A probiotic for improving bacterial vaginosis and composition and application thereof

Abstract

The application discloses probiotics for improving bacterial vaginosis and a composition and application thereof, characterized in that the probiotics are at least one of a fermented Mucilaginis lactobacillus QS150 strain with a preservation number of CGMCC No. 34373, a curled lactobacillus QS204 strain with a preservation number of CGMCC No. 34371 and a Roy's Mucilaginis lactobacillus QS020 strain with a preservation number of CGMCC No. 34372, and the composition is a mixture of the three strains in a proportion of 1:1:1 in terms of viable bacterial quantity, and has an application in preparing products for preventing, relieving or treating bacterial vaginosis, and has the advantages of having antioxidant properties, producing acid and hydrogen peroxide, and inhibiting female vaginal pathogenic bacteria, and improving bacterial vaginosis.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a probiotic for improving bacterial vaginosis, its composition, and its application. Background Technology

[0002] Vaginitis can affect reproductive health, increase the risk of sexually transmitted infections, and induce other gynecological inflammations. It can also affect fertility, increasing the risk of miscarriage, premature birth, infertility, and ectopic pregnancy. Approximately 26%-33% of women are infected with vaginitis, with a recurrence rate of about 72%. The recurrence rate of vulvovaginal candidiasis (VVC) is as high as 89%. Among these, bacterial vaginosis (BV), which accounts for the largest proportion of vaginitis cases, and VVC, are primarily caused by Gardnerella vaginalis (…). Gardnerella vaginalis Once it adheres and colonizes in the vaginal environment, it aggregates with other pathogens to form a biofilm, resisting the host's immune defenses and exacerbating vaginitis symptoms. Furthermore, due to vaginal flora imbalance, it can also lead to the growth and reproduction of pathogens such as Escherichia coli and Staphylococcus aureus, further intensifying the inflammatory response.

[0003] When the vagina is invaded by a large number of pathogens, the body can regulate itself through the vagina's self-regulation mechanisms. These mechanisms mainly include: secreting acidic secretions to expel some pathogens; vaginal innate immune cells recognizing and engulfing pathogens to initiate an immune response and prevent the spread of infection; and lactobacilli in the vagina creating and maintaining an acidic environment by producing lactic acid, hydrogen peroxide, and other substances, thereby inhibiting the growth of other harmful bacteria. However, the body's self-regulation is limited and cannot completely eliminate pathogens from the vagina, requiring treatment intervention. Currently, the treatment of vaginal infections is mainly divided into drug therapy and biological therapy. Drug therapy, while inhibiting pathogenic bacteria, can adversely affect the vaginal lactobacillus flora, easily leading to recurrence and chronic infection. Biological therapies, such as lactobacillus therapy, inhibit the invasion and overgrowth of exogenous and opportunistic pathogens, offering significant advantages such as low recurrence rates, high safety, few side effects, and no drug resistance. For the health of the vaginal microecology, there is an urgent need to develop lactobacilli and their combinations that can improve vaginitis. However, there are currently few types and products of vaginal probiotics on the market, so it is of great significance to explore probiotics that can be used to treat vaginal inflammation. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a probiotic with antioxidant properties, acid production and hydrogen peroxide production, and inhibition of pathogenic bacteria in the female vagina, which improves bacterial vaginosis, and the composition and application thereof.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] This invention provides a probiotic for improving bacterial vaginosis, wherein the probiotic is *Lactobacillus fermentum* with accession number CGMCC No. 34373. Limosilactobacillus fermentum Lactobacillus curvature, strain QS150, preservation number CGMCC No. 34371. Lactobacillus crispatus Strain QS204 and Lactobacillus reuteri with accession number CGMCC No. 34372 Limosilactobacillus reuteri At least one of the QS020 strains.

[0007] This invention also provides a probiotic composition for improving bacterial vaginosis, wherein the probiotic composition comprises *Lactobacillus fermentum* with accession number CGMCC No. 34373. Limosilactobacillus fermentum Lactobacillus curvature, strain QS150, preservation number CGMCC No. 34371. Lactobacillus crispatus Strain QS204 and *Lactobacillus reuteri* with accession number CGMCC No. 34372 ( Limosilactobacillus reuteri Composition of QS020 strain.

[0008] Furthermore, the mixing volume ratio of the *Lactobacillus mucinus* QS150 bacterial suspension, the *Lactobacillus curvature* QS204 bacterial suspension, and the *Lactobacillus reuteri* QS020 bacterial suspension is 1:1:1, and the OD of the bacterial suspension is... 600 nm = 1-2.

[0009] The present invention also provides a probiotic agent for improving bacterial vaginosis, wherein the strain in the probiotic agent includes the probiotics described in claim 1.

[0010] Furthermore, the dosage form of the probiotic agent includes lyophilized powder, capsules, tablets, or granules.

[0011] The present invention also provides the use of the above-mentioned probiotics or the above-mentioned probiotic composition or the above-mentioned probiotic agent in the preparation of products for the prevention, relief or treatment of bacterial vaginosis.

[0012] Furthermore, the pathogens causing bacterial vaginosis are Escherichia coli, Staphylococcus aureus, and Gardnerella vaginalis.

[0013] This invention also provides the application of the above-mentioned probiotics in the preparation of antioxidant products, wherein the probiotics are *Lactobacillus fermentum* (…). Limosilactobacillus fermentum QS150 strain, Lactobacillus curvature ( Lactobacillus curly QS204 strain and Lactobacillus reuteri ( Limosilactobacillus reuteri One of the QS020 strains.

[0014] This invention also provides the application of the above-mentioned probiotics in the preparation of products for regulating vaginal microecology, wherein the probiotics are *Lactobacillus curvaturei* (…). Lactobacillus crispatus QS204 strain and Lactobacillus reuteri ( Limosilactobacillus reuteri One of the QS020 strains.

[0015] This invention also provides the application of the above-mentioned probiotics in the preparation of antibacterial products, wherein the probiotics are Lactobacillus curlis ( Lactobacillus crispatus ) QS204 strain.

[0016] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a lactic acid bacteria and its composition and application for inhibiting pathogenic bacteria causing bacterial vaginosis. The lactic acid bacteria include *Lactobacillus fermentum* isolated from the intestines of infants, *Lactobacillus curvaturei* from the intestines of healthy, long-lived elderly women, and *Lactobacillus reuteri* from *Myrica rubra*. These bacteria possess antioxidant properties and exhibit the effects of producing acid and hydrogen peroxide, as well as inhibiting pathogenic bacteria in the female vagina. Further combination of probiotics, through synergistic effects of multiple strains, can comprehensively cover multiple targets of host defense: for example, *Lactobacillus fermentum* inhibits the growth and reproduction of pathogenic bacteria by producing lactic acid or acetic acid, while *Lactobacillus curvaturei*, as a dominant bacterium in the normal female vaginal flora, can produce hydrogen peroxide and other substances to maintain vaginal flora balance. This multidimensional regulatory strategy not only rebuilds the microecological barrier but also reduces the risk of drug resistance, providing a sustainable solution for long-term health management. Furthermore, specific strain combinations can significantly improve the inhibition rate against pathogenic bacteria causing bacterial vaginosis, exhibiting a synergistic effect.

[0017] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum Strain QS150 was deposited on April 27, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34373. The depository is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0018] Lactobacillus curvaturei ( Lactobacillus crispatus Strain QS204 was deposited on April 27, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34371. The depository is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0019] Lactobacillus reuteri ( Limosilactobacillus reuteri Strain QS020 was deposited on April 27, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34372. The depository is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0020] Figure 1 The antibacterial effects of *Lactobacillus fermentatus* QS150, *Lactobacillus curvatureus* QS204, *Lactobacillus reuteri* QS020, and their combination on *Escherichia coli* ATCC 25922 were investigated.

[0021] Figure 2 The antibacterial effects of Lactobacillus fermentum QS150, Lactobacillus curvatureus QS204, Lactobacillus reuteri QS020 and their combination on Staphylococcus aureus ATCC 6538 were studied.

[0022] Figure 3 The antibacterial effects of *Lactobacillus fermentum* QS150, *Lactobacillus curvularia* QS204, *Lactobacillus reuteri* QS020, and their combination on *Gardnerella vaginalis* ATCC 14018 were investigated. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0024] Example 1: Isolation and identification of strains.

[0025] 1. Strain Isolation: 1g of infant intestinal samples, intestinal samples from healthy long-lived women, and bayberry samples were placed in centrifuge tubes containing 9mL of sterile physiological saline, vortexed to mix, and set aside. Dilute to 10⁻⁶ using a serial dilution method. -6 From 10 respectively -4 10 -5 10 -6 100 μL of each of the three gradients was evenly spread onto MRS solid medium. The petri dishes were placed in an anaerobic workstation at 37°C for incubation. After 48 hours, the culture dishes were removed, and the colony morphology was observed and recorded. Single colonies with typical lactic acid bacteria characteristics were picked up using an inoculation loop and streaked onto MRS solid medium. The colonies were then anaerobically incubated at 37°C for 48 hours to obtain pure colonies. The pure colonies obtained from the plates were inoculated into MRS liquid medium and anaerobically incubated at 37°C for 12-18 hours. The pure strains were then mixed with a 30%-50% (v / v) glycerol solution in equal proportions and stored at -80°C.

[0026] Ultimately, a total of 236 bacterial strains were isolated from infant intestinal samples, 30 strains from healthy long-lived women's intestinal samples, and 2 strains from bayberry samples. The isolated strains were then tested for their inhibitory effects on *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, and *Gardnerella vaginalis* ATCC 14018. The three strains with the strongest inhibitory effects were: strain QS150 from infant intestinal samples, strain QS204 from healthy long-lived women's intestinal samples, and strain QS020 from bayberry samples.

[0027] 2. Strain identification: The bacterial genomic DNA obtained in step 1 was extracted using a rapid bacterial genomic DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd., Shanghai). The bacterial genomic DNA was then amplified using 16S rDNA universal primers 27F and 1492R, followed by sequencing to identify the strain species.

[0028] The primer sequences for the universal 16S rDNA primers 27F and 1492R are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'; PCR system components: 1 μL DNA template, 1 μL 27F, 1 μL 1492R, 22 μL sterile water, 25 μL 2×Taq PCR Master Mix (PCR premix).

[0029] The PCR program was as follows: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for 30 cycles; final extension at 72℃ for 10 min. The amplified PCR product was removed, and 1.5-2 μL of the PCR product was added to an agarose gel. The electrophoresis apparatus voltage was set to 99-100V, and the gel was run for 20-30 min. After the gel was removed, the DNA bands were observed using a gel imaging system, and the target fragment length was determined to be 1500 bp.

[0030] 16S rDNA sequencing: The validated PCR products were sent to Shanghai Saiheng Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequence was BLASTed on NCBI and compared with the sequencing information of known strains for homology to identify the strains.

[0031]

[0032] The strain QS204 screened from the intestinal samples of healthy and long-lived women was identified as Lactobacillus crispatus by morphological and 16S rDNA identification. Its 16S rDNA sequence is shown in SEQ ID NO.2: CATCATCGAGCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCGGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTAAAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTACCGAAGTAAATCTGTTAGTTCCGCTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGATAAAAACTCAT。

[0033]

[0034] The above-mentioned strain was deposited in a cultural heritage institution, and the information is as follows: *Lactobacillus fermentum* (… Limosilactobacillus leaven Strain QS150 was deposited on April 27, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34373. The depository is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0035] Lactobacillus curvaturei ( Lactobacillus crispatus Strain QS204 was deposited on April 27, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34371. The depository is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] Lactobacillus reuteri ( Limosilactobacillus reuteri Strain QS020 was deposited on April 27, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34372. The depository is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0037] Example 2: Antibacterial activity test against Escherichia coli, Staphylococcus aureus, and Gardnerella vaginalis.

[0038] Preparation of probiotic suspension: *Lactobacillus fermentum* QS150, *Lactobacillus cremastrae* QS204, and *Lactobacillus reuteri* QS020, stored at -80℃, were inoculated into MRS liquid medium at a volume ratio of 5% and cultured for 3 generations. The culture was then centrifuged at 8000 rpm for 5 minutes at 4℃. The bacterial cells were collected, washed 2-3 times with PBS, and then resuspended in PBS. The OD of the probiotic suspension was adjusted. 600 nm=0.8. The composition consists of three probiotic suspensions: Lactobacillus fermentum QS150, Lactobacillus curvatureus QS204, and Lactobacillus reuteri QS020, mixed in a volume ratio of 1:1:1. The combination of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 consists of two probiotic suspensions mixed in a volume ratio of 1:1.

[0039] Experimental methods: The antibacterial effects of *Lactobacillus fermentans* QS150, *Lactobacillus crassifolius* QS204, *Lactobacillus reuteri* QS020, and their combinations were detected using the agar diffusion method. *Escherichia coli* and *Staphylococcus aureus* were added to LB liquid medium, and *Gardnerella vaginalis* was added to BHI liquid medium supplemented with 5% serum to form a concentration of 1×10⁻⁶. 8 A CFU / mL suspension of pathogenic bacteria was prepared. Then, 100 μL of the pathogenic bacterial suspension was evenly spread onto a solid agar plate. Wells were punched, and 120 μL of a probiotic suspension or a combined bacterial suspension was added to each well. The solid plates were then incubated at 37°C for 12 h, and the antibacterial activity of the inhibition zone was assessed by the diameter of the inhibition zone. Uninoculated MRS liquid medium served as a negative control, and commercially available *Lactobacillus rhamnosus* GR-1, *Lactobacillus reuteri* RC-14, and their combinations served as positive controls.

[0040] 1. Inhibitory effects of *Lactobacillus fermentans* QS150, *Lactobacillus curvature* QS204, *Lactobacillus reuteri* QS020, and their combination on *Escherichia coli* ATCC 25922.

[0041] The results are as follows Figure 1 As shown, both single bacteria and combinations exhibit significant inhibitory effects against *Escherichia coli* ATCC2 5922. Specifically, the inhibition zone diameters of *Lactobacillus fermentum* QS150 and *Lactobacillus curvatureus* QS204 against *Escherichia coli* ATCC 25922 were 40 mm and 46 mm, respectively, significantly higher than the 33 mm and 23 mm of *Lactobacillus rhamnosus* GR-1 and *Lactobacillus reuteri* RC-14. The inhibition zone diameter of *Lactobacillus reuteri* QS020 against *Escherichia coli* ATCC25922 was 30 mm, significantly higher than *Lactobacillus reuteri* RC-14 and similar to that of *Lactobacillus rhamnosus* GR-1. Furthermore, the combination of *Lactobacillus fermentum* QS150, *Lactobacillus curvature* QS204, and *Lactobacillus reuteri* QS020 exhibited an inhibition zone diameter of 52 mm against *Escherichia coli* ATCC 25922, which was significantly higher than the combination of *Lactobacillus rhamnosus* GR-1 and *Lactobacillus reuteri* RC-14 (inhibition zone diameter was 31 mm).

[0042] 2. Inhibitory effects of *Lactobacillus fermentata* QS150, *Lactobacillus curvature* QS204, *Lactobacillus reuteri* QS020, and their combination on *Staphylococcus aureus* ATCC 6538.

[0043] The results are as follows Figure 2As shown, both single strains and combinations exhibit significant inhibitory effects against Staphylococcus aureus ATCC 6538. Specifically, the inhibition zone diameters of *Lactobacillus fermentum* QS150, *Lactobacillus curvature* QS204, and *Lactobacillus reuteri* QS020 against *S. aureus* ATCC 6538 were 19, 19, and 28 mm, respectively, all higher than the 17 mm diameter of the inhibition zone of *Lactobacillus reuteri* RC-14. Furthermore, the inhibition zone diameter of the combination of *Lactobacillus fermentum* QS150, *Lactobacillus curvature* QS204, and *Lactobacillus reuteri* QS020 against *S. aureus* ATCC 6538 was 60 mm, significantly higher than the combination of *Lactobacillus rhamnosus* GR-1 and *Lactobacillus reuteri* RC-14 (inhibition zone diameter 25 mm).

[0044] 3. Inhibitory effects of *Lactobacillus fermentum* QS150, *Lactobacillus curvature* QS204, *Lactobacillus reuteri* QS020, and their combination on *Gardnerella vaginalis* ATCC 14018.

[0045] The results are as follows Figure 3 As shown, both single bacteria and mixtures exhibited significant inhibitory effects against Gardnerella vaginalis ATCC 14018. Specifically, the inhibition zone diameters of *Lactobacillus fermentum* QS150 and *Lactobacillus reuteri* QS020 against *Gardnerella vaginalis* ATCC 14018 were 40 mm and 40 mm, respectively, both higher than the 37 and 38 mm of *Lactobacillus rhamnosus* GR-1 and *Lactobacillus reuteri* RC-14, while the inhibition zone diameter of *Lactobacillus curvaturei* QS204 against *Gardnerella vaginalis* ATCC 14018 was 25 mm. Furthermore, the inhibition zone diameter of the combination of *Lactobacillus fermentum* QS150, *Lactobacillus curvaturei* QS204, and *Lactobacillus reuteri* QS020 against *Gardnerella vaginalis* ATCC 14018 was 60 mm, significantly higher than the inhibition zone diameter of the combination of *Lactobacillus rhamnosus* GR-1 and *Lactobacillus reuteri* RC-14 (35 mm).

[0046] Example 3: In vitro antioxidant evaluation of the strain

[0047] Lactobacillus fermentans QS150, Lactobacillus curvatureii QS204, and Lactobacillus reuteri QS020, stored at -80℃, were taken out and inoculated into MRS liquid medium at a volume ratio of 5% for 3 generations. After activation culture, the medium was centrifuged at 8000 rpm / min for 5 min at 4℃. The supernatant was filtered through a 0.22 μM filter membrane and collected for in vitro antioxidant assay.

[0048] 1. Evaluation of total antioxidant capacity of the strains: According to the instructions of the total antioxidant capacity kit (Grace, Biosciences), the total antioxidant capacity of the fermentation supernatant of Lactobacillus mucilaginosus QS150, Lactobacillus curvatureus QS204, and Lactobacillus reuteri QS020 was determined to be 1.83, 0.77, and 2.45 μmol Trolox / mL, respectively.

[0049] 2. Evaluation of DPPH free radical scavenging ability of the strains: Following the instructions of the DPPH free radical scavenging ability kit (Grace Biotech, Suzhou), the DPPH free radical scavenging abilities of the fermentation supernatants of *Lactobacillus mucinus* QS150, *Lactobacillus crenulata* QS204, and *Lactobacillus reuteri* QS020 were determined to be 31.51 μg Trolox / mL, 29.71 μg Trolox / mL, and 31.33 μg Trolox / mL, respectively. This indicates that all three strains possess good DPPH free radical scavenging ability.

[0050] 3. Evaluation of hydroxyl radical scavenging ability of the strains: According to the instructions of the hydroxyl radical scavenging ability kit (Grace Biotech, Suzhou), the hydroxyl radical scavenging abilities of the fermentation supernatants of *Lactobacillus mucinus* QS150, *Lactobacillus crenulata* QS204, and *Lactobacillus reuteri* QS020 were determined to be 94.86%, 99.52%, and 43.7%, respectively. This shows that all three strains possess good hydroxyl radical scavenging ability.

[0051] 4. Evaluation of Superoxide Anion Scavenging Rate of the Strains: Following the instructions of the superoxide anion scavenging ability kit (Grace Biotech, Suzhou), the superoxide anion scavenging rates of the fermentation supernatants of *Lactobacillus myxitis* QS150, *Lactobacillus crenulata* QS204, and *Lactobacillus reuteri* QS020 were determined to be 95.84%, 98.37%, and 84.7%, respectively. This indicates that all three strains possess good superoxide anion scavenging ability.

[0052] 5. Evaluation of ABTS free radical scavenging ability of the strains: Following the instructions of the ABTS free radical scavenging ability kit (Grace Biotech, Suzhou), the ABTS free radical scavenging abilities of the fermentation supernatants of *Lactobacillus mucilaginosus* QS150, *Lactobacillus crenulata* QS204, and *Lactobacillus reuteri* QS020 were determined to be 192.31 μg Trolox / mL, 143.18 μg Trolox / mL, and 137.47 μg Trolox / mL, respectively. This indicates that the three strains possess good ABTS free radical scavenging ability.

[0053] Lactobacillus fermentum QS150, Lactobacillus curvatureus QS204, and Lactobacillus reuteri QS020, which have excellent antioxidant functions, can effectively scavenge free radicals, reduce reactive oxygen species released by vaginal inflammation, help protect the vaginal mucosal barrier, and create a favorable environment for the colonization of lactic acid bacteria.

[0054] Example 4: Determination of lactic acid production capacity

[0055] Take out the fermenting Lactobacillus mucinus QS150 and Lactobacillus curvature QS204 stored at -80℃, inoculate them into MRS liquid medium at a volume ratio of 5%, activate and culture for 3 generations, centrifuge at 8000 rpm / min for 5 min at 4℃, filter through a 0.22 μM filter membrane, and collect the supernatant.

[0056] Preparation of standard curve: Weigh an appropriate amount of lactic acid, dissolve it in ultrapure water, and dilute to 100 mL to obtain a lactic acid standard solution with a final concentration of 1 mg / mL. For determination, dilute with 0.1% phosphate buffer to obtain lactic acid solutions of 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL. Chromatographic conditions: Injection volume: 20 μL; Column: Agilent ZORBAX SB-Aq (4.6 × 150 mm, 3.5 μm); Mobile phase: 0.1% phosphate solution: methanol = 97.5:2.5 (V / V), isocratic elution; Flow rate: 2 mL / min; Column temperature: 40℃; Detection wavelength: 210 nm. The lactic acid content in the supernatant of *Lactobacillus fermentans* QS150 and *Lactobacillus curvature* QS204 was determined by UPLC.

[0057] The results showed that the lactic acid standard curve was Y = 3481.8X - 3.3238, R0 2 =0.9997. The lactic acid content in the fermentation supernatant of fermented Lactobacillus mucilaginosus QS150 and Lactobacillus curvature QS204 was 8.23 ​​and 8.55 g / L, respectively.

[0058] Lactobacillus fermentum QS150 and Lactobacillus curvature QS204 can produce lactic acid through metabolism, which lowers the vaginal pH and inhibits the growth and reproduction of vaginal pathogens such as Escherichia coli and Staphylococcus aureus.

[0059] Example 5: Determination of short-chain fatty acids.

[0060] Take out the fermenting Lactobacillus mucinus QS150 and Lactobacillus curvature QS204 stored at -80℃, inoculate them into MRS liquid medium at a volume ratio of 5%, activate and culture for 3 generations, centrifuge at 8000 rpm / min for 5 min at 4℃, filter through a 0.22 μM filter membrane, and collect the supernatant.

[0061] Preparation of standard curves: Weigh appropriate amounts of acetic acid, propionic acid, and butyric acid, dissolve them in ultrapure water, and dilute to 100 mL to obtain standard solutions of acetic acid, propionic acid, and butyric acid with a final concentration of 1 mg / mL. For determination, dilute with 0.1% phosphate buffer to obtain acetic acid, propionic acid, and butyric acid solutions of 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively. Chromatographic conditions: Injection volume: 20 μL; Column: Agilent ZORBAX SB-Aq (4.6 × 150 mm, 3.5 μm); Mobile phase: 0.1% phosphate solution: methanol = 97.5:2.5 (V / V), isocratic elution; Flow rate: 2 mL / min; Column temperature: 40℃; Detection wavelength: 210 nm. The contents of acetic acid, propionic acid, and butyric acid in the supernatants of fermenting *Lactobacillus mucilaginosus* QS150 and *Lactobacillus curvature* QS204 were determined by UPLC.

[0062] The results showed that the standard curve for acetic acid was Y = 2504.9X - 16.907, R0 2 =0.9992; the standard curve for propionic acid is Y=2575.7-32.716, R0 2 =0.9985; the standard curve for butyric acid is Y=2477.1X-0.1678, R0 2 =0.9999. The acetic acid content in the fermentation supernatant of *Lactobacillus mucinus* QS150 and *Lactobacillus curvature* QS204 was 1.44 g / L and 2.72 g / L, respectively; the propionic acid content in the fermentation supernatant of *Lactobacillus mucinus* QS150 and *Lactobacillus curvature* QS204 was 0.79 g / L and 16.1 g / L, respectively; and the butyric acid content in the fermentation supernatant of *Lactobacillus curvature* QS204 was 0.08 g / L.

[0063] Fermenting Lactobacillus mucosa QS150 and Lactobacillus curvature QS204 can metabolize to produce acetic acid, propionic acid, and butyric acid, which can alleviate vaginal inflammation through multiple mechanisms, including antibacterial, anti-inflammatory, immunomodulatory, and mucosal barrier repair. Acetic acid can disrupt the cell membrane integrity of Gardnerella vaginalis, achieving an antibacterial effect; propionic acid can enhance immunomodulation; and butyric acid can inhibit the formation of biofilms by pathogenic bacteria and provide nutrients to vaginal mucosal cells, promoting mucosal repair and maintaining the integrity of the mucosal barrier.

[0064] Example 6: Determination of hydrogen peroxide production.

[0065] Lactobacillus curvatureii QS204, stored at -80℃, was inoculated into MRS liquid medium at a volume ratio of 5% and cultured for 3 generations. After centrifugation at 8000 rpm / min for 5 min at 4℃, the supernatant was collected after filtration through a 0.22 μM filter. The hydrogen peroxide content in the fermentation supernatant of the three strains was determined using a hydrogen peroxide assay kit (colorimetric method) (Nanjing Jiancheng Bioengineering Institute, Nanjing) according to the manufacturer's instructions. The hydrogen peroxide content in the fermentation supernatant of Lactobacillus curvatureii QS204 was found to be 0.25 mmol / L. Hydrogen peroxide can destroy the biofilm structure of pathogenic bacteria, leading to their death. Furthermore, it can synergistically lower vaginal pH with lactic acid, further inhibiting the growth and reproduction of pathogenic bacteria.

[0066] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A probiotic composition for improving bacterial vaginosis, characterized in that: The probiotic composition described herein consists of *Lactobacillus fermentum* with accession number CGMCC No. 34373. Limosilactobacillus fermentum Lactobacillus curvature, strain QS150, preservation number CGMCC No. 34371. Lactobacillus crispatus Strain QS204 and *Lactobacillus reuteri* with accession number CGMCC No. 34372 ( Limosilactobacillus reuteri The strain composition is as follows: the mixed volume ratio of the *Lactobacillus fermentatus* QS150 suspension, the *Lactobacillus curvatureus* QS204 suspension, and the *Lactobacillus reuteri* QS020 suspension is 1:1:

1. The OD of the bacterial suspension is... 600nm =1-2.

2. A probiotic agent for improving bacterial vaginosis, characterized in that, The strains in the probiotic agent include the probiotic composition of claim 1.

3. The probiotic agent for improving bacterial vaginosis according to claim 2, characterized in that, The dosage forms of the probiotic agent include lyophilized powder, capsules, tablets, or granules.

4. The use of the probiotic composition of claim 1 or the probiotic agent of claim 2 in the preparation of products for the prevention, relief or treatment of bacterial vaginosis.

5. The application according to claim 4, characterized in that: The pathogens causing bacterial vaginosis are Escherichia coli, Staphylococcus aureus, and Gardnerella vaginalis.

6. The use of the probiotic composition of claim 1 in the preparation of antioxidant products.

7. The use of the probiotic composition according to claim 1 in the preparation of products for regulating vaginal microecology.

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