus gasseri* CUL09 to produce hydrogen peroxide was semi-quantitatively grade 3. In terms of H2O2 production capacity, *Lactobacillus gasseri* CUL09 showed a significant advantage over other strains.

[0047] Example 4

[0048] In this embodiment, the application of lactobacillus in inhibiting the activity of Gardnerella vaginalis (ATCC14018) was demonstrated. The specific procedure was as follows: 10 μL of freshly cultured lactobacillus suspension was taken, i.e., more than 10 μL of the suspension. 8 CFU / ml samples were spotted onto the surface of MRS agar and anaerobically cultured at 37°C for 48 hours. 100 μL of freshly cultured Candida albicans suspension was mixed thoroughly with 5 mL of soft BHI agar (0.7% agar) in a 50°C water bath and poured onto the previously spotted lactobacillus plates. After solidification, the plates were anaerobically cultured at 37°C for 12-48 hours. The inhibition of Gardnerella vaginalis activity was assessed by observing the growth of Gardnerella vaginalis. The results are shown in Table 3.

[0049] Table 3. Inhibition of Gardnerella vaginalis activity

[0050] strain Inhibits Gardnerella vaginalis activity Lactobacillus gasseri CUL09 +++ Lactobacillus gasseri CUL36 + Lactobacillus acidophilus CUL60 ++ Lactobacillus acidophilus CUL21 +

[0051] In Table 3, the inhibitory activity was assessed as follows: +++, high inhibitory activity against Gardnerella vaginalis (complete inhibition of Gardnerella vaginalis growth); ++, moderate inhibitory activity against Gardnerella vaginalis; +, low inhibitory activity against Gardnerella vaginalis; 0, no inhibitory activity (Gardnerella vaginalis grows normally). The results indicate that *Lactobacillus gasseri* CUL09 exhibits superior inhibitory activity against vaginal Gardnerella vaginalis compared to *Lactobacillus gasseri* CUL36, and also superior to *Lactobacillus acidophilus* CUL60 and CUL21. *Lactobacillus gasseri* CUL09 demonstrates a significant advantage over other strains in inhibiting vaginal Gardnerella vaginalis activity.

[0052] Example 5

[0053] In this embodiment, the application of Lactobacillus to inhibit the activity of Candida albicans (ATCC90028) was demonstrated. The specific process was as follows: 10 μL of freshly cultured Lactobacillus suspension was taken (more than 10 μL). 8 CFU / ml samples were inoculated onto the surface of MRS agar and anaerobically cultured at 37°C for 48 hours. 100 μL of freshly cultured Candida albicans was mixed thoroughly with 5 mL of soft YM agar (0.7% agar) 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 assessed by observing yeast growth. The results are shown in Table 4.

[0054] Table 4. Inhibition of Candida albicans activity

[0055] strain Inhibits Gardnerella vaginalis activity Lactobacillus gasseri CUL09 +++ Lactobacillus gasseri CUL36 + Lactobacillus acidophilus CUL60 ++ Lactobacillus acidophilus CUL21 +

[0056] In Table 4, the inhibitory activity was assessed as follows: +++, high inhibitory activity against Candida albicans (complete inhibition of Candida albicans growth); ++, moderate inhibitory activity against Candida albicans; +, low inhibitory activity against Candida albicans; 0, no inhibitory activity (Candida albicans grows normally). The results indicate that Lactobacillus gasseri CUL09 exhibits superior inhibitory activity against Candida albicans compared to Lactobacillus gasseri CUL36, and also superior to Lactobacillus acidophilus CUL60 and Lactobacillus acidophilus CUL21. In terms of inhibiting Candida albicans activity, Lactobacillus gasseri CUL09 demonstrates a significant advantage over other strains.

[0057] Example 6

[0058] In this embodiment, the adhesion ability of lactobacilli to intestinal epithelial cells was applied. 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 procedure was as follows: Four strains of lactobacilli were cultured in MRS liquid medium for 48 hours (greater than 10...) 8 The cells were centrifuged (CFU / ml), washed three times with PBS, and resuspended in DMEM complete culture medium for later use. Meanwhile, Caco-2 cells were adjusted to 600,000 cells / mL and seeded into 6-well plates. After 48 hours of culture, a monolayer was formed. The cells were washed twice with sterile PBS, and the cells in the first well were digested with trypsin. The cell count was determined using a hemocytometer. Additionally, 1 mL of the previously prepared Lactobacillus and a blank control PBS were added to each of the other five wells. After 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 48 hours of anaerobic culture, the number of Lactobacillus on each plate was counted, and the number of adherent Lactobacillus was calculated. The adhesion rate (%) was calculated as: (Number of adherent Lactobacillus / Total number of cells) × 100. The results are shown in Table 5.

[0059] Table 5 Adhesion ability to intestinal epithelial cells

[0060] strain Adhesion rate to intestinal epithelial cells (%) Lactobacillus gasseri CUL09 178.95 Lactobacillus gasseri CUL36 28.68 Lactobacillus acidophilus CUL60 130.77 Lactobacillus acidophilus CUL21 32.56

[0061] The results showed that *Lactobacillus gasseri* CUL09 exhibited a higher adhesion rate to intestinal epithelial cells than *Lactobacillus gasseri* CUL36, and also superior to *Lactobacillus acidophilus* CUL60 and CUL21. In terms of intestinal epithelial cell adhesion ability, *Lactobacillus gasseri* CUL09 demonstrated a significant advantage over other strains.

[0062] Example 7

[0063] In this embodiment, the adhesion ability of lactobacilli to vaginal epithelial cells was applied. 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 procedure 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 cell count was determined using a hemocytometer. Additionally, 1 mL of the previously prepared lactobacilli and 1 mL of blank control PBS were added to each of the other 5 wells. After co-incubation for 2 hours, unadhered lactobacilli were 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 culture for 48 hours, the number of lactobacilli on each plate was counted, and the number of adherent lactobacilli was calculated. Adhesion rate % = Number of adherent lactobacilli / Total number of cells × 100. The results are shown in Table 6.

[0064] Table 6 shows the adhesion ability of vaginal epithelial cells.

[0065] strain Adhesion rate to vaginal epithelial cells (%) Lactobacillus gasseri CUL09 86.58 Lactobacillus gasseri CUL36 32.45 Lactobacillus acidophilus CUL60 45.21 Lactobacillus acidophilus CUL21 36.78

[0066] The results showed that *Lactobacillus gasseri* CUL09 exhibited a higher adhesion rate to vaginal epithelial cells than *Lactobacillus gasseri* CUL36, and also superior to *Lactobacillus acidophilus* CUL60 and CUL21. *Lactobacillus gasseri* CUL09 demonstrated a significant advantage over other strains in its ability to adhere to vaginal epithelial cells.

[0067] Example 8

[0068] In this embodiment, an animal experiment was conducted to investigate the reduction of the risk of Gardnerella vaginalis infection by oral administration of a probiotic composition containing Lab4 + Lactobacillus gasseri CUL09. The risk of bacterial vaginosis was determined by inhibiting the number of Gardnerella vaginalis and increasing the number of lactobacilli in the animals' vaginas. Specifically, 50 female BALB / c mice were randomly divided into 5 groups of 10 mice each: blank control group, model group, Lab4 group (Lab4), Lactobacillus gasseri CUL09 group (CUL09), and Lab4 + Lactobacillus gasseri CUL09 probiotic group (Lab4 + CUL09).

[0069] An animal model of bacterial vaginosis was established based on the model described by Sabbatini et al. Three days before infection (day -3) and on the day of infection (day 0), all mice except the control group received a subcutaneous injection of 100 μL of estradiol valerate (0.5 mg dissolved in 100 μL of medium-chain glycerides). On day 0, the vaginal inoculation contained 10 live bacteria. 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. On day 6 post-infection, except for the model group, the control group received 200 μL of PBS buffer via gavage once daily, while the other three groups received 200 μL of PBS buffer at a concentration of 10 CFU / mL via gavage once daily. 10 The probiotic suspension at CFU / mL was administered by gavage once daily until the end of the experiment, for a total of 14 days.

[0070] Detection of vaginal flora in mice: Using a micropipette containing 50 μL of physiological saline, the vaginas of mice were repeatedly flushed 5-6 times at the following time periods: after modeling (day 5), day 1 after gavage (day 19), and day 6 after gavage (day 25). 30 μL of the vaginal irrigation fluid was collected for Gardnerella vaginalis and Lactobacillus colony counting, and compared with the model group and the blank control group. The percentages of Gardnerella vaginalis and Lactobacillus in each group were calculated. Specifically: if the Lactobacillus percentage in the blank control group was 100%, then the Lactobacillus percentage in the model group = (Lactobacillus count in the model group × 100) / (Lactobacillus count in the blank control group). Similarly, if the Gardnerella vaginalis percentage in the model control group was 100%, then the Gardnerella vaginalis percentage 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.

[0071] Table 7. Percentages of Gardnerella vaginalis and Lactobacillus in vaginal lavage fluids of each experimental group

[0072]

[0073] As shown in Table 7, on day 1 after gavage, the number of Gardnerella vaginalis colonizations in the vaginas of mice in the three probiotic groups was significantly lower than that in the model group: 30.44% in the Lab4 group, 10.65% in the CUL09 group, and 4.21% in the Lab4+CUL09 group. Conversely, the number of Lactobacillus colonizations in the vaginas was significantly higher than that in the control group: 1265.89%, 1399.13%, and 1785.20%, 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 indicates that oral administration of individual Lab4, CUL09, and Lab4+CUL09 combinations can inhibit Gardnerella vaginalis infection while increasing the number of Lactobacillus. The comparison of the three probiotic groups shows that the Lab4+Lactobacillus gasseri CUL09 combination is superior to either Lab4 or Lactobacillus gasseri CUL09 alone in inhibiting Gardnerella vaginalis infection.

[0074] Example 9

[0075] In this embodiment, two probiotic composition products prepared according to the present invention are used respectively. One is a probiotic composition (Lab4 and Lactobacillus gasseri CUL09, each 5.0 × 10⁻⁶). 9 CFU / day, total viable bacteria count is 1.0 × 10⁻⁶ 10 One product contains 10 billion CFU / day of probiotics (CFU / day) in combination with vitamin B6 (2.1 mg / day), cranberry extract (110 mg / day, 36:1), and a conventional carrier. Another option is a low-dose formulation of this probiotic combination (Lab4 and Lactobacillus gasseri CUL09 at 1.0 × 10⁻⁶ CFU / day). 8 CFU / day, total viable bacteria count is 2.0 × 10⁻⁶ 8 A product containing 200 million CFU / day of probiotics (CFU / day) and vitamin B6 (2.1 mg / day), cranberry extract (110 mg / day, 36:1), and a conventional carrier combination, was used to treat bacterial vaginosis. The Amsel "gold standard" and Gram staining Nugent scoring criteria for the clinical diagnosis of bacterial vaginosis (BV) were used to evaluate changes in BV-related indicators before and after oral administration of the probiotic composition of this invention. The results are shown in Table 8.

[0076] Table 82 shows the therapeutic effects of 82 probiotic compositions on BV.

[0077]

[0078]

[0079] In addition, the results of the vaginal lactobacillus count are shown in Table 9:

[0080] Table 9. Number of vaginal lactobacilli

[0081]

[0082] The lactobacillus count was scored based on the average number of lactobacillus-like bacteria observed per 10 oil immersion fields: 0+, no lactobacillus observed; 1+, <1 lactobacillus; 2+, 1-4 lactobacillus; 3+, 5-30 lactobacillus; 4+, >30 lactobacillus. Referring to Tables 8 and 9, based on the Amsel "gold standard" for clinical diagnosis and the Gram staining Nugent scoring criteria, patients with bacterial vaginosis showed improvement in both clinical symptoms and lactobacillus count after taking the 10 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, specifically 1.0 × 10⁻⁶ for Lab4 and 1.0 × 10⁻⁶ for Lactobacillus gasseri CUL09. 8 CFU / day, total viable bacteria count is 2.0 × 10⁻⁶ 8 CFU / day, also after 4 weeks of use, showed a significantly lower effect on improving bacterial vaginosis compared to the probiotic composition described in this invention. The results indicate that the live bacteria content of Lab4 and Lactobacillus gasseri CUL09 in the probiotic composition described in this invention is not less than 5.0 × 10⁻⁶. 9 CFU / day, total viable count not less than 1.0 × 10⁻⁶ 10 CFU / day.

[0083] Therefore, the above-mentioned probiotic composition using Lab4 and Lactobacillus gasseri CUL09 and its application in reducing the risk of vaginal infections, comprising Lab4 probiotics and the novel strain Lactobacillus gasseri CUL09, achieves vaginal flora reconstruction via oral administration, effectively improving vaginal pH, eliminating odor, and increasing lactobacillus colonization. This invention also relates to the combination of the probiotic composition with vitamin B6, cranberry extract, and conventional carriers, 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, vaginitis, vaginal candidiasis, and urethral infections. Furthermore, this invention, through the combination of Lab4 and Lactobacillus gasseri CUL09, achieves the inhibition of harmful vaginal bacteria, reconstruction of vaginal flora to reduce the risk of vaginal infections, and can also prevent and treat urethral infections.

[0084] 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.

[0085] 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 of Lab4 and Lactobacillus gasseri CUL09, characterized in that, The probiotic composition contains Lab4 and Lactobacillus gasseri CUL09, wherein Lab4 contains four probiotic strains, namely Lactobacillus acidophilus CUL60, Lactobacillus acidophilus CUL21, Bifidobacterium animalis subsp. lactis CUL34 and Bifidobacterium bifidum CUL20.

2. The probiotic composition of Lab4 and Lactobacillus gasseri CUL09 according to claim 1, characterized in that, In the probiotic composition, the viable counts of Lab4 and Lactobacillus gasseri CUL09 are both greater than 1.0 × 10⁻⁶. 9 CFU / ml(g).

3. The use of a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 according to any one of claims 1 to 2 in the reconstruction of vaginal flora.

4. The use of a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 according to any one of claims 1 to 2, combined with vitamin B6, cranberry extract and a conventional carrier, in the preparation of food, supplements or medicines for the prevention and treatment of vaginal infections.

5. The use of a probiotic composition of Lab4 and Lactobacillus gasseri CUL09 according to any one of claims 1 to 2, combined with vitamin B6, cranberry extract and a conventional carrier, in the preparation of food, supplements or medicines for the prevention and treatment of urinary tract infections.

6. A strain of Lactobacillus gasseri CUL09, characterized in that, The strain number of Lactobacillus gasseri CUL09 is CUL09, the deposit date is March 14, 2003, the deposit location is the British Microbiological Culture Collection (NCIMB), and the biological accession number is NCIMB 30155.

7. The use of Lactobacillus gasseri CUL09 as described in claim 6 in the generation of hydrogen peroxide.

8. The use of Lactobacillus gasseri CUL09 as described in claim 6 in adherent intestinal epithelial cells and vaginal epithelial cells.

9. The use of Lactobacillus gasseri CUL09 as described in claim 6 in inhibiting the proliferation of Gardnerella vaginalis.

10. The use of Lactobacillus gasseri CUL09 as described in claim 6 in inhibiting the proliferation of Candida albicans.

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