Probiotics for improving bacterial vaginitis and composition and application thereof

Abstract

The invention discloses probiotics for improving bacterial vaginitis as well as a composition and application thereof, and is characterized in that the probiotics are at least one of a lactobacillus mucus fermentation QS150 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.34373, a lactobacillus crispatus QS204 strain with the preservation number of CGMCC No.34371 and a lactobacillus reuteri mucus QS020 strain with the preservation number of CGMCC No.34372; the composition is formed by mixing three strains according to the viable count ratio of 1: 1: 1, can be applied to preparation of products for preventing, relieving or treating the bacterial vaginitis, and has the advantages of having the anti-oxidation characteristic, producing acid and hydrogen peroxide, inhibiting female vaginal pathogenic bacteria and improving the bacterial vaginitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a probiotic for improving bacterial vaginosis, a composition thereof and an application thereof. Background Art

[0002] Vaginitis affects reproductive health, increases the risk of STDs, and can trigger other gynecological inflammation, affect conception, and increase the risk of miscarriage, premature birth, infertility, and ectopic pregnancy. Approximately 26% to 33% of women are infected, with a recurrence rate of approximately 72% for vaginitis and 89% for vulvovaginal candidiasis (VVC). Bacterial vaginosis (BV), which accounts for the largest proportion of vaginitis, and the main pathogenic bacteria of VVC are Gardnerella vaginalis ( Gardnerella vaginalis After colonizing the vaginal environment, the bacteria gather other pathogens to form a biofilm, which resists the host's immune defenses and exacerbates vaginitis symptoms. Furthermore, vaginal flora imbalance can lead to the growth and reproduction of pathogens such as Escherichia coli and Staphylococcus aureus, further exacerbating the inflammatory response.

[0003] When the vagina is invaded by a large number of pathogens, the body can regulate itself through its own mechanisms. These include: acidic secretions secreted by the vagina expel some pathogens; innate vaginal immune cells that recognize and phagocytize pathogens, initiating an immune response and preventing the spread of infection; and lactic acid bacteria in the vagina, which create and maintain an acidic environment by producing substances such as lactic acid and hydrogen peroxide, thereby inhibiting the growth of other harmful bacteria. However, the body's self-regulatory mechanisms are limited and cannot completely eliminate pathogens in the vagina, necessitating therapeutic intervention. Currently, treatments for vaginal infections are primarily divided into medications and biological therapies. While medications suppress pathogens, they can negatively impact the vaginal lactobacillus flora, leading to recurrence and chronic infection. Biological therapies, such as lactobacillus therapy, inhibit the invasion and overgrowth of exogenous bacteria and opportunistic pathogens, offering distinct advantages such as low recurrence rates, high safety, minimal side effects, and no resistance to drug resistance. To maintain a healthy vaginal microbiome, there is an urgent need to develop lactobacilli and their compositions 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 discover 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 having antioxidant properties, the ability to produce acid and hydrogen peroxide, and the ability to inhibit female vaginal pathogens for improving bacterial vaginosis, as well as a composition and application thereof.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: The present invention provides a probiotic for improving bacterial vaginosis, wherein the probiotic is fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain, Lactobacillus crispatus with the deposit number of CGMCC No.34371 ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri with the deposit number of CGMCC No.34372 ( Limosilactobacillus reuteri ) at least one of the QS020 strains.

[0006] The present invention also provides a probiotic composition for improving bacterial vaginosis, wherein the probiotic composition is composed of fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain, Lactobacillus crispatus with the deposit number of CGMCC No.34371 ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri with the deposit number of CGMCC No.34372 ( Limosilactobacillus reuteri )QS020 strain composition.

[0007] Furthermore, the mixing volume ratio of the fermented Lactobacillus mucilaginosus QS150 bacterial suspension, the crispatus Lactobacillus QS204 bacterial suspension and the reuteri Lactobacillus mucilaginosus QS020 bacterial suspension is 1:1:1, and the OD of the bacterial suspension is 600 nm=1-2.

[0008] The present invention also provides a probiotic for improving bacterial vaginosis, wherein the strain in the probiotic comprises the probiotic according to claim 1.

[0009] Furthermore, the dosage form of the probiotic includes freeze-dried powder, capsule, tablet or granule.

[0010] The present invention also provides use of the above-mentioned probiotics or the above-mentioned probiotic composition or the above-mentioned probiotic agent in preparing a product for preventing, alleviating or treating bacterial vaginosis.

[0011] Furthermore, the pathogenic bacteria of bacterial vaginosis are Escherichia coli, Staphylococcus aureus and Gardnerella vaginalis.

[0012] The present invention also provides the use of the above-mentioned probiotics in the preparation of antioxidant products, wherein the probiotics are fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain, Lactobacillus crispatus ( Lactobacillus curly ) QS204 strain and Lactobacillus reuteri ( Limosilactobacillus reuteri ) One of the QS020 strains.

[0013] The present invention also provides the use of the above-mentioned probiotics in the preparation of a product for regulating vaginal microecology, wherein the probiotics are Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri ( Limosilactobacillus reuteri ) One of the QS020 strains.

[0014] The present invention also provides the use of the above-mentioned probiotics in the preparation of antibacterial products, wherein the probiotics are Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain.

[0015] Compared with existing technologies, the present invention offers advantages in that: Lactobacillus fermentans, its compositions, and applications for inhibiting bacterial vaginosis pathogens, comprise Lactobacillus mucilaginosus isolated from the intestines of infants, Lactobacillus crispatus from the intestines of healthy, long-lived women, and Lactobacillus mucilaginosus reuteri from bayberry. These lactic acid bacteria possess antioxidant properties, producing acid and hydrogen peroxide, and inhibiting female vaginal pathogens. Further combining probiotics through multi-strain synergistic synergy can comprehensively address multiple host defense targets. For example, Lactobacillus mucilaginosus produces lactic acid or acetic acid to inhibit the growth and reproduction of pathogens, while Lactobacillus crispatus, as a dominant bacteria in the normal female vaginal flora, produces hydrogen peroxide and other factors to maintain vaginal flora balance. This multi-dimensional 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 bacterial vaginosis pathogens, demonstrating a synergistic effect.

[0016] Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on April 27, 2025, with the deposit number CGMCC No.34373. The depository unit is: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on April 27, 2025, with the deposit number CGMCC No.34371. The depository unit is: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] Lactobacillus reuteri ( Limosilactobacillus reuteri ) QS020 strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on April 27, 2025, with the deposit number CGMCC No.34372. The depository unit is: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The antibacterial effect of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020 and the combination on Escherichia coli ATCC 25922; Figure 2 The antibacterial effect of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020 and the combination on Staphylococcus aureus ATCC 6538; Figure 3 The antibacterial effect of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020 and the combination on Gardnerella vaginalis ATCC 14018; DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0021] Example 1. Isolation and identification of strains.

[0022] 1. Isolation of strains: Place 1g of infant intestinal sample, healthy and long-lived female intestinal sample, and bayberry sample in a centrifuge tube containing 9mL of sterile saline, shake and mix, and set aside. Use gradient dilution method to dilute to 10 -6 . From 10 -4 , 10 -5 , 10 -6 100 μL of each of the three gradients was evenly spread on MRS solid medium. The culture dish was placed in an anaerobic workstation at 37°C for incubation. After 48 hours, the colony morphology was observed and recorded. Single colonies with typical lactic acid bacteria characteristics were picked with an inoculation loop and streaked on MRS solid medium. Then, the culture was anaerobically incubated at 37°C for 48 hours to obtain pure colonies. The pure colonies obtained on the plate were inoculated into MRS liquid medium. After anaerobically incubated at 37°C for 12-18 hours, a glycerol solution with a volume concentration of 30%-50% was mixed with the pure strain in equal proportions and stored in a -80°C refrigerator.

[0023] Ultimately, a total of 236 strains were isolated from infant intestinal samples, 30 strains were isolated from intestinal samples of healthy, long-lived women, and two strains were isolated from bayberry samples. The isolated strains were tested for their inhibitory effects against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, and Gardnerella vaginalis ATCC 14018. The strains QS150, QS204, and QS020, which were isolated from infant intestinal samples, healthy, long-lived women, and bayberry samples, were found to have the strongest inhibitory effects.

[0024] 2. Strain identification: The genomic DNA of the strain obtained in step 1 was extracted using a bacterial genomic DNA rapid extraction kit (Shanghai Sangon Biotech Co., Ltd., Shanghai). The genomic DNA of the strain was then amplified using 16S rDNA universal primers 27F and 1492R, and then sequenced to identify the strain species.

[0025] The primer sequences of 16S rDNA universal 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).

[0026] The PCR procedure was as follows: 30 cycles of initial denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 10 minutes. The amplified PCR product was removed and 1.5-2 μL of the product was loaded onto an agarose gel. The electrophoresis instrument was set to 99-100 V and run for 20-30 minutes. After the gel was removed, DNA bands were visualized using a gel imaging system, indicating that the target fragment was 1500 bp in length.

[0027] 16S rDNA sequencing: The verified PCR products were sent to Shanghai Saiheng Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequences were BLASTed on NCBI and compared with the sequencing information of known strains for homology to identify the strains.

[0028]

[0029] The strain QS204 screened from the intestinal samples of healthy and long-lived women was identified as Lactobacillus crispatus by morphology and 16S rDNA identification. Its 16S rDNA sequence is shown in SEQ ID NO.2: CATCATCGAGCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCGGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTAAAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTACCGAAGTAAATCTGTTAGTTCCGCTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGATAAAAACTCAT。

[0030]

[0031] The above strains were sent to a depository institution for preservation, and the information is as follows: Lactobacillus mucilaginosus ( Limosilactobacillus leaven ) QS150 strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on April 27, 2025, with the deposit number CGMCC No.34373. The depository unit is: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on April 27, 2025, with the deposit number CGMCC No.34371. The depository unit is: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Lactobacillus reuteri ( Limosilactobacillus reuteri ) QS020 strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on April 27, 2025, with the deposit number CGMCC No.34372. The depository unit is: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] Example 2: Antibacterial activity test on Escherichia coli, Staphylococcus aureus and Gardnerella vaginalis.

[0035] Preparation of probiotic suspension: fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 stored at -80°C were taken out and inoculated into MRS liquid medium at a volume ratio of 5% for activation culture for 3 generations. After centrifugation at 4°C, 8000 rpm / min for 5 minutes, the cells were collected, washed with PBS 2-3 times, and then resuspended in PBS to adjust the OD value of the probiotic suspension. 600 nm=0.8. The composition of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 is a mixture of three probiotic suspensions at a volume ratio of 1:1:1, and the combination of Lactobacillus rhamnosus GR-1 and Lactobacillus mucilaginosus RC-14 is a mixture of two probiotic suspensions at a volume ratio of 1:1.

[0036] Experimental method: The antibacterial effect of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020 and the combination was tested by 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 8 CFU / mL of pathogenic bacteria suspension. Subsequently, 100 μL of the pathogenic bacteria suspension was evenly spread on a solid agar plate. Use a hole punch to punch corresponding holes, and add 120 μL of the probiotic suspension or the combination suspension into the holes. The solid plate was then incubated at 37°C for 12 hours, and the antibacterial ability of the antibacterial liquid was evaluated by the diameter of the inhibition zone. Uninoculated MRS liquid culture medium was used as a negative control, and commercially available Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and their combination were used as positive controls.

[0037] 1. Inhibitory effects of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020, and their combination on Escherichia coli ATCC 25922 The results are as follows Figure 1 As shown, both single bacteria and combinations have significant inhibitory effects on E. coli ATCC25922. The inhibition zone diameters of Lactobacillus fermentans QS150 and Lactobacillus crispatus QS204 against E. coli ATCC25922 were 40 mm and 46 mm, respectively, significantly higher than those of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 (33 mm and 23 mm). The inhibition zone diameter of Lactobacillus reuteri QS020 against E. coli ATCC25922 was 30 mm, significantly higher than that of Lactobacillus reuteri RC-14 and similar to that of Lactobacillus rhamnosus GR-1. In addition, the combination of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 had 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 mucilaginosus RC-14 (inhibition zone diameter of 31 mm).

[0038] 2. Inhibitory effects of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020, and their combination on Staphylococcus aureus ATCC 6538 The results are as follows Figure 2As shown, both single bacteria and combinations exhibited significant inhibitory effects against Staphylococcus aureus ATCC 6538. The inhibition zone diameters for Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 against Staphylococcus aureus ATCC 6538 were 19, 19, and 28 mm, respectively, all exceeding the 17 mm inhibition zone diameter for Lactobacillus mucilaginosus RC-14. Furthermore, the combination of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 against Staphylococcus aureus ATCC 6538 exhibited an inhibition zone diameter of 60 mm, significantly exceeding the 25 mm inhibition zone diameter for the combination of Lactobacillus rhamnosus GR-1 and Lactobacillus mucilaginosus RC-14.

[0039] 3. Inhibitory effects of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, Lactobacillus mucilaginosus QS020, and their combination on Gardnerella vaginalis ATCC 14018 The results are as follows Figure 3 As shown, both single bacteria and mixtures exhibited significant inhibitory effects against Gardnerella vaginalis ATCC 14018. The inhibition zone diameters of fermented Lactobacillus mucilaginosus QS150 and Lactobacillus mucilaginosus QS020 against Gardnerella vaginalis ATCC 14018 were 40 mm and 40 mm, respectively, both exceeding the 37 mm and 38 mm values ​​for Lactobacillus rhamnosus GR-1 and Lactobacillus mucilaginosus RC-14, respectively. The inhibition zone diameter of Lactobacillus crispatus QS204 against Gardnerella vaginalis ATCC 14018 was 25 mm. Furthermore, the combination of fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 against Gardnerella vaginalis ATCC 14018 had an inhibition zone diameter of 60 mm, significantly exceeding the 35 mm inhibition zone diameter for the combination of Lactobacillus rhamnosus GR-1 and Lactobacillus mucilaginosus RC-14.

[0040] Example 3: In vitro antioxidant evaluation of bacterial strains The fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus mucilaginosus QS020 stored at -80°C were taken out and inoculated into MRS liquid culture medium at a volume ratio of 5% for activation culture for three generations. The culture was then centrifuged at 4°C, 8000 rpm / min for 5 minutes, and filtered using a 0.22 μM filter membrane. The supernatant was collected for in vitro antioxidant assay.

[0041] 1. Evaluation of the total antioxidant capacity of the strains: According to the instructions of the total antioxidant capacity kit (Greis, Biological), the total antioxidant capacity of the fermentation supernatant of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus reuteri QS020 was determined to be 1.83, 0.77, and 2.45 μmol Trolox / mL, respectively.

[0042] 2. Evaluation of the DPPH Radical Scavenging Ability of the Strains: Following the instructions of the DPPH Radical Scavenging Ability Kit (Greis Biotech, Suzhou), the DPPH radical scavenging abilities of the fermentation supernatants of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus reuteri QS020 were measured, yielding 31.51 μgTrolox / mL, 29.71 μgTrolox / mL, and 31.33 μgTrolox / mL, respectively. These results indicate that all three strains possess excellent DPPH radical scavenging abilities.

[0043] 3. Evaluation of the hydroxyl radical scavenging ability of the strains: The hydroxyl radical scavenging ability of the fermentation supernatants of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus reuteri QS020 was determined according to the instructions of the hydroxyl radical scavenging kit (Greis Biotech, Suzhou). The results were 94.86%, 99.52%, and 43.7%, respectively. This indicates that all three strains possess excellent hydroxyl radical scavenging abilities.

[0044] 4. Evaluation of Superoxide Anion Scavenging Efficiency of the Strain: According to the instructions for the Superoxide Anion Scavenging Efficiency Test Kit (Greis Biotech, Suzhou), the superoxide anion scavenging efficiencies of the fermentation supernatants of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus reuteri QS020 were measured to be 95.84%, 98.37%, and 84.7%, respectively. This indicates that all three strains possess excellent superoxide anion scavenging abilities.

[0045] 5. Evaluation of ABTS Free Radical Scavenging Ability of the Strains: The ABTS Free Radical Scavenging Ability Test Kit (Greis Biotech, Suzhou) was used to determine the ABTS free radical scavenging ability of the fermentation supernatants of Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus reuteri QS020. The results were 192.31 μg Trolox / mL, 143.18 μg Trolox / mL, and 137.47 μg Trolox / mL, respectively. These results indicate that the three strains possess excellent ABTS free radical scavenging ability.

[0046] Fermented Lactobacillus mucilaginosus QS150, Lactobacillus crispatus QS204, and Lactobacillus reuteri QS020, which have excellent antioxidant functions, can effectively scavenge free radicals, reduce the active oxygen released by vaginal inflammation, help protect the vaginal mucosal barrier, and create a favorable environment for the colonization of lactic acid bacteria.

[0047] Example 4, Lactic Acid Production Capacity Determination The fermented Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 stored at -80°C were taken out and inoculated into MRS liquid culture medium at a volume ratio of 5%. After activation culture for 3 generations, the culture was centrifuged at 4°C and 8000 rpm / min for 5 minutes, filtered using a 0.22 μM filter membrane, and the supernatant was collected.

[0048] Standard curve preparation: Weigh an appropriate amount of lactic acid and dissolve it in ultrapure water, then dilute to 100 mL to obtain a final concentration of 1 mg / mL lactic acid standard solution. For determination, dilute with 0.1% phosphate buffer to obtain lactic acid solutions at concentrations 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 ZORBAXSB-Aq (4.6 × 150 mm, 3.5 μm); mobile phase: 0.1% phosphoric acid solution: methanol = 97.5:2.5 (v / v), isocratic elution; flow rate: 2 mL / min; column temperature: 40°C; detection wavelength: 210 nm. Lactic acid content in the supernatants of fermentations of Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 was determined using UPLC.

[0049] The results showed that the standard curve of lactic acid was Y=3481.8X-3.3238, R 2 =0.9997. The lactic acid contents in the fermentation supernatants of Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 were 8.23 ​​and 8.55 g / L, respectively.

[0050] Fermented Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 can produce lactic acid through metabolism, lowering the vaginal pH and inhibiting the growth and reproduction of vaginal pathogens such as Escherichia coli and Staphylococcus aureus.

[0051] Example 5: Determination of short-chain fatty acid production.

[0052] The fermented Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 stored at -80°C were taken out and inoculated into MRS liquid culture medium at a volume ratio of 5%. After activation culture for 3 generations, the culture was centrifuged at 4°C and 8000 rpm / min for 5 minutes, filtered using a 0.22 μM filter membrane, and the supernatant was collected.

[0053] To prepare a standard curve, appropriate amounts of acetic acid, propionic acid, and butyric acid were weighed and dissolved in ultrapure water, and the volume was adjusted to 100 mL to obtain a final concentration of 1 mg / mL for each standard solution. For determination, the solutions were diluted with 0.1% phosphate buffer to obtain 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively. Chromatographic conditions included: injection volume, 20 μL; column, Agilent ZORBAX SB-Aq (4.6 × 150 mm, 3.5 μm); mobile phase, 0.1% phosphoric acid solution: methanol (97.5:2.5 v / v), isocratic elution; flow rate, 2 mL / min; column temperature, 40°C; detection wavelength, 210 nm. The acetic acid, propionic acid, and butyric acid contents in the supernatants of fermentations of Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 were determined using UPLC.

[0054] The results showed that the standard curve of acetic acid was Y=2504.9X-16.907, R 2 =0.9992; the standard curve of propionic acid is Y=2575.7-32.716, R 2 =0.9985; the standard curve of butyric acid is Y=2477.1X-0.1678, R 2 =0.9999. The acetic acid content in the fermentation supernatant of Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 was 1.44 and 2.72 g / L, respectively; the propionic acid content in the fermentation supernatant of Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 was 0.79 g / L and 16.1 g / L, respectively; and the butyric acid content in the fermentation supernatant of Lactobacillus crispatus QS204 was 0.08 g / L.

[0055] Fermented Lactobacillus mucilaginosus QS150 and Lactobacillus crispatus QS204 metabolize to produce acetic acid, propionic acid, and butyric acid, alleviating vaginal inflammation through antibacterial, anti-inflammatory, immunomodulatory, and mucosal barrier repair. Acetic acid disrupts the cell membrane integrity of Gardnerella vaginalis, achieving an antibacterial effect; propionic acid enhances immune regulation; and butyric acid inhibits biofilm formation of pathogenic bacteria while providing nutrients to vaginal mucosal cells, promoting mucosal repair and maintaining mucosal barrier integrity.

[0056] Example 6: Determination of hydrogen peroxide production.

[0057] Lactobacillus crispatus QS204 strains stored at -80°C were removed and inoculated into MRS liquid medium at a 5% inoculum volume ratio for activation for three generations. The culture was then centrifuged at 8000 rpm / min at 4°C for 5 minutes and filtered through a 0.22 μM filter membrane to collect the supernatant. 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 crispatus QS204 was 0.25 mmol / L. Hydrogen peroxide can destroy the biofilm structure of pathogens, killing them. Furthermore, it synergizes with lactic acid to lower vaginal pH, further inhibiting the growth and reproduction of pathogens.

[0058] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A probiotic for improving bacterial vaginosis, characterized by: The probiotics are fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain, Lactobacillus crispatus with the deposit number of CGMCC No.34371 ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri with the deposit number of CGMCC No.34372 ( Limosilactobacillus reuteri ) at least one of the QS020 strains.

2. A probiotic composition for improving bacterial vaginosis, characterized in that: The probiotic composition is made of fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain, Lactobacillus crispatus with the deposit number of CGMCC No.34371 ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri with the deposit number of CGMCC No.34372 ( Limosilactobacillus reuteri )QS020 strain composition.

3. A probiotic composition for improving bacterial vaginosis according to claim 2, characterized in that: The mixing volume ratio of the fermented Lactobacillus mucilaginosus QS150 bacterial suspension, the crispatus Lactobacillus QS204 bacterial suspension and the reuteri Lactobacillus mucilaginosus QS020 bacterial suspension is 1:1:1, and the OD of the bacterial suspension is 0. 600 nm=1-2.

4. A probiotic for improving bacterial vaginosis, characterized in that: The strains in the probiotics include the probiotics according to claim 1.

5. A probiotic for improving bacterial vaginosis according to claim 4, characterized in that: The dosage form of the probiotics includes freeze-dried powder, capsules, tablets or granules.

6. Use of the probiotics according to claim 1, the probiotic composition according to claim 2, or the probiotic agent according to any one of claims 3 to 5 in the preparation of a product for preventing, alleviating or treating bacterial vaginosis.

7. The use according to claim 6, characterized in that: The pathogenic bacteria of bacterial vaginosis are Escherichia coli, Staphylococcus aureus and Gardnerella vaginalis.

8. Use of the probiotics according to claim 1 in preparing an antioxidant product, characterized in that: The probiotics are fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) QS150 strain, Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri ( Limosilactobacillus reuteri ) One of the QS020 strains.

9. Use of the probiotics according to claim 1 in preparing a product for regulating vaginal microecology, characterized in that: The probiotics are Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain and Lactobacillus reuteri ( Limosilactobacillus reuteri ) One of the QS020 strains.

10. Use of the probiotics according to claim 1 in preparing an antibacterial product, characterized in that: The probiotics are Lactobacillus crispatus ( Lactobacillus crispatus ) QS204 strain.

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