A vaginal probiotic composition for the relief of a variety of vaginitis

Abstract

The application discloses a vaginal probiotic composition for relieving multiple vaginitis, and belongs to the technical field of microorganisms. The combination of Lactobacillus crispatus CCFM1298, Lactobacillus gasseri CCFM1201 and fructooligosaccharide is selected based on the growth characteristics of the strains, the synergistic symbiosis of the combined strains is realized, and the effect of multiple relief of vaginitis is realized. The probiotic composition can relieve vaginal tissue inflammation, regulate the local immune system of the vagina, and help relieve and improve bacterial vaginitis and candidal vaginitis of women. Moreover, since the probiotics are all derived from the vagina of healthy women, the probiotics are used in the preparation of products and sanitary products for female vaginitis, help relieve and improve female vaginitis, and have a good application prospect.

Description

Technical Field

[0001] This invention relates to a vaginal probiotic composition for relieving various types of vaginitis, belonging to the field of microbial technology. Background Technology

[0002] Unlike the highly diverse gastrointestinal tract, the female reproductive tract has a lower microbiome diversity and changes dynamically throughout the menstrual cycle. A healthy vaginal flora is dominated by Lactobacillus species, primarily including *Lactobacillus curvaturei*, *Lactobacillus gasseri*, *Lactobacillus jenny*, and *Lactobacillus indolentus*. When exogenous pathogens interfere or endogenous imbalances occur, infections such as vaginitis, cervicitis, and pelvic inflammatory disease can develop. Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) are the most common vaginal infections in women of reproductive age. Furthermore, BV and / or VVC are associated with numerous complications, including premature birth, pelvic inflammatory disease, infertility, ectopic pregnancy, pelvic abscess, spontaneous abortion, menstrual disorders, increased susceptibility to HIV infection, and other chronic health problems. Given the prevalence of BV and VVC and their associated complications, BV and VVC are considered significant global public health issues.

[0003] BV (vaginal vaginitis) is characterized by increased vaginal microbiome diversity, decreased lactobacilli, and increased BV-associated anaerobic bacteria. Common BV-associated bacteria include Gardnerella vaginalis and Prevotella vaginalis. Clinically, BV primarily presents with increased vaginal discharge with a fishy odor, sometimes accompanied by mild vulvar itching or burning. The discharge is grayish-white, homogeneous, and thin. The initial cure rate for BV is 70%–90%, with a recurrence rate of up to 40% after 3 months and as high as 60% after 12 months. The most common bacteria associated with VVC are Candida species, particularly Candida albicans, Candida glabrata, and Candida tropicalis. Clinical symptoms of VVC are nonspecific and can be associated with various vaginal infections, such as bacterial vaginosis. The most common clinical manifestations are vulvar itching and burning, accompanied by vaginal soreness and irritation, leading to difficulty urinating. Vulvar and vaginal erythema, edema, and fissures are also common. It is estimated that approximately 10-15% of asymptomatic women are infected with Candida, 50% of women with a first infection will experience a second VVC event, and 5-10% will develop recurrent candidal vaginitis. Currently, the main clinical treatment for BV and VVC is antibiotic therapy. However, antibiotics indiscriminately kill both pathogenic and non-pathogenic bacteria, ultimately leading to dysbiosis and resulting in poor treatment efficacy and high recurrence rates.

[0004] Probiotics are widely used to promote gastrointestinal health, and they may also be beneficial in the prevention and treatment of gynecological diseases. Currently, the probiotics reported in China suitable for women are mainly Lactobacillus delbrueckii DM8909, a single lactobacillus preparation screened from vaginal secretions of healthy women by Professor Kang Bai of Dalian Medical University (Microbiological Study of Lactobacillus delbrueckii DM8909 strain [J]. Chinese Journal of Microecology, 2001). This is primarily for external vaginal application, and lactobacilli are not common bacteria in the reproductive tract of healthy women. Furthermore, some patents disclose vaginal probiotic mixtures (CN113384601A, CN111281896, CN109674826A), mostly targeting single vaginitis, primarily for external vaginal application, with unknown effects after oral administration. Moreover, the combination of probiotic mixtures does not consider factors that may reduce efficacy, such as antagonism between strains, competition for nutrients in the gastrointestinal tract, and adhesion sites. It is also unclear whether they can stimulate the host's immune system in vivo to alleviate and / or treat vaginitis. Therefore, there is currently a lack of vaginal probiotic compositions that have undergone comprehensive evaluation and have a clear effect on various types of vaginitis after oral administration. Summary of the Invention

[0005] To address the existing problems, this invention provides a vaginal probiotic composition that can synergistically regulate bacterial vaginosis and candidal vaginitis. The key point is that this composition can enhance the immunomodulatory effect of single probiotic preparations.

[0006] This invention provides a strain of Lactobacillus curvaturei ( Lactobacillus crispatus CCFM1298 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 9, 2022, with accession number GDMCC No: 62275.

[0007] The present invention provides *Lactobacillus curvatureii* ( Lactobacillus crispatus CCFM1298 was derived from vaginal swabs from healthy women in Jiangsu Province. The genome of CCFM1298 was extracted, and the 16S rDNA of strain CCFM1298 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.; the nucleotide sequence of the amplified 16S rDNA from CCFM1298 is shown in SEQ ID NO.1). This sequence was then compared with the nucleotide sequence in NCBI, and the results showed that the strain was *Lactobacillus curlis*, and it was named *Lactobacillus curlis*. Lactobacillus crispatus (CCFM1298)

[0008] This invention provides a vaginal probiotic composition, which, based on the total number of colonies, contains 1 part of Lactobacillus curvaturei CCFM1298 and 1 part of Lactobacillus gasseri CCFM1201, and also contains prebiotic fructooligosaccharides.

[0009] The present invention provides a composition containing probiotics and prebiotics, wherein the probiotics are Lactobacillus curvaturei CCFM1298 and Lactobacillus gasseri CCFM1201; and the prebiotics are fructooligosaccharides.

[0010] The Lactobacillus gasseri CCFM1201 is described in the Chinese invention patent application with publication number CN 114250186 A and accession number GDMCC No:62136.

[0011] In one embodiment of the present invention, *Lactobacillus curvatureii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 are added to the composition at a live count ratio of 1:1; the amount of probiotics added to the composition is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0012] In one embodiment of the present invention, the fructooligosaccharide was purchased from Quantum Hi-Tech (Guangdong) Biotechnology Co., Ltd.

[0013] In one embodiment of the present invention, the amount of fructooligosaccharide added to the composition is at least 2 g / Kg bw.

[0014] In one embodiment of the present invention, the content of both *Lactobacillus curvaturei* and *Lactobacillus gasseri* in the composition is not less than 10%. 6 CFU / mL or 10 6 CFU / g.

[0015] The present invention also provides a product comprising the above-described composition.

[0016] In one embodiment of the present invention, the product is food, medicine, or hygiene product.

[0017] In one embodiment of the present invention, the food includes dietary supplements, ordinary food, and health products.

[0018] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0019] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0020] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0021] In one embodiment of the present invention, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0022] In one embodiment of the present invention, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0023] The present invention also provides the use of the above composition in the preparation of products for relieving and / or treating bacterial and candidal vaginitis.

[0024] In one embodiment of the present invention, the relief and / or treatment of bacterial vaginosis and candidal vaginitis includes relieving vaginal tissue inflammation and regulating local vaginal immunity; the local vaginal immune system includes reducing the expression of myeloperoxidase (MPO) and TNF-α.

[0025] In one embodiment of the present invention, the product is a pharmaceutical or hygiene product.

[0026] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0027] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0028] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0029] In one embodiment of the present invention, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0030] In one embodiment of the present invention, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0031] The present invention also provides the application of the above composition in the preparation of products that reduce vaginal epithelial shedding, alleviate vaginal tissue inflammation, and regulate local vaginal immunity.

[0032] In one embodiment of the present invention, the product is a pharmaceutical or hygiene product.

[0033] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0034] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0035] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0036] In one embodiment of the present invention, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0037] In one embodiment of the present invention, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0038] Beneficial effects

[0039] (1) This invention provides a strain of Lactobacillus curvaturei ( Lactobacillus crispatus ) CCFM1298 and a strain of Lactobacillus gasseri ( Lactobacillus gasseri The composition of CCFM1201, based on the Lactobacillus curvature ( Lactobacillus crispatus CCFM1298 and Lactobacillus gasseri ( Lactobacillus gasseri CCFM1201 utilizes the growth characteristics of substrates to select optimal combinations of strains and prebiotics, ensuring the synergistic symbiosis of combined strains and truly realizing the multiple effects corresponding to the combined strains.

[0040] (2) The present invention provides a vaginal probiotic composition composed of Lactobacillus curvaturei CCFM1298, Lactobacillus gasseri CCFM1201 and prebiotics. The vaginal probiotic composition can effectively relieve vaginal tissue inflammation, regulate the local vaginal immune system, and help relieve and improve female bacterial vaginosis and candidal vaginitis.

[0041] Preservation of biological materials

[0042] A strain of Lactobacillus curvature ( Lactobacillus crispatus CCFM1298, taxonomically named: Lactobacillus crispatus It was deposited on December 9, 2022 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62275. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology. Attached Figure Description

[0043] Figure 1 CCFM1201 and CCFM1298 were co-cultured in different proportions in MRS liquid medium.

[0044] Figure 2The generation time for CCFM1201 and CCFM1298 using different carbon sources.

[0045] Figure 3 CCFM1201 and CCFM1298 were co-cultured in different proportions in MRS-FOS liquid medium.

[0046] Figure 4 Flowchart for designing animal experimental protocols for BV.

[0047] Figure 5 Effects of probiotics on myeloperoxidase (MPO) in vaginal tissue of BV mice; *: p <0.05, **: p <0.01, ***: p <0.001.

[0048] Figure 6 Effects of probiotics on TNF-α in vaginal tissue of BV mice; *: p <0.05, **: p <0.01.

[0049] Figure 7 Figure 1 shows the histopathological evaluation of probiotics on vaginal tissue in BV mice: (A): blank control group; (B): bacterial vaginosis model group; (C): Lactobacillus gasseri CCFM1201 group; (D): Lactobacillus curvaturei CCFM1298 group; (E): probiotic composition group 1; (F): probiotic composition group 2.

[0050] Figure 8 The effect of probiotics on the concentration of sialyl sidase in vaginal secretions of BV mice;*: p <0.05, **: p <0.01, ***: p <0.001.

[0051] Figure 9 Flowchart for designing animal experimental protocols for VVC.

[0052] Figure 10 The effect of probiotics on myeloperoxidase (MPO) in vaginal tissue of VVC mice; **: p <0.01.

[0053] Figure 11 The effect of probiotics on TNF-α in vaginal tissue of VVC mice; *: p <0.05.

[0054] Figure 12The images show the histopathological evaluation of probiotics on the vaginal tissue of VVC mice; (A): blank control group; (B): candidal vaginitis model group; (C): Lactobacillus gasseri CCFM1201 group; (D): Lactobacillus curvularis CCFM1298 group; (E): probiotic composition group 1; (F): probiotic composition group 2. Detailed Implementation

[0055] The Gardnerella vaginalis involved in the following examples was Gardnerella vaginalis ATCC 14018, purchased from the Guangdong Institute of Microbiology Culture Collection Center (GDMCC). The Candida albicans involved in the following examples was isolated from the vaginal secretions of patients with candidal vaginitis and deposited at the Culture Collection Center of the Food Biotechnology Research Center of Jiangnan University.

[0056] The culture media involved in the following examples are as follows:

[0057] MRS liquid culture medium ( / L): peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 20.0 g, anhydrous sodium acetate 2.0 g, dipotassium hydrogen phosphate trihydrate 2.6 g, diammonium hydrogen citrate 2.0 g, manganese sulfate monohydrate 0.25 g, magnesium sulfate heptahydrate 0.5 g. After adding 1 mL of Tween 80, add 1000 mL of distilled water to adjust the pH to 6.2~6.4, and autoclave at 115℃ for 20 min.

[0058] MRS solid medium: Add 1.5%~2% agar to the prepared MRS liquid medium and autoclave at 115℃ for 20 min.

[0059] Selective medium ( / L): Add 39 g NaCl to the prepared MRS liquid medium, add 1.5%~2% agar, and autoclave at 115℃ for 20 min.

[0060] BHI liquid culture medium ( / L): Tryptic extract 10.0 g, bovine heart extract 17.5 g, sodium chloride 5.0 g, disodium hydrogen phosphate 2.5 g, glucose 2.0 g, plus 1% yeast extract, 0.1% maltose, and 0.1% glucose. Adjust the pH to 7.4. Autoclave at 115℃ for 20 min. Before use, add 10% sterile fetal bovine serum to the culture medium.

[0061] YPD liquid culture medium ( / L): peptone 20.0 g, glucose 20.0 g, yeast extract 10.0 g. Autoclave at 115℃ for 20 min.

[0062] Plain agar solid medium ( / L): Add 2% agar to every 1 L of water and autoclave at 115℃ for 20 min.

[0063] The detection methods involved in the following embodiments are as follows:

[0064] Determination of TNF-α and MPO concentrations:

[0065] At the end of the experiment, mice were euthanized by cervical dislocation, and vaginal tissue was dissected. The vaginal tissue sample (20 mg) was placed in 200 μL of RIPA lysis buffer containing a 2% (v / v) mixture of protease inhibitors and a 2% (v / v) mixture of phosphatase inhibitors. The tissue was homogenized using a high-throughput tissue homogenizer (65 Hz, 45 s / time, 6 times) to obtain a vaginal tissue homogenate. The homogenate was centrifuged at 12000×g for 15 min at 4°C, and the vaginal tissue supernatant was used to determine the concentrations of MPO and TNF-α according to the kit instructions.

[0066] Detection of sialidase content

[0067] Vaginal lavage fluid was collected from mice on day 18 and centrifuged at 3600×g for 3 min at 4°C to remove cell debris. 10 μL of vaginal lavage fluid was extracted from the vaginal supernatant for experimental use. Sialidase levels were measured using enzyme-linked immunosorbent assay (ELISA), and OD values ​​were calculated. 450 nm Compare the values ​​with the standard curve. Follow the kit instructions for specific procedures.

[0068] Example 1: Lactobacillus curvature ( Lactobacillus crispatus CCFM1298 Separation and Identification Method

[0069] (1) Separation

[0070] Using vaginal swabs from healthy women in Jiangsu Province as samples, the samples were pretreated and stored in 30% glycerol at -80°C. After thawing, 0.5 mL of the sample was mixed and then serially diluted tenfold in 4.5 mL of 0.9% physiological saline. Appropriate serial dilutions were plated onto MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus curlis* were picked and streaked onto MRS solid medium for purification. Single colonies were then picked and enlarged on MRS liquid medium. The bacterial resuspended in 30% glycerol for preservation, yielding strain CCFM1298. The typical colonies of *Lactobacillus curlis* were small, white, and round, with a plump center and diffuse periphery.

[0071] (2) Identification

[0072] The genome of CCFM1298 was extracted, and the 16S rDNA of strain CCFM1298 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.; the nucleotide sequence of the 16S rDNA obtained from CCFM1298 amplification is shown in SEQ ID NO.1). The sequence was aligned to the nucleotide sequence in NCBI, and the results showed that the strain was Lactobacillus curlis, and it was named Lactobacillus curlis. Lactobacillus crispatus (CCFM1298)

[0073] Example 2: Co-culture of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298

[0074] To observe whether *Lactobacillus gasseri* CCFM1201 and *Lactobacillus curvature* CCFM1298 strains can stably coexist when co-cultured, we co-cultured *Lactobacillus gasseri* CCFM1201 and *Lactobacillus curvature* CCFM1298 at different ratios and observed changes in community structure. The specific steps are as follows:

[0075] (1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298

[0076] Both *Lactobacillus curlii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 were preserved in 30% glycerol at -80°C before activation. Small amounts of *Lactobacillus curlii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 bacterial suspensions were streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h. Single colonies were obtained, and each colony was inoculated into MRS liquid medium. Then, a 2% (v / v) inoculation was performed into MRS medium, and the culture was continued at 37°C for 24 h. After two generations of activation, *Lactobacillus curlii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 bacterial suspensions were obtained, with bacterial concentrations of 4.07 × 10⁻⁶ and 4.07 × 10⁻⁶, respectively. 8 CFU / mL, 1.84×10 8 CFU / mL.

[0077] (2) The activated second-generation Lactobacillus curli CCFM1298 and Lactobacillus gasseri CCFM1201 bacterial suspensions obtained in step (1) were inoculated into 5 mL of MRS medium in any proportion and incubated in a constant temperature incubator at 37 ℃ for 24 h to prepare cultures. Wherein, any proportion means: Lactobacillus gasseri CCFM1201 and Lactobacillus curli CCFM1298 bacterial suspensions were mixed in volume ratios of 1:1, 1:9 and 9:1 respectively to prepare cultures.

[0078] (3) The cultures obtained in step (2) were inoculated into MRS medium at an inoculation rate of 2% (v / v) and cultured again at 37 °C for 24 h. This transfer was repeated twice.

[0079] As described above, growth measurements were performed for 72 hours, with samples collected every 24 hours. The total number of bacteria in the co-culture was counted in MRS medium, and the viable count of *Lactobacillus gasseri* CCFM1201 in the co-culture system was counted in selective medium. Changes in relative abundance were observed, and the results are as follows: Figure 1 As shown.

[0080] The results showed that when Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298 were co-cultured at different ratios, Lactobacillus gasseri CCFM1201 was the dominant bacterium in the co-culture system. This indicates that Lactobacillus gasseri CCFM1201 has a competitive advantage in the co-culture system, which leads to growth imbalance and makes it impossible to achieve stable coexistence of the combined strains.

[0081] Example 3: Determination of the antibacterial activity of Lactobacillus gasseri CCFM1201 against Lactobacillus curvature CCFM1298

[0082] Interference competition exists between microorganisms in co-culture systems, and this interaction is crucial in the composition and maintenance of the microbial community. The generation of antimicrobial compounds plays a significant role in this interference competition. Therefore, to verify whether *Lactobacillus gasseri* CCFM1201 inhibits the growth of *Lactobacillus curvatureii* CCFM1298 by producing secondary metabolites, we determined the antimicrobial effect of *Lactobacillus gasseri* CCFM1201 on *Lactobacillus curvatureii* CCFM1298. The specific steps are as follows:

[0083] (1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298

[0084] Both *Lactobacillus curvature* CCFM1298 and *Lactobacillus gasseri* CCFM1201 were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus* suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h to obtain single colonies. These single colonies were then inoculated into MRS liquid medium and cultured again. The resulting culture was inoculated at 2% (v / v) onto MRS medium and incubated at 37°C for 24 h, activating for three generations to prepare *CCFM1298* and *Lactobacillus gasseri* CCFM1201 bacterial suspensions, respectively.

[0085] (2) Preparation of antibacterial substances

[0086] The activated Lactobacillus geostriatus CCFM1201 suspension obtained in step 1 was centrifuged at 4°C and 15000×g for 1 min. The supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain cell-free supernatant of Lactobacillus geostriatus CCFM1201.

[0087] (3) Double-layer agar antibacterial test

[0088] Antimicrobial substances were determined using a double-layer agar containing *Lactobacillus curvatureii* CCFM1298 in step (1) and the cell-free supernatant obtained in step (2). Specifically:

[0089] Pour MRS solid medium containing 2% Lactobacillus curlis CCFM1298 suspension into a dry plain agar plate and dry at room temperature for 15 min.

[0090] Each antibacterial substance obtained in step (2) was dropped into the top of the agar and dried until completely absorbed. MRS liquid culture medium was selected as a negative control and streptomycin sulfate at 640 μg / mL as a positive control. The results are shown in Table 1.

[0091] Table 1: Determination of antimicrobial substances in Lactobacillus gasseri CCFM1201

[0092]

[0093] Note: (+) indicates positive; (-) indicates negative.

[0094] The results showed that Lactobacillus gasseri CCFM1201 did not produce antibacterial substances that inhibited Lactobacillus curvature CCFM1298.

[0095] Example 4: Generation time of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298 under different carbon sources

[0096] According to the competition-related hypothesis, the closer the relationship between two organisms, the more likely they are to compete and exclude each other due to overlapping ecological niches. In the reproductive tract, α-amylase breaks down glycogen into smaller carbohydrates (such as maltose, maltotriose, maltopentose, and maltodextrin) to provide energy for lactobacilli in the vagina. Both *Lactobacillus curvatureii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 originate from the vagina, suggesting that *Lactobacillus gasseri* CCFM1201 may be more competitive for carbohydrates than *Lactobacillus curvatureii* CCFM1298, leading to the rapid proliferation of *Lactobacillus gasseri* CCFM1201 and its dominance. Therefore, we measured the glucose utilization time of *Lactobacillus gasseri* CCFM1201 and *Lactobacillus curvatureii* CCFM1298.

[0097] The specific steps are as follows:

[0098] 1. Preparation of culture media with different carbon sources:

[0099] The culture medium is MRS liquid medium, the difference being that glucose is replaced with different carbon sources (inulin, resistant dextrin, galactooligosaccharide, fructooligosaccharide, xylooligosaccharide, and mannose), all at a concentration of 20.0 g / L, and the initial pH is adjusted to 7.0.

[0100] 2. Determination of time

[0101] (1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298

[0102] Both *Lactobacillus curvatureii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus curvatureii* suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h to obtain single colonies. These single colonies were then inoculated into MRS liquid medium and cultured again. The resulting culture was inoculated at 2% (v / v) onto MRS medium and incubated at 37°C for 24 h. After three generations of activation, *Lactobacillus curvatureii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 suspensions were obtained.

[0103] (2) The activated Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298 obtained in step (1) were separately inoculated at a rate of 2% (v / v) into the culture media with different carbon sources obtained in step (1) (5 mL / tube), in triplicate, and incubated in a constant temperature incubator at 37°C. The OD of the bacterial culture was measured every 2 h. 600 Value. Then, with time as the x-axis, log2OD. 600 The value is plotted on the ordinate to create a growth curve of the strain. The generation time is calculated, and the results are as follows: Figure 2 As shown.

[0104] The results showed that the glucose utilization time of Lactobacillus gasseri CCFM1201 (2.03±0.16 h) was less than that of Lactobacillus curvature CCFM1298 (2.43±0.10 h), and the difference was statistically significant. p < 0.01 Therefore, when Lactobacillus gasseri CCFM1201 is co-cultured with Lactobacillus curvature CCFM1298, it will preferentially utilize glucose, thus making Lactobacillus gasseri CCFM1201 the dominant bacterium.

[0105] To alleviate the competition for nutritional niches between *Lactobacillus gasseri* CCFM1201 and *Lactobacillus curvature* CCFM1298, we sought suitable oligosaccharides with the goal of balancing the generation times of *Lactobacillus gasseri* CCFM1201 and *Lactobacillus curvature* CCFM1298. For example... Figure 2 As shown, when glucose was replaced with fructooligosaccharides, the generation time of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298 was shortened and there was no significant difference.

[0106] Example 5: Effect of fructooligosaccharides on the co-culture of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298

[0107] To observe the effect of fructooligosaccharides on the community composition of *Lactobacillus gasseri* CCFM1201 and *Lactobacillus crispa* CCFM1298 co-cultured in any ratio, we replaced glucose in the MRS medium with an equal amount of fructooligosaccharides. The specific steps are as follows:

[0108] (1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298

[0109] Both *Lactobacillus curlii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus* suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h to obtain single colonies. These single colonies were then inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and incubated at 37°C for 24 h. After three generations of activation, bacterial suspensions of *Lactobacillus curlii* CCFM1298 and *Lactobacillus gasseri* CCFM1201 were prepared, with concentrations of 7.2 × 10⁻⁶ and 7.2 × 10⁻⁶, respectively. 8 CFU / mL, 3.9×10 8 CFU / mL.

[0110] (2) The activated second-generation Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298 obtained in step (1) were inoculated into 5 mL of MRS-FOS medium (with glucose in MRS replaced by an equal amount of fructooligosaccharides) in any ratio, and incubated in a 37 ℃ constant temperature incubator for 24 h to prepare cultures. MRS liquid medium was used as a control. "Any ratio combination" means that Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298 bacterial suspensions were mixed at volume ratios of 1:1, 1:9, and 9:1 to prepare cultures.

[0111] (3) The culture obtained in step (2) is inoculated into the culture medium described in step (2) at an inoculation amount of 2% (v / v) and cultured again at 37 °C for 24 h. This transfer is repeated twice.

[0112] As described above, growth measurements were performed for 72 hours, with samples collected every 24 hours. The total number of bacteria co-cultured was counted in MRS medium, and the viable count of *Lactobacillus gasseri* CCFM1201 during co-culture was counted in selective medium. The changes in the relative abundance of the two strains were observed, and the results are as follows: Figure 3 As shown.

[0113] The results showed that when co-cultured at different ratios, fructooligosaccharides could effectively alleviate the competition between Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298, promote the growth of Lactobacillus curvature CCFM1298, and avoid the occurrence of growth imbalance.

[0114] Example 6: Application of vaginal probiotic composition in alleviating bacterial vaginosis in mice

[0115] The specific implementation method is as follows:

[0116] (1) Preparation of bacterial suspension and composition

[0117] Lactobacillus curlifron CCFM1298 bacterial suspension: Lactobacillus curlifron CCFM1298, stored at -80℃ with 30% glycerol, was activated in MRS liquid medium (activation method as described in Example 2). After three generations of activation, the suspension was centrifuged at 8000 g for 15 min, and the bacterial sludge was collected. The sludge was washed twice with PBS and resuspended in PBS at a 1:100 ratio. Finally, a concentration of 1×10⁻⁶ was obtained. 10 A CFU / mL suspension of Lactobacillus curlis CCFM1298 was used for intervention experiments.

[0118] Gardnerella vaginalis suspension: Gardnerella vaginalis, preserved in 30% glycerol at -80℃, was activated in BHI liquid medium. The method was as follows: 2% (v / v) inoculum was inoculated into BHI medium. The first generation was anaerobically cultured at 37℃ for 48 h, and the second and third generations were anaerobically cultured at 37℃ for 24 h. After three generations of activation, the bacterial sludge was collected by centrifugation at 8000 g for 15 min. The sludge was washed twice with PBS and resuspended in PBS at a 1:10 ratio. Finally, a concentration of 1×10⁻⁶ was obtained. 10 A CFU / mL vaginal Gardnerella vaginalis suspension was used for infection experiments.

[0119] Lactobacillus gasseri CCFM1201 bacterial suspension: Lactobacillus gasseri CCFM1201 preserved in an -80°C refrigerator with 30% glycerol was activated in MRS liquid medium (the activation method is referred to Example 2). After three generations of activation, it was centrifuged at 8000 g for 15 min, and the bacterial sludge of the Lactobacillus gasseri bacterial suspension after three generations of activation was collected. The bacterial sludge was washed twice with PBS and resuspended in PBS at a ratio of 1:100. Finally, a Lactobacillus gasseri CCFM1201 bacterial suspension with a concentration of 1×10 10 CFU / mL was obtained for the intervention experiment.

[0120] Probiotic composition 1: The obtained Lactobacillus crispatus CCFM1298 and the Lactobacillus gasseri CCFM1201 bacterial suspension were compounded at a viable bacteria count ratio of 1:1, and finally a probiotic composition 1 with a viable bacteria count of 1×10 10 CFU / mL was obtained.

[0121] (2) Animal experiments

[0122] Experimental animals and experimental strains:

[0123] SPF-grade 7-week-old female BALB / c mice, weighing 17 - 19 g, were raised in an IVC system for one week to adapt. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license number SCXK(Beijing)2012 - 0001). During the feeding period, each mouse had free access to water and was fed SPF mouse feed (Co 60 disinfected). Raising environment: Lighting with a half-cycle of day and night, temperature 22 - 24°C, humidity about 45% - 55%.

[0124] Experimental procedure Figure 4 As shown, the experimental protocol and grouping: The mice were randomly divided into 6 groups according to their body weights, as shown in Table 2.

[0125] Table 2: Grouping of mouse experiments

[0126]

[0127] The experimental period was 22 days (days -3 to 18). Before the start of the experiment, the mice were in a one-week adaptation phase. Three days before and on the day of infection, except for the blank group, the remaining mice were subcutaneously injected with 100 μL of estradiol valerate (0.5 mg of estradiol valerate dissolved in 100 μL of sesame oil) to induce estrus in the mice. This was done every morning, and then once every 7 days until the end of the experiment.

[0128] Infection experiment (modeling period): On day 0, the mice in the blank control group were inoculated vaginally with 20 μL of PBS buffer, and the remaining mice were inoculated vaginally with 20 μL of a bacterial suspension with a viable bacteria count of 10 10CFU / mL Gardnerella vaginalis suspension. After inoculation, mice were inverted for 1-2 minutes to prevent bacteria from flowing out. Vaginal Gardnerella vaginalis inoculation was continued for 7 days until the 6th day.

[0129] Intervention experiment: Starting from day 7, the details are as follows:

[0130] Blank control group: 200 μL of normal saline was administered by gavage once a day;

[0131] Model group mice: 200 μL of physiological saline was administered by gavage once a day;

[0132] Lactobacillus gasseri CCFM1201 group: Mice were administered 200 μL once daily by gavage. 9 CFU Lactobacillus gasseri CCFM1201 bacterial suspension;

[0133] Lactobacillus curlis CCFM1298 group: Mice were administered 200 μL once daily by gavage. 9 CFU Lactobacillus curlis CCFM1298 bacterial suspension;

[0134] Probiotic composition group 1: 200 μL once daily by gavage, 10 9 CFU compound bacterial suspension;

[0135] Two groups of probiotic compositions: 200 μL once daily by gavage, 10 9 CFU probiotic composition 1, simultaneously administered by gavage of 2 g / Kg bw of fructooligosaccharide complex.

[0136] The mice in each group were administered the medication by gavage once a day for 12 consecutive days.

[0137] At the end of infection (day 7) and the end of intervention (day 18), 50 μL of PBS buffer solution was aspirated from the vagina of mice using a pipette tip each time. A total of 300 μL of vaginal lavage fluid was collected to determine the sialidase content. Simultaneously, on day 19, all experimental mice were euthanized and vaginal tissue was dissected for subsequent histopathological analysis, measuring the secretion of myeloperoxidase (MPO) and the inflammatory factor TNF-α.

[0138] (3) Experimental results:

[0139] 1) Effects of vaginal probiotic composition on local vaginal immunity in mice with bacterial vaginosis

[0140] At the end of the experiment, mice were euthanized by cervical dislocation, and vaginal tissue was dissected. The vaginal tissue supernatant was used to determine the concentrations of MPO and TNF-α according to the kit instructions. The results are as follows: Figures 5-6 As shown, Figure 5 and Figure 6The concentrations of MPO and the pro-inflammatory factor TNF-α are shown respectively.

[0141] The results showed that in the blank group, the concentrations of MPO and TNF-α were 19.3 ng / L and 115.8 pg / mg, respectively.

[0142] Model group: After infection with Gardnerella vaginalis, the body experienced oxidative stress, and the myeloperoxidase (MPO) level significantly increased to 23.15 ng / L. p <0.001);

[0143] After 12 days of intervention with Lactobacillus gasseri CCFM1201, Lactobacillus curvature CCFM1298, and probiotic combination 1, the MPO content decreased to 21.40 ng / L. p <0.05), 22.63 ng / L, 21.78 ng / L, and probiotic composition 2 reduced MPO content (significantly reduced to 20.28 ng / L). p <0.01)) The effect is significantly better than CCFM1201, CCFM1298 and probiotic composition 1.

[0144] Similarly, in the model group: infection with Gardnerella vaginalis stimulated the host's immune system, and the expression of the pro-inflammatory factor TNF-α significantly increased to 378.6 pg / mg. p <0.01).

[0145] After 12 days of intervention with Lactobacillus gasseri CCFM1201, the expression of the pro-inflammatory factor TNF-α decreased to 266.3 pg / mg, with no significant difference. Compared with CCFM1201, the combination of CCFM1298 and probiotics significantly reduced the expression of TNF-α (expression levels were 177.9 pg / mg and 240.3 pg / mg, respectively), with inhibition rates of 53% and 53%, respectively. p <0.01), 36.5% ( p <0.05. Probiotic composition 2 (expression level: 204.1 pg / mg) showed no significant difference in efficacy compared to Lactobacillus curlis CCFM1298, but was superior to Lactobacillus gasseri CCFM1201 and probiotic composition 1. p <0.01)

[0146] 2) Histopathological analysis of mouse vaginal tissue

[0147] Mouse vaginal tissue was fixed and preserved in 4% paraformaldehyde solution, embedded in paraffin, and sectioned to a thickness of 5 mm. The sections were then stained with hematoxylin and eosin (H&E). The sections were observed under a pathological slide scanner at 30x magnification. The results are as follows: Figure 7 As shown, Figure 7A, 7B, 7C, 7D, 7E and 7F show HE staining images of the blank control group, bacterial vaginosis model group, CCFM1201 group, CCFM1298 group and probiotic composition intervention group, respectively.

[0148] The results showed that in the blank group, the surface of the spinous layer of vaginal tissue was smooth and continuous, without necrosis or sloughing, and no obvious inflammatory cell infiltration was found. Furthermore, the intercellular connections of the spinous layer cells were tight, and no obvious inter-bridging structures were found.

[0149] Model group: Compared with the blank control, the surface of the stratum spinosum of the vaginal tissue was severely damaged, with obvious necrosis and shedding, and a large number of neutrophils infiltrated to form microabscesses. Spina spinosum cells were decomposed, showing obvious interbridging structures. The dermis showed extensive inflammatory cell infiltration.

[0150] CCFM1201 group and CCFM1298 group: After 12 days of intervention with CCFM1201 and CCFM1298, compared with the model group, the inflammatory cell infiltration phenomenon was significantly improved, the continuity was significantly improved, and the intercellular structure of spinous layer cells gradually became tighter.

[0151] Group 1 of probiotic composition: The effect of probiotic composition 1 in relieving inflammation was not obvious compared with Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298, and a small amount of inflammatory cell infiltration and interbridging structures were still present.

[0152] Probiotic Composition 2: Probiotic Composition 2 is similar to Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298, with significantly improved continuity of the surface of spinous cells and significantly improved inflammatory cell infiltration, and its effect is better than that of Probiotic Composition 1.

[0153] 3) Effect of vaginal probiotic composition on sialidase content in mice with bacterial vaginosis

[0154] Vaginal irrigation fluid was collected from mice on day 18, and sialidase content was measured using enzyme-linked immunosorbent assay (ELISA). OD200 was then converted to sialidase. 450 nm Compare the values ​​with the standard curve. Follow the kit instructions for specific procedures. Results are as follows: Figure 8 As shown.

[0155] The results showed that the sialidase content in the vaginal secretions of mice in the model group was significantly increased to 59.7 ng / L after infection with Gardnerella vaginalis. p <0.001).

[0156] Compared with the model group, after 12 days of intervention with Lactobacillus gasseri CCFM1201 and Lactobacillus curlis CCFM1298, the sialidase levels were significantly reduced (49.4 ng / L and 50.7 ng / L, respectively), with reduction rates of 17.3% and 15.1%, respectively.p <0.01).

[0157] Compared with Lactobacillus gasseri CCFM1201 and Lactobacillus curvature CCFM1298, the probiotic composition 1 had a less significant effect on reducing sialidase levels. p <0.05), which is 53.5 ng / L.

[0158] The effect of probiotic composition 2 was the opposite of that of probiotic composition 1, with a significant decrease in sialidase content to 44.6 ng / L and an inhibition rate of 25.3%. p <0.001).

[0159] Example 7: Application of vaginal probiotic composition in alleviating candidal vaginitis in mice

[0160] The specific implementation method is as follows:

[0161] (1) Preparation of bacterial suspension and composition

[0162] Lactobacillus curlifron CCFM1298 bacterial suspension: Lactobacillus curlifron CCFM1298, stored at -80℃ with 30% glycerol, was activated in MRS liquid medium (activation method as described in Example 2). After three generations of activation, the suspension was centrifuged at 8000 g for 15 min, and the bacterial sludge was collected. The sludge was washed twice with PBS and resuspended in PBS at a 1:100 ratio. Finally, a concentration of 1×10⁻⁶ was obtained. 10 A CFU / mL suspension of Lactobacillus curlis CCFM1298 was used for intervention experiments.

[0163] Candida albicans suspension: Candida albicans stored at -80℃ with 30% glycerol was activated in YPD liquid medium. The method was as follows: 2% (v / v) inoculum was added to YPD medium, and the first generation was cultured at 37℃ for 48 h, and the second and third generations were cultured at 37℃ for 24 h. After three generations of activation, the culture was centrifuged at 8000 g for 15 min, and the mycelial sludge was collected. The mycelial sludge was washed twice with PBS and resuspended in PBS at a 1:10 ratio. Finally, a concentration of 1×10⁻⁶ was obtained. 8 CFU / mL Candida albicans suspension was used for infection experiments.

[0164] Lactobacillus gasseri CCFM1201 bacterial suspension: Lactobacillus gasseri CCFM1201 preserved in an -80 °C refrigerator with 30% glycerol was activated in MRS liquid medium (the activation method is referred to Example 2). After three generations of activation, it was centrifuged at 8000 g for 15 min, and the bacterial sludge of the Lactobacillus gasseri bacterial suspension after three generations of activation was collected. The bacterial sludge was washed twice with PBS and resuspended in PBS at a ratio of 1:100. Finally, a Lactobacillus gasseri CCFM1201 bacterial suspension with a concentration of 1×10 10 CFU / mL was obtained for the intervention experiment.

[0165] Probiotic composition 1: The obtained Lactobacillus crispatus CCFM1298 and the Lactobacillus gasseri CCFM1201 bacterial suspension were compounded at a viable bacteria count ratio of 1:1, and finally, probiotic composition 1 with a concentration of 1×10 10 CFU / mL was obtained.

[0166] (2) Animal experiments

[0167] Experimental animals and experimental strains:

[0168] SPF-grade 7-week-old female BALB / c mice, weighing 17 - 19 g, were raised in an IVC system for one week to adapt. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license number SCXK(Beijing)2012 - 0001). During the feeding period, each mouse had free access to water and was fed SPF mouse feed (Co 60 disinfected). Raising environment: Lighting with a half-cycle of day and night, temperature 22 - 24 °C, humidity about 45% - 55%.

[0169] Experimental procedure Figure 9 As shown, experimental protocol and grouping: The mice were randomly divided into 6 groups according to body weight, as shown in Table 3.

[0170] Table 3 Grouping of mouse experiments

[0171]

[0172] The experimental period was 22 days (days -3 to 18). Before the start of the experiment, the mice were in a one-week adaptation stage. Three days before infection and on the day of infection, except for the blank group, the remaining mice were subcutaneously injected with 100 μL of estradiol valerate (0.5 mg of estradiol valerate dissolved in 100 μL of sesame oil) to induce estrus in the mice. This was done every morning, and then once every 7 days until the end of the experiment.

[0173] Infection experiment (model establishment period): On day 0, the mice in the blank control group were inoculated vaginally with 20 μL of PBS buffer, and the remaining mice were inoculated vaginally with 20 μL of viable bacteria with a count of 10 8CFU / mL Candida albicans suspension. After inoculation, mice were inverted for 1-2 minutes to prevent the bacterial suspension from flowing out. This was repeated for 7 consecutive days until the end of the 6th day.

[0174] Intervention experiment: Starting from day 7, the details are as follows:

[0175] Blank control group: 200 μL of normal saline was administered by gavage once a day;

[0176] Model group mice: 200 μL of physiological saline was administered by gavage once a day;

[0177] Lactobacillus gasseri CCFM1201 group: Mice were administered 200 μL once daily by gavage. 9 CFU Lactobacillus gasseri CCFM1201 bacterial suspension;

[0178] Lactobacillus curlis CCFM1298 group: Mice were administered 200 μL once daily by gavage. 9 CFU Lactobacillus curlis CCFM1298 bacterial suspension;

[0179] Probiotic composition group 1: 200 μL once daily by gavage, 10 9 CFU compound bacterial suspension;

[0180] Two groups of probiotic compositions: 200 μL once daily by gavage, 10 9 CFU probiotic composition 1, simultaneously administered by gavage of 2 g / Kg bw of fructooligosaccharide complex.

[0181] The mice in each group were administered the medication by gavage once a day for 12 consecutive days.

[0182] On day 19, all experimental mice were euthanized and vaginal tissue was dissected for subsequent histopathological analysis, and the expression of myeloperoxidase (MPO) and inflammatory factor TNF-α was measured.

[0183] (3) Experimental results:

[0184] 1) Effects of vaginal probiotic composition on local vaginal immunity in mice with candidal vaginitis

[0185] At the end of the experiment, mice were euthanized by cervical dislocation, and vaginal tissue was dissected. The vaginal tissue supernatant was used to determine the concentrations of MPO and TNF-α according to the kit instructions. The results are as follows: Figures 10-11 As shown, Figure 10 and Figure 11 The expression of myeloperoxidase (MPO) and pro-inflammatory factor TNF-α were shown, respectively.

[0186] The results showed that in the model group, when Candida albicans infected the vagina, the expression of MPO was significantly increased compared with the blank group.p <0.01), which is 24.3 ng / L.

[0187] After 12 days of intervention with probiotic composition 1, the expression of MPO in CCFM1201 and CCFM1298 groups did not decrease significantly, remaining at 22.6 ng / L, 24.2 ng / L, and 24.4 ng / L, respectively. After 12 days of intervention with probiotic composition 2, the MPO content significantly decreased to 19.2 ng / L. p <0.01), the effect was significantly better than the other three groups.

[0188] The expression level of the pro-inflammatory factor TNF-α in the model group was 1.8 times higher than that in the blank control group (115.8 pg / mg). p <0.001), which is 211.9 pg / mg.

[0189] After 12 days of intervention with Lactobacillus gasseri CCFM1201 and probiotic combination, the expression level of TNF-α was significantly reduced (136.9 pg / mg and 138.7 pg / mg, respectively), but the difference was not statistically significant.

[0190] Compared with Lactobacillus gasseri CCFM1201 and probiotic composition 2, CCFM1298 and probiotic composition 1 had less significant effects on regulating the expression of inflammatory factors (195.8 pg / mg and 211.1 pg / mg, respectively).

[0191] 2) Histopathological analysis of mouse vaginal tissue

[0192] Mouse vaginal tissue was fixed and preserved in 4% paraformaldehyde solution, embedded in paraffin, and sectioned to a thickness of 5 mm. The sections were then stained with hematoxylin and eosin (H&E). The sections were observed under a pathological slide scanner at 30x magnification. The results are as follows: Figure 12 As shown, Figure 12 A, 12B, 12C, 12D, 12E, and 12F show HE staining images of the blank control group, the candidal vaginitis model group, the CCFM1201 group, the CCFM1298 group, and the probiotic composition group, respectively.

[0193] The results showed that in the control group, the surface of the spinous layer of vaginal tissue was smooth and continuous, without necrosis or shedding, and no obvious inflammatory cell infiltration was found. Furthermore, the spinous layer cells were tightly connected, and no obvious inter-bridging structures were found.

[0194] Model group: Compared with the blank control, the vaginal tissue showed significant edema of the spinous layer, degeneration and destruction of keratinocytes into a reticular pattern, and a large number of eosinophils, neutrophils and lymphocytes distributed in it. The spinous layer was also loose and the number of intercellular bridges increased.

[0195] CCFM1201 group and CCFM1298 group: After 12 days of intervention with CCFM1201 and CCFM1298, respectively, the continuity was significantly improved compared with the model group, with only a small amount of inflammatory cell infiltration and a small amount of interbridging structures.

[0196] Group 1 of probiotic composition: Compared with the CCFM1201 group and the CCFM1298 group, the improvement effect was not obvious, and there was still a lot of inflammatory cell infiltration.

[0197] Group 2 of probiotic composition: Compared with Lactobacillus gasseri CCFM1201, Lactobacillus curvature CCFM1298 and probiotic composition 1, probiotic composition 2 showed significant improvement in inflammatory cell infiltration and intercellular bridge structure.

[0198] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A composition, characterized in that, The composition contains probiotics and prebiotics, wherein the probiotics are Lactobacillus curvaturei (Lactobacillus curvaturei). Lactobacillus crispatus CCFM1298 and Lactobacillus gasseri CCFM1201, wherein the prebiotic is fructooligosaccharide; Lactobacillus curvature CCFM1298 and Lactobacillus gasseri ( Lactobacillus gasseri CCFM1201 was added at a live bacteria ratio of 1:

1. The *Lactobacillus curlis* CCFM1298 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 9, 2022, with accession number GDMCC No: 62275; the *Lactobacillus gasseri* CCFM1201 has accession number GDMCC No: 62136.

2. The composition according to claim 1, characterized in that, In the composition, the amount of probiotics added is at least 10. 6 CFU / mL or 10 6 CFU / g.

3. A product characterized in that, The product contains the composition according to claim 1 or 2; the product is food, medicine or hygiene product.

4. The product according to claim 3, wherein the food is a dietary supplement, ordinary food, or health product; the medicine is the composition according to claim 1 or 2 and a pharmaceutically permissible carrier; and the hygiene product is a sanitary wipe, sanitary tampon, sanitary cotton, or vaginal wash.

5. The product according to claim 3, characterized in that, The hygiene product in question is a feminine antibacterial / bacteriostatic wash.

6. The product according to claim 3, characterized in that, The sanitary product in question is a panty liner.

7. The product according to claim 3, characterized in that, The hygiene product in question is a sanitary napkin.

8. Use of the composition according to claim 1 or 2 in the preparation of a medicament for relieving and / or treating bacterial and candidal vaginitis.

9. The use of the composition according to claim 1 or 2 in the preparation of a medicine for reducing vaginal epithelial shedding, relieving vaginal tissue inflammation, and regulating local vaginal immunity.

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