A probiotic composition for regulating the microecological balance of the female vagina and its use

Abstract

The application belongs to the field of biological medicine and microbial technology, and specifically discloses a probiotic composition for regulating the microecological balance of the female vagina and application thereof. Lactobacillus crispatus ​ The probiotic composition comprises Chinese native vagina-derived Lactobacillus crispatus (L. crispatus) with a preservation number of GDMCC No. 67862 and GDMCC No. 67864 as the main active ingredient. The strains in the composition are derived from women in the northeast of China and have excellent adaptability and colonization ability to the reproductive tract environment of Chinese women. Experiments show that the strains have significant high expression and high secretion characteristics of S-layer proteins, can effectively down-regulate pro-inflammatory factors (such as TNF-α, IL-1β and TGF-β1) and up-regulate anti-inflammatory factors (such as IL-10), and regulate the vaginal flora structure of mice. The application also provides application of the composition, which can be used for developing drugs, external preparations or health foods for preventing or adjuvant treating vaginitis, bacterial vaginosis and vaginal flora disorder, and has important significance for improving the reproductive health of women.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and microbial technology, specifically relating to a probiotic composition for regulating the vaginal microecological balance in women and its application. Background Technology

[0002] The vaginal microbiota of healthy women (including those of reproductive age and during pregnancy) is dominated by Lactobacillus. According to the Community State Types (CSTs) classification, a vaginal microenvironment dominated by *Lactobacillus curlii* (CST type I) is considered the healthiest vaginal flora state. *Lactobacillus curlii* maintains vaginal microenvironment homeostasis through multiple mechanisms: it utilizes glycogen released from shed vaginal epithelial cells to produce large amounts of lactic acid, maintaining the vaginal pH within the physiological range of 3.8-4.5, thereby inhibiting the invasion of pathogens such as *Gardnerella vaginalis*, *Prevotella vaginalis*, and *Candida albicans*. The hydrogen peroxide and bacteriocins produced by this bacterium have a good bactericidal effect against anaerobic pathogens. Furthermore, the S-layer protein on the surface of *Lactobacillus curlii*, in addition to participating in adhesion to vaginal epithelial cells, also participates in the body's immune regulation. These findings indicate its potential as a probiotic beneficial for women.

[0003] Currently, most commercially available vaginal probiotic products use strains derived from European and American populations or gut microbiota, thus lacking compatibility with the reproductive tracts of Chinese women. To address this, there is an urgent need to establish a standardized strain resource bank with localized characteristics for China. Population studies indicate that the detection rate and dominance of *Lactobacillus curvaturei* in the vagina of women in northern China are significantly higher than in women in southern China, with CST type I being more prevalent in the northern population. This regional microbial distribution pattern suggests that *Lactobacillus curvaturei* strains obtained from women in Northeast China may have stronger environmental adaptability and local colonization advantages. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a probiotic composition for regulating the vaginal microecological balance in women and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a probiotic composition comprising vaginal-derived Lactobacillus curvaturei (Lactobacillus curvaturei). Lactobacillus crispatus As an active ingredient, the *Lactobacillus curvifolius* is selected from at least one of *Lactobacillus curvifolius* with accession number GDMCC No. 67862 and accession number GDMCC No. 67864, which are preserved at the Guangdong Provincial Center for Microbial Culture Collection.

[0006] Based on the above technical solution, the 16S rDNA sequence of the Lactobacillus curvature with accession number GDMCC NO.67862 is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1: Based on the above technical solution, the 16S rDNA sequence of the Lactobacillus curvature with accession number GDMCC NO.67864 is shown in SEQ ID NO.2.

[0008] SEQ ID NO.2: Based on the above technical solution, the *Lactobacillus curvatureensis* was further isolated from the vaginal secretions of healthy women in Northeast China.

[0009] Based on the above technical solution, the *Lactobacillus curvature* further exhibits a high ability to express and secrete S-layer protein; preferably, the concentration of free S-layer protein in the liquid culture supernatant of strain GDMCC No. 67862 is not less than 1.09 g / L, and the concentration of free S-layer protein in the liquid culture supernatant of strain GDMCC No. 67864 is not less than 0.86 g / L.

[0010] Secondly, the present invention provides a microbial agent, drug, topical preparation, hygiene product or health food prepared from the probiotic composition described above.

[0011] Thirdly, the present invention provides the application of the described Lactobacillus curvature or the described probiotic composition in the preparation of products that regulate the female reproductive tract flora and restore the vaginal microecological balance.

[0012] Based on the above technical solution, the product is further used to prevent or assist in the treatment of vaginitis, bacterial vaginosis, or vaginal flora imbalance.

[0013] To this end, the present invention proposes a technical solution: 137 strains of *Lactobacillus curvatureii* were isolated and purified from vaginal secretions collected from healthy women of childbearing age (22-35 years old) and women in their second trimester (14-27 weeks of gestation) in Northeast China. The strains were identified by morphological and molecular biological identification (16S rRNA gene sequencing). The identified strains were cryopreserved in glycerol at -80℃ according to a standardized procedure. Each strain was assigned a unique number, and a database was established to record the strain name, 16S rRNA gene sequence, and identification results.

[0014] The strain resource library of this invention has the following beneficial effects: it enriches the resources of probiotics specifically for women; the included vaginal-derived *Lactobacillus curvatureensis* strains have natural advantages and can effectively restore and maintain the vaginal microecological balance; the strains have undergone rigorous identification and safety testing, ensuring high application safety; the strains in the library are genetically stable, each with complete and registered 16S rRNA sequence information, facilitating scientific research and functional development; and it establishes a standardized preservation and management system, supporting long-term preservation, quality control, and shared application of the strains. Based on this strain resource library, it can be used to develop and prepare various vaginal probiotic compositions for the prevention or adjunctive treatment of vaginitis, bacterial vaginosis, vaginal flora imbalance, and other diseases, improving women's reproductive health.

[0015] Fourthly, the present invention provides an application of the described curly Lactobacillus in the preparation of S-layer protein products.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The *Lactobacillus curvatureii* strain used in the probiotic composition of this invention has the preservation number GDMCC NO.67862. GDMCC NO.67864 is derived from vaginal secretions of healthy women in a specific region of China and exhibits good adaptability and colonization ability in the reproductive tract environment of Chinese women, more effectively restoring and maintaining a healthy vaginal microecological environment of CST type I. Attached Figure Description To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0017] Figure 1 The image shows the expression levels of S-layer proteins in two strains of Lactobacillus curvature and the Western blot (WB) results of free S-layer proteins in the culture supernatant.

[0018] Figure 2 These are pathological sections of the vagina in mice after intervention with two strains of Lactobacillus curvature.

[0019] Figure 3 The results of ELISA quantification of inflammatory factors in mice after intervention with two strains of Lactobacillus curvature.

[0020] Figure 4 The results show the vaginal flora of mice after intervention with two strains of Lactobacillus curvature. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0022] Example 1 Sample Collection and Ethical Compliance: This step was conducted after obtaining written informed consent from the participants and obtaining ethical review approval. Vaginal secretion samples were collected from healthy women of childbearing age and mid-pregnancy (14-27 weeks of gestation) aged 22-35 years in Northeast China. All samples met the requirements of the "Regulations on the Management of Human Genetic Resources of the People's Republic of China," and the source of the samples was truthfully stated. Secretions from the upper vaginal wall or posterior fornix were collected from each participant by a clinician using disposable sterile swabs and stored at -80℃ after collection.

[0023] Isolation and culture of the bacterial strain: After removing the vaginal swab and allowing it to return to room temperature, transfer it to 1 ml of physiological saline solution and mix thoroughly. Spread 50 μL of the stock solution evenly onto Lactobacillus selective solid medium (LBS medium) and incubate at 37°C with 5% CO2 for 24–48 hours. Select typical Lactobacillus colonies (smooth surface, regular edges, opaque, white or grayish-white round colonies, approximately 1–2 mm in diameter), transfer them to De Man, Rogosa and Sharp (MRS) solid medium for streaking and purification, and pick single colonies for enrichment culture in MRS broth medium.

[0024] Strain identification: The purified strain was Gram-stained and its morphology was observed under a microscope. 1 mL of liquid culture in the logarithmic growth phase was centrifuged at 8000 rpm for 5 min to collect the cells, and genomic DNA was initially extracted using cell lysis buffer. The 16S rRNA gene was then amplified using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'). After amplification, the amplified product was identified by agarose gel electrophoresis (target band approximately 1500 bp) and sent to a professional institution for Sanger bidirectional sequencing. After removing low-quality sequences at both ends, the sequencing results were analyzed for similarity with the model strain sequence using the NCBI BLAST algorithm. Cultures with ≥99% similarity to the *Lactobacillus curvatureii* model strain were selected for further preservation treatment.

[0025] The present invention ultimately isolated, screened and identified two representative Lactobacillus curvature strains, which were deposited at the Guangdong Provincial Microbial Culture Collection Center on February 11, 2026, with accession numbers GDMCC NO.67862 and GDMCC NO.67864, respectively.

[0026] Example 2 Two strains of *Lactobacillus curvatureii* from the above examples were inoculated into 15 mL of MRS liquid medium at a ratio of 10% (v / v%) and anaerobically cultured for 24 h. One mL of the bacterial culture was then centrifuged at 6000 rpm for 5 min to collect the bacterial pellet, which was then washed three times with an equal volume of sterile PBS buffer. Simultaneously, three randomly selected *Lactobacillus curvatureii* strains from our laboratory were treated in the same manner as positive controls. Total RNA was extracted from the bacteria according to the Trizol reagent (Ciscotech) instructions. The concentration (ng / μL) and purity (A260 / A280) were determined using a nucleic acid detector (Nano-300, Hangzhou Aosheng Instruments). The RNA was then reverse transcribed into cDNA according to the RNA reverse transcription kit (Kangrun Biotechnology) instructions. The 16S rRNA (V3-V4) gene fragments of each strain were amplified using the following primers (Forward: 5'-CCTACGGGAGGCAGCA-3', Reverse: 5'-GGACTACCGGGTATCTAATC-3') as internal control genes, and the S-layer protein gene was amplified using the following primers (Forward: 5'-GTACCAAGCCAAAGCAAGAC-3', Reverse: 5'-GTTTGAAGCCTTTACGTAAGTC-3'). The expression levels of the S-layer protein gene in each *Lactobacillus curvatureii* strain were detected using quantitative real-time PCR (qPCR), with the relative expression level of the control strain serving as the baseline. The expression levels were then analyzed using a 2^... -ΔΔCT The relative expression level of the S-layer protein gene of Lactobacillus curvature was calculated using a method.

[0027] Since the S-layer protein of *Lactobacillus curvatureensis* easily detaches from the cell surface, the supernatant was extracted after centrifugation of the culture after 24 h of culture and placed on ice. Trichloroacetic acid solution was slowly added to the supernatant at a ratio of 10% (v / v%), and the mixture was allowed to stand overnight. After the formation of a white precipitate, the mixture was centrifuged at 15,000 rpm for 5 min at 4°C, the supernatant was discarded, and anhydrous ethanol solution cooled to 4°C was added. After thorough vortexing, the precipitate was collected again under the same centrifugation conditions. This process was repeated twice, and the protein precipitate was allowed to stand until the ethanol completely evaporated. The dried protein precipitate was dissolved in a mixed solution containing 1% SDS and 0.1M NaOH, and its quantification was performed using the BCA method.

[0028] A 20 μg protein solution was denatured at 95°C for 5 min. After separation by SDS-PAGE, the gel was stained with Coomassie Brilliant Blue for 2 h or transferred to a PVDF membrane. The transferred PVDF membrane was incubated with anti-S layer protein antibody (BS3797, Beijing Bio-Sens Biotechnology Co., Ltd.) at a concentration of 1 μg / mL in TBS-T buffer containing 5% BSA. The secondary antibody was HRP-labeled anti-rabbit IgG antibody (1:2000, ICH005, Beijing Bio-Sens Biotechnology Co., Ltd.). ECL signals were recorded using a Bio-Rad ChemiDoc XRS+ chemiluminescence imaging system.

[0029] The results showed that the expression level of S-layer protein in the two *Lactobacillus curvature* strains was significantly higher than that in the control strain (GDMCC NO. 67862). p <0.001; GDMCC NO.67864 compared with the control strain, p =0.0059), and a large amount of S-layer protein was present in the culture supernatant. The concentrations of S-layer protein in the supernatant of GDMCC NO.67864 and GDMCC NO.67862 were 0.86 g / L and 1.09 g / L, respectively.

[0030] Example 3 The effects of the above-mentioned probiotic composition on vaginal microecological regulation were evaluated by constructing CST type III and IV vaginal flora structure mouse models. Six-week-old female ICR mice were selected and their vaginas were perfused with a concentration of 10% for 10 consecutive days. 8 CFU / mL inert lactobacilli or Gardnerella vaginalis suspensions were used to establish CST III or CST IV vaginal flora models, respectively. After model establishment, mice were administered CFU / mL of the suspension for 10 consecutive days. 8 A 1:1 mixture of the two types of *Lactobacillus curvatureus* suspensions (CFU / mL) was used for intervention. On the second day after model establishment and the day after intervention, 200 μL of physiological saline was instilled into the vagina of mice, and the vaginal irrigation fluid was collected for vaginal flora analysis. Simultaneously, mice were sacrificed, and vaginal tissue was collected for pathological examination and detection of vaginal inflammatory factors.

[0031] The results showed that the vaginal swelling and redness of CST IV vaginal structure mice were alleviated after topical administration of the above-mentioned probiotic composition. p =0.0017), vaginal histopathological sections showed a significant reduction in inflammatory cell infiltration ( p =0.0119). A remission trend was observed in CST III mice, but no statistically significant difference was observed. Among various inflammatory cytokines, TNF-α and TGF-β1 were significantly reduced in CST IV mice after administration of this probiotic complex (respectively...). p =0.0056;p =0.0213). CSTIII mice, on the other hand, showed decreased pro-inflammatory cytokine IL-1β and significantly increased anti-inflammatory cytokine IL-10 (respectively). p =0.0082; p =0.0269). The above results indicate that the application of the above probiotic complex can improve inflammatory manifestations in both mouse models.

[0032] Example 4 Total bacterial DNA was extracted from vaginal lavage fluid samples from mice according to the QlAamp DNA mini Kit instructions. The concentration and purity were determined using a nucleic acid concentration analyzer, followed by PCR amplification and Illumina MiSeq sequencing. The V3–V4 region of the 16S rRNA gene was amplified using the following universal primers: 338 F: 5'-ACTCCTACGGGAGGCAGCA-3' 806 R: 5'-GGACTACHVGGGTWTCTAAT-3' After initial quality control, paired-end sequencing data were processed using the 16S rRNA gene workflow of the dada2 R package (v1.1) to obtain amplicon sequence variants (ASVs). Subsequently, taxonomic annotation of the species was performed using the Taxminer R package and the Sliva database.

[0033] The results showed that after the probiotic composition was applied externally to the vagina of mice in both model groups, the live bacteria were able to colonize the vagina and increase in abundance, thus regulating the vaginal flora structure and demonstrating the regulatory ability of the probiotic composition on the vaginal microecological structure.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probiotic composition, characterized in that, The composition contains vaginal-derived *Lactobacillus curvaturei* (… Lactobacillus crispatus As an active ingredient, the *Lactobacillus curvature* is selected from at least one of *Lactobacillus curvature* with accession number GDMCC No. 67862 and accession number GDMCC No. 67864, which are preserved at the Guangdong Provincial Center for Microbial Culture Collection.

2. The probiotic composition according to claim 1, characterized in that, The curly lactobacillus is selected from at least one of (1) or (2) below: (1) The 16S rDNA sequence of Lactobacillus curvature with accession number GDMCC No. 67862 is shown in SEQ ID NO. 1; (2) The 16S rDNA sequence of Lactobacillus curvature with accession number GDMCC No. 67864 is shown in SEQ ID NO.

2.

3. The probiotic composition according to claim 1, characterized in that, The *Lactobacillus curvatureensis* was isolated from vaginal secretions of healthy women in Northeast China.

4. The probiotic composition according to claim 1, characterized in that, The *Lactobacillus curvature* has the ability to highly express and secrete S-layer protein; preferably, the concentration of free S-layer protein in the liquid culture supernatant of strain GDMCC No. 67862 is not less than 1.09 g / L, and the concentration of free S-layer protein in the liquid culture supernatant of strain GDMCC No. 67864 is not less than 0.86 g / L.

5. A microbial agent, drug, topical preparation, hygiene product or health food prepared from the probiotic composition according to any one of claims 1 to 4.

6. The use of the *Lactobacillus curvature* as described in any one of claims 1 to 4 or the probiotic composition as described in any one of claims 1 to 4 in the preparation of products that regulate the female reproductive tract flora and restore the vaginal microecological balance.

7. The application according to claim 6, characterized in that, The product is used to prevent or assist in the treatment of vaginitis, bacterial vaginosis, or vaginal flora imbalance.

8. The use of the *Lactobacillus curvature* according to any one of claims 1 to 4 in the preparation of S-layer protein products.

Images