Lactobacillus gasseri with efficacy of preventing and treating vaginitis in women and application thereof

Abstract

The present application relates to the functional microorganism screening and application technical field, specifically provide a lactobacillus gasseri with the efficacy of preventing and treating female vaginitis and application thereof.The lactobacillus gasseri is preserved in CCTCC NO: M2023609, can produce bacteriostatic substance H2O2, and can inhibit the growth of vagina gardner and candida albicans under the condition of mixed culture, and the inhibition rates are 97% and 100% respectively, and can be applied to the prevention and adjuvant therapy of female bacterial vaginitis and mycotic vaginitis.

Description

Technical Field

[0001] This invention relates to the field of probiotic screening and application technology, specifically to a strain of Lactobacillus gasseri that has the effect of preventing and treating vaginitis in women and its application. Background Technology

[0002] The vagina of healthy women harbors a diverse microbial community, primarily composed of lactobacilli, with *Lactobacillus curvatureii*, *Lactobacillus gasseri*, *Lactobacillus johnsonii*, and *Lactobacillus japonicus* being the most common species. These bacteria metabolize glycogen secreted by the vaginal epithelium, producing organic acids to maintain the normal acidic environment of the vagina (pH < 4.5). They interact and coordinate with the host and environment, maintaining the dynamic balance of the vaginal microecological system. If this microecological balance is disrupted, pathogens can proliferate, leading to various gynecological diseases. The most common gynecological diseases are bacterial vaginosis and yeast infections. *Gardnerella vaginalis* is the main pathogen causing bacterial vaginosis. This bacterium is part of the normal vaginal flora, detectable in the vaginas of 20%-40% of normal women, and generally does not pose a threat. However, excessive growth of *Gardnerella vaginalis* and anaerobic bacteria in the vagina can lead to an increase in vaginal pH, causing inflammation, resulting in itching, odor, and abnormal vaginal discharge. Bacterial vaginosis has become a significant global issue affecting women's daily lives, quality of sexual life, and public health.

[0003] In recent years, there has been increasing attention paid to probiotics, and their application areas are constantly expanding. Intravaginal or oral administration of probiotics, primarily lactobacilli, provides a new treatment direction for bacterial vaginosis. Lactobacilli are part of the normal vaginal flora. Some lactobacilli can produce organic acids, hydrogen peroxide, or bacteriocins to inhibit the growth of pathogenic bacteria, or competitively adhere to vaginal epithelial cells, thus rejecting the adhesion and growth of Gardnerella vaginalis. Clinical trials have confirmed that probiotics, alone or in combination with antibiotics, can improve the clinical symptoms of bacterial vaginosis, reduce the recurrence rate, and shorten the cure time. Currently, there are relatively few probiotic products on the market specifically targeting bacterial vaginosis, and the efficacy of different strains varies. Therefore, screening for dominant vaginal strains and developing probiotic products containing these strains for the prevention and treatment of vaginitis in women is a research hotspot in this field. Summary of the Invention

[0004] The purpose of this invention is to obtain a new strain of Lactobacillus gasseri through screening. Lactobacillus gasseri (This is used to prevent or treat vaginitis in women.)

[0005] This invention relates, in one aspect, to a strain of Lactobacillus gasseri ( Lactobacillus gasseri (), its accession number is CCTCCNO: M2023609.

[0006] The 16S rDNA sequence of the Lactobacillus gasseri strain VHProbi E09 is SEQ ID NO:1.

[0007] One aspect of this invention relates to an antibacterial agent comprising the Lactobacillus gasseri VHProbi E09 strain.

[0008] This invention also relates to the use of the Lactobacillus gasseri VHProbi E09 strain in the preparation of a medicine having the function of preventing or treating vaginitis in women.

[0009] This invention also relates to the use of the Lactobacillus gasseri VHProbi E09 strain in the preparation of pharmaceuticals with functions of preventing or treating intestinal diseases.

[0010] The present invention also relates to a composition for use in the vagina, comprising live bacteria, inactivated bacteria or fermentation products of the Lactobacillus gasseri VHProbi E09 strain.

[0011] The composition is a non-therapeutic vaginal health product, including vaginal health care products, vaginal cleansing products, vaginal care products, vaginal cosmetics, and vaginal hygiene products.

[0012] The composition is a therapeutic vaginal health product, including vaginal medicines.

[0013] The composition is a vaginal medical device.

[0014] The *Lactobacillus gasseri* VHProbi E09 provided by this invention is sensitive to common antibiotics and has good biocompatibility; it can tolerate high salinity, with a maximum tolerated salt concentration of 5%; it can grow at 45℃ and has a certain degree of heat resistance; it has strong antioxidant function, with a DPPH free radical scavenging rate of 19.87%±1.95% and a HRS free radical scavenging rate of 56.30%±1.83%; its supernatant has an anti-lipid peroxidation inhibition rate of 43.70%±0.40%, its cell has an anti-lipid peroxidation inhibition rate of 10.52%±0.64%, and its intracellular extract has an anti-lipid peroxidation inhibition rate of 7.66%±0.28%.

[0015] The Lactobacillus gasseri VHProbi E09 strain exhibits strong hydrophobicity, with an in vitro cell surface hydrophobicity of 20.64%. Simultaneously, at the cellular level, its adhesion index to human vaginal epithelial cells is 7.0, indicating that this strain has strong adhesion to human vaginal epithelial cells and can colonize the vagina.

[0016] The Lactobacillus gasseri VHProbi E09 can produce the antibacterial substance H2O2 and can inhibit the growth of Gardnerella vaginalis and Candida albicans under mixed culture conditions, with inhibition rates of 97% and 100%, respectively. It can be used for the prevention and adjunctive treatment of bacterial vaginosis and fungal vaginitis.

[0017] The Lactobacillus gasseri VHProbi E09 can also tolerate the digestion of gastric acid and intestinal fluid, and inhibit intestinal pathogens such as Escherichia coli and Salmonella enteritidis. Therefore, it can be taken orally into the intestines and has a certain preventive and therapeutic effect on intestinal diseases.

[0018] The Lactobacillus gasseri VHProbi E09 and its metabolites can be made into antibacterial agents or drugs, or added to feminine hygiene products for the prevention or treatment of vaginitis, with broad application prospects. Attached Figure Description

[0019] Figure 1 MALDI-TOF-MS protein fingerprint of Lactobacillus gasseri VHProbi E09;

[0020] Figure 2 RAPD fingerprint of Lactobacillus gasseri VHProbi E09;

[0021] Figure 3 The rep-PCR fingerprint of Lactobacillus gasseri VHProbi E09;

[0022] Figure 4 This is a colony morphology diagram of hydrogen peroxide-producing Lactobacillus gasseri VHProbi E09. Detailed Implementation

[0023] The *Lactobacillus gasseri* VHProbi E09 strain provided by this invention meets regulatory requirements and, through polyphasic taxonomic identification, is a newly discovered strain. The *Lactobacillus gasseri* VHProbi E09 strain provided by this invention exhibits strong tolerance to the gastrointestinal environment and can effectively inhibit pathogens such as *Gardnerella vaginalis* and *Candida albicans*, thus possessing significant application value in the prevention and treatment of vaginal infections caused by bacteria or fungi in women.

[0024] Lactobacillus gasseri VHProbi E09 was deposited on April 24, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2023609.

[0025] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0026] Example 1: Isolation and screening of Lactobacillus gasseri VHProbi E09

[0027] 1.1 Initial screening of lactic acid bacteria

[0028] In accordance with the 2019 edition of the "Ethical Guidelines for Human Genetic Resource Banks," and after signing a project commitment letter and informed consent form with the sample provider, vaginal secretions from healthy women who had not consumed probiotic preparations within the past six months were collected according to the standard operating procedures for biobanks. The secretions were then serially diluted, and 10... -1 10 -2 10 -3 100 μL of each of the three dilution gradients was spread onto MRS selective medium and anaerobic incubated at 37°C for 48 h. After single bacteria grew on the plates, the lactic acid bacteria with the shape of bacilli were selected by microscopic examination, and a total of 9 strains were named E01, E02, ..., E09.

[0029] 1.2 Rescreening against Gardnerella vaginalis strains

[0030] (1) Preparation of Gardnerella vaginalis culture

[0031] Gardnerella vaginalis BNCC337545 and BNCC354890 were activated by streaking on Columbia blood agar plates. Single colonies were then picked and transferred to modified BHI broth (BHI with 5% serum) and cultured aerobically at 37°C for 24 hours. The colonies were then transferred to fresh modified BHI broth at a ratio of 1% and cultured aerobically at 37°C for 24 hours to obtain fresh bacterial suspension. The fresh bacterial suspension was then mixed in equal volumes at a 1:1 ratio to obtain Gardnerella vaginalis bacterial suspension.

[0032] The Gardnerella vaginalis culture described in the following examples is the same as that in this example.

[0033] Using two different Gardnerella vaginalis strains as indicator strains can better demonstrate the antibacterial ability of the selected lactic acid bacteria against Gardnerella vaginalis.

[0034] (2) Oxford Cup Antibacterial Experiment

[0035] Spread 50 μL of the prepared Lactobacillus Gardnerella bacterial suspension onto a Columbia blood agar plate, place an Oxford cup on top, add 100 μL of the initial screening Lactobacillus fermentation suspension to each well, incubate at 37°C for 48 h, and observe for the presence or absence of inhibition zones.

[0036] The results showed that among the nine lactobacillus strains obtained in the initial screening of this invention, strain E09 had the most significant inhibitory effect on Lactobacillus gardeninae, with an inhibition zone diameter of 1.47±0.11 mm.

[0037] Example 2 Identification of Lactobacillus gasseri VHProbi E09

[0038] 2.1 Colony morphology identification

[0039] Single colonies of strain E09 are translucent, dull, and have an uneven surface, with a diameter of 2-3 mm. Under a microscope, strain E09 appears as rod-shaped cells.

[0040] 2.2 Identification of Physiological and Biochemical Characteristics

[0041] In this embodiment, the inoculum solution is prepared as follows: Under aseptic conditions, take an appropriate amount of fresh E09 strain culture, centrifuge at 5000 rpm / min for 5 min, wash twice with PBS buffer, resuspend with the same volume of PBS buffer, and dilute 50 times to obtain the inoculum solution.

[0042] 2.2.1 Temperature growth range experiment

[0043] Under aseptic conditions, the inoculum was inoculated into 10 mL of MRS liquid culture medium at a 10% inoculation rate. 10 mL of uninoculated MRS liquid culture medium was used as a control. The culture was placed in a 15℃ constant temperature shaking incubator for 7 days and a 45℃ constant temperature shaking incubator for 2 days, respectively, and the culture medium was observed to see if it became turbid.

[0044] The results showed that after 7 days of constant temperature incubation at 15℃, the culture medium remained clear; after 2 days of constant temperature incubation at 45℃, the culture medium became turbid. This indicates that strain E09 cannot grow at 15℃ but can grow normally at 45℃, demonstrating strong heat resistance.

[0045] 2.2.2 Glucose Acid and Gas Production Test

[0046] The culture medium formulation used in this embodiment is as follows:

[0047] Peptone 0.5g; yeast extract 0.3g; Tween 80 0.1mL; salt solution A 0.5ml; salt solution B 0.5ml; sodium acetate 0.5g; glucose 2.5g; 2% bromocresol green (w / v) 0.05mL; distilled water 100ml; pH 6.8~7.0.

[0048] Dispense the prepared culture medium into large test tubes containing inverted small test tubes, 3 mL / tube, and autoclave at 121℃ for 15 min.

[0049] Salt solution A consists of 10g KH2PO4 and 1.0g K2HPO4, dissolved in distilled water and brought to a final volume of 100mL.

[0050] Salt solution B consists of: 11.5g MgSO4·7H2O, 2.4g MnSO4·2H2O, and 0.68g FeSO4·7H2O, dissolved in distilled water and brought to a final volume of 100mL.

[0051] Under aseptic conditions, the inoculum was inoculated into the culture medium at a rate of 10%, with an uninoculated medium serving as a control. The top was then sealed with 2 mL of sterile liquid paraffin and incubated at 37°C. The medium was incubated continuously for 6 days, with daily observation for any changes in color.

[0052] The results showed that after 6 days of incubation at 37℃, the culture medium changed from green to yellow, and there was no gas in the small inverted tube, indicating that strain E09 fermented glucose to produce acid but not gas.

[0053] 2.2.3 Carbon source metabolism experiment

[0054] The phenol red-containing basal culture medium formula used in this embodiment is as follows:

[0055] Peptone 1.5g; yeast extract 0.6g; Tween 80 0.1g; salt solution 0.5mL; phenol red 18mg; distilled water 100mL; pH 7.4±0.2. Salt solution composition: MgSO4·7H2O 11.5g, MnSO4·4H2O 28g, distilled water 100mL.

[0056] Prepare 10 g / mL solutions of sugars, alcohols, and glycosides, and filter them using a 0.22 μm sterile filter. Under aseptic conditions, add 20 μL of the sterilized carbohydrate solution to each well of a 96-well plate, with four replicates for each carbohydrate. Then add 170 μL of sterilized basal medium containing phenol red, followed by 10 μL of inoculum. Leave wells uninoculated as a control. Add 50 μL of liquid paraffin to each well to prevent moisture evaporation during culture. Incubate at 37°C and observe the color change of the medium using phenol red as an indicator.

[0057] The results showed that strain E09 could utilize cellobiose, melibiose, raffinose, amygdalin, sucrose, galactose, lactose, maltose, mannose, and salicin; but could not utilize mannose, trehalose, D-arabinitol, gluconate, mesotriose, ribose, sorbitol, L-xylose, and rhamnose.

[0058] 2.3 Molecular biological identification

[0059] Single colonies of strain E09 were picked from the plate and placed in MRS broth medium. The culture was carried out at 37°C for 24 hours. Then, 500 μL of fermentation broth was taken and the genome of the strain was obtained by following the procedure of Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302). The genome was used for subsequent molecular biological identification.

[0060] 1. Identification of 16S rDNA gene sequence

[0061] 16S rDNA gene amplification

[0062] 1) Primer sequence:

[0063] 27F: AGAGTTTGATCCTGGCTCA;

[0064] 1492R: GGTTACCTTGTTACGACTT.

[0065] 2) Reaction system (50 μL)

[0066] Table 1 16S rDNA PCR amplification system

[0067] Element reaction volume 10×PCR buffer 5μL dNTPs 4μL 27F 2μL 1492R 2μL DNA 2.5μL rTaq 0.5μL ddH2O 34μL

[0068] 3) Electrophoresis verification: When the nucleic acid electrophoresis result of the PCR product is around 1500bp, it meets the requirements.

[0069] 4) Sequencing of PCR products

[0070] Sequencing results showed that the 16S rDNA sequence of strain E09 is SEQ ID NO:1. The specific sequence is as follows:

[0071]

[0072] The sequence SEQ ID NO:1 was BLAST-aligned with the NCBI database, and it matched that of *Lactobacillus gasseri* (…). Lactobacillus gasseri The E09 strain showed the highest similarity to *Lactobacillus gasseri*. Therefore, it was preliminarily identified as *Lactobacillus gasseri*. Lactobacillus gasseri ).

[0073] 2. Protein spectroscopy identification

[0074] Using a toothpick, transfer a single colony of strain E09 from the plate onto a mass spectrometry plate. Then, use the toothpick to spread the bacterial slurry evenly across the disc of the mass spectrometry plate. The slurry does not need to be too thick. Next, add 1 μL of the matrix solution from the mass spectrometry sample pretreatment kit to cover the sample, as per the instructions of the mass spectrometry kit. Allow the plate to air dry at room temperature. After drying, place the mass spectrometry plate on a Motitof proteometry instrument for identification.

[0075] Protein spectroscopy identification results as follows Figure 1 As shown, strain E09 was identified as Lactobacillus gasseri, with a matching rate of 96%.

[0076] 3. RAPD fingerprint identification

[0077] 1) Primer sequence:

[0078] M13(5'- GAGGGTGGCGGTTCT-3');

[0079] 2) RAPD reaction system

[0080] Table 2 RAPD Reaction System

[0081] Element reaction volume Taq DNA polymerase (5 U / μL) 0.2 μL <![CDATA[10×Buffer (containing Mg 2+ )]]> 2 μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 0.8 μL DNA template 2 μL sterile double-distilled water 14 μL Total volume 20 μL

[0082] 3) Electrophoresis

[0083] A 1.5% agarose gel plate was prepared, and a DL2000 DNA Marker was used as a result control. Electrophoresis was performed at a constant voltage of 100V for 80 minutes. Finally, the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of strain E09 is shown below. Figure 2 As shown.

[0084] A search revealed no relevant information in existing publicly available reports. Figure 2 The perfectly matching RAPD fingerprint pattern indicates that strain E09 is a novel Lactobacillus gasseri strain.

[0085] 4. Rep-PCR fingerprint identification

[0086] 1) rep-PCR primers

[0087] GTGGTGGTGGTGGTG.

[0088] 2) Rep-PCR reaction system

[0089] Table 3 Reaction system for rep-PCR

[0090] Reactive components volume r Taq DNA polymerase 0.2μL <![CDATA[10×Ex Taq DNA Buffer (containing Mg 2+ )]] 2μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 2μL DNA template 2μL sterile double-distilled water 12.8 μL

[0091] 3) Electrophoresis

[0092] The DL2000 DNA Marker was used as a result control. Amplification results were detected at 100 V for 80 min. The rep-PCR fingerprint of strain E09 is shown below. Figure 3 As shown.

[0093] A search revealed no relevant information in existing publicly available reports. Figure 3 The perfectly matching rep-PCR fingerprint pattern indicates that strain E09 is a novel Lactobacillus gasseri strain.

[0094] 2.4 Whole genome sequencing

[0095] The E09 strain was inoculated into 500 mL of MRS broth at a volume ratio of 1% and cultured at 37°C for 22 h. The cells were then collected by centrifugation at 8000 rpm for 10 min. The cells were sent to a sequencing center to obtain the complete genome sequence, which was uploaded to the NCBI gene database with accession number CP129028. The entire genome is 1864621 bp long, with a GC content of 35.23% and a total of 1752 genes.

[0096] In summary, based on the colony morphology, physiological and biochemical characteristics, and molecular biological identification results of strain E09, strain E09 was identified as a novel *Lactobacillus gasseri*, and named *Lactobacillus gasseri*. Lactobacillus gasseri VHProbi E09.

[0097] Example 3: Salinity tolerance test of Lactobacillus gasseri VHProbi E09

[0098] Under aseptic conditions, the inoculum was inoculated at a rate of 10% into 5 mL of MRS liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 5 mL of uninoculated MRS liquid culture medium was used as a control. The medium was incubated at 37°C with shaking, and the condition of the medium was observed to determine whether it became turbid.

[0099] The results showed that Lactobacillus gasseri VHProbi E09 grew at salt concentrations of 1%–5% but did not grow at salt concentrations of 6%–8%, and the maximum salt concentration that Lactobacillus gasseri VHProbi E09 could tolerate was 5%.

[0100] Example 4: Antibiotic resistance test of Lactobacillus gasseri VHProbi E09

[0101] Ampicillin, clindamycin, erythromycin, streptomycin, tetracycline, and gentamicin were all prepared into stock solutions of 2048 μg / mL and stored at -20℃ for later use. Before use, the stock solutions were serially diluted 2-fold with MRS liquid medium to prepare the working solutions, with 11 gradient concentrations ranging from 1 to 1024 μg / mL.

[0102] Take an appropriate amount of fresh bacterial culture (24-48h, cultured at 40℃), centrifuge at 5000rpm for 5 min, wash once with sterile physiological saline, resuspend in the same volume of physiological saline, and dilute 50 times to obtain the inoculum.

[0103] The minimum inhibitory concentration (MIC) of antibiotics against Lactobacillus gasseri VHProbi E09 was determined using the microbroth dilution method.

[0104] In a 96-well plate, MRS liquid medium without antibiotics was added to the first column as a negative control. 190 μL of MRS liquid medium containing different concentrations of antibiotics was added sequentially to columns 2 through 12. Then, 10 μL of the above inoculum was inoculated into each well, creating three parallel wells. One well was left as a blank. 50 μL of paraffin oil was added to cover the wells and prevent moisture evaporation. After incubating the 96-well plate at 37°C with shaking for 48 hours, the plate was removed, and the bacterial suspension became turbid. The MIC values ​​of the antibiotics against the bacterial strains were calculated. The results are shown in Table 4.

[0105] Table 4. Antibiotic MIC values ​​of Lactobacillus gasseri VHProbi E09

[0106] Note: MIC is measured in μg / mL.

[0107] As can be seen from the results in Table 4, the Lactobacillus gasseri VHProbi E09 provided by this invention is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline and clindamycin, and slightly sensitive to streptomycin and gentamicin, with good overall biosafety.

[0108] Example 5: Determination of the antioxidant function of Lactobacillus gasseri VHProbi E09

[0109] 1. Determination of DPPH and hydroxyl radical (HRS) scavenging capacity

[0110] 1) Preparation of bacterial suspension

[0111] A single colony of *Lactobacillus gasseri* VHProbi E09 in excellent growth condition was inoculated into 3 ml of MRS liquid medium and cultured at 37°C for 18-20 h. Using this culture as the inoculum, 2% of the colony was inoculated into 50 ml of MRS liquid medium and incubated statically for 18 h to obtain the bacterial suspension. 1 mL of the bacterial suspension was centrifuged to collect the bacterial cells. The cells were then washed twice with 1 mL of PBS buffer, and finally resuspended in 2 mL of PBS solution for later use.

[0112] 2) Determination of DPPH free radical scavenging ability

[0113] Take 1 mL of Lactobacillus gasseri VHProbi E09 bacterial suspension, add 1 mL of 0.4 mM freshly prepared DPPH free radical solution, mix well, and incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm. 样本 The test was performed in triplicate. The control group sample was prepared with an equal volume of PBS solution and DPPH-ethanol mixture, and the blank was zeroed with an equal volume of Lactobacillus gasseri VHProbiE09 suspension and ethanol mixture.

[0114] The clearance rate is calculated using the following formula:

[0115] Clearance rate = [1 - (A)] 样品 -A 空白 ) / A 对照 ]×100%.

[0116] The results showed that the Lactobacillus gasseri VHProbi E09 provided by this invention had a DPPH free radical scavenging rate of up to 19.87% ± 1.95%.

[0117] 3) Determination of hydroxyl radical scavenging capacity (HRS)

[0118] Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water, and 200 μL of Lactobacillus gasseri VHProbi E09 suspension, then add 100 μL of hydrogen peroxide solution (3 mM). After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm.

[0119] The hydroxyl radical scavenging rate is calculated using the following formula:

[0120] Clearance rate (%) = (A 样品 -A 控制 ) / (A 空白 -A 控制 ) × 100%.

[0121] Among them: A 控制 A was used as a substitute for deionized water in the sample.空白 Deionized water was used to replace the sample and H2O2.

[0122] The results showed that the Lactobacillus gasseri VHProbi E09 provided by this invention had a scavenging rate of up to 56.30% ± 1.83% against HRS free radicals.

[0123] 2. Determination of anti-lipid peroxidation capacity

[0124] 1) Preparation of bacterial strain culture and fermentation supernatant, bacterial cells, and intracellular extracts:

[0125] Lactobacillus gasseri VHProbi E09 was cultured in MRS liquid medium at 37°C for 24 h, passaged 3 times, centrifuged at 6000 r / min at 4°C for 10 min, and the supernatant was collected as the fermentation supernatant.

[0126] The collected bacterial cells were centrifuged in PBS buffer (pH 7.4) at 6000 r / min for 10 min and washed three times. The bacterial cells were then resuspended in PBS buffer and the concentration was adjusted to 1.0 × 10⁻⁶ cells / mL. 9 The bacterial suspension was obtained by measuring cells / mL.

[0127] The suspension of Lactobacillus gasseri VHProbi E09 was ultrasonically disrupted for 2 minutes to obtain an intracellular extract.

[0128] 2) Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0129] 3) Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture. Incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 r / min for 15 min, collect the supernatant, and measure the absorbance at OD532 nm to obtain A. The control group is A0, which is obtained by replacing the sample with 0.5 mL of distilled water.

[0130] Inhibition rate (%) = (A0 - A) / A0 × 100%.

[0131] The specific results are shown in Table 5.

[0132] Table 5. Anti-lipid peroxidation inhibition rate of Lactobacillus gasseri VHProbi E09

[0133] Example 6 Hydrophobic cell surface assay of Lactobacillus gasseri VHProbi E09

[0134] Purified Lactobacillus gasseri VHProbi E09 colonies were picked and inoculated into MRS liquid medium and cultured at 40°C with shaking for 24–48 h. Then, the colonies were transferred to MRS liquid medium at a volume ratio of 1% and cultured at 40°C with shaking for another 24–48 h. The colonies were centrifuged at 6000×g for 10 min, the bacterial cells were collected, washed twice with sterile physiological saline, and then resuspended in 1 mL of sterile 0.1 M KNO3 solution to obtain a bacterial suspension.

[0135] Add 50 μL of the above bacterial suspension to 2450 μL of 0.1 M KNO3 solution and record the OD600 as A0. Mix 1.5 mL of the bacterial suspension with 500 μL of xylene and let stand at room temperature for 10 min (at which point a two-phase system is formed). Vortex the two-phase system for 2 min and let it stand for 20 min to reform the aqueous and organic phases. Carefully pipette the aqueous phase (avoiding the organic phase) and measure the absorbance A1 at 600 nm.

[0136] Cell hydrophobicity is calculated using the following formula. The average value is taken from three experiments.

[0137] Hydrophobicity (%) = (A0-A1) / A1×100%.

[0138] The results showed that the surface hydrophobicity of Lactobacillus gasseri VHProbi E09 cells provided by this invention was 20.64%.

[0139] Example 7: Tolerance of Lactobacillus gastroenteritis VHProbi E09 to artificial gastric and intestinal fluids

[0140] 1. Preparation of bacterial culture:

[0141] The cryopreserved Lactobacillus gasseri VHProbi E09 strain was streaked onto MRS solid medium and cultured at 37°C for 24–48 h. After one subculture on MRS liquid medium, Lactobacillus gasseri VHProbi E09 was inoculated into fresh MRS liquid medium at a 5% inoculum and cultured at 40°C with shaking for 24–48 h to obtain fresh bacterial culture.

[0142] 2. Preparation of artificial gastric juice

[0143] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, and 2g of NaCl, add them to 1000mL of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0144] 3. Preparation of artificial intestinal fluid

[0145] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH₂PO₄, and 3.0g of ox bile salts, respectively. Add them to 77mL of 0.2 mol / L NaOH solution, and bring the volume to 1000mL. Adjust the pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 115℃ for 20min. Before use, add 1g of trypsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to simulate human body temperature.

[0146] 4. Test methods

[0147] Take 2 mL of fresh bacterial culture, centrifuge at 5000 rpm for 5 min to collect bacterial cells, wash the cells three times with physiological saline, and then resuspend them in 2 mL of physiological saline as the inoculum. Take 1 mL of the inoculum and add it to 9 mL of artificial gastric fluid that has been warmed for 1 h. Place the mixture in a 37℃ water bath shaker at 200 rpm for 2 h. Take 1 mL samples at 0 h and 2 h to detect the viable bacterial count. Then take 1 mL of artificial gastric fluid after 2 h of digestion and add it to 24 mL of artificial intestinal fluid. Place the mixture in a 37℃ water bath shaker (200 rpm) for 3 h. Take 1 mL samples to detect the viable bacterial count.

[0148] The viable bacteria count method was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination, and the viable bacteria count of the strain after passing through artificial gastric fluid and artificial intestinal fluid was calculated as Log (CFU / mL).

[0149] The specific results are shown in Table 6.

[0150] Table 6. Viable bacterial count after digestion in artificial gastrointestinal tract / Log (CFU / mL)

[0151] As shown in Table 6, the bacterial count of Lactobacillus gastrosae VHProbi E09 hardly decreased after digestion with artificial gastric juice; after digestion with artificial intestinal juice, the bacterial count decreased by only 0.63 logCFU / mL, indicating that this strain has strong tolerance to the gastrointestinal environment and can be used orally as an intestinal probiotic to exert its probiotic function.

[0152] Example 8: Determination of the hydrogen peroxide production capacity of Lactobacillus gasseri VHProbi E09

[0153] Tetramethylaniline was prepared with ethanol to a concentration of 0.1 g / 100 mL, and then filtered at a concentration of 0.22 μL; horseradish peroxidase was dissolved in water to a concentration of 0.01 g / 100 mL, and then filtered at a concentration of 0.22 μL.

[0154] Add 1% (w / v) glucose to MRS medium, then autoclave at 121°C. When the temperature drops to 50°C, add tetramethylaniline and horseradish peroxidase to achieve final concentrations of 25 mg / 100 mL for tetramethylaniline and 1 mg / 100 mL for horseradish peroxidase. Then, dilute a fresh Lactobacillus gasseri VHProbi E09 culture, take 50 μL, spread it onto the plate, and incubate at 37°C for 48 hours. Observe whether blue colonies are formed.

[0155] The results are as follows Figure 4 As shown, blue colonies are formed around Lactobacillus gasseri VHProbi E09 colonies, indicating the production of hydrogen peroxide. Hydrogen peroxide is toxic to microorganisms and thus effectively inhibits their reproduction.

[0156] Example 9: Antibacterial effect of Lactobacillus gasseri VHProbi E09

[0157] 1. Inhibitory effect of Lactobacillus gasseri VHProbi E09 on Gardnerella vaginalis

[0158] The experiment was divided into two groups: the experimental group and the control group.

[0159] (1) Experimental group: Take 100 μL of Lactobacillus gasseri VHProbi E09 bacterial culture (10 9 CFU / mL) and 100 μL Gardnerella vaginalis culture (10 9 CFU / mL) was inoculated into a test tube containing 5 mL of modified BHI liquid medium, shaken well, and the test tube was incubated in a 37℃ incubator for 24 h to obtain the experimental group culture medium;

[0160] (2) Control group: 100 μL of MRS liquid culture medium and 100 μL of Gardnerella vaginalis culture were inoculated into a test tube containing 5 mL of modified BHI medium, shaken evenly, and the test tube was incubated in a 37℃ incubator for 24 h to obtain the control group culture medium.

[0161] After diluting the culture medium of the experimental group and the control group with sterile physiological saline, 100 μL of each medium was spread onto Columbia blood agar solid medium and incubated upside down in a 37°C incubator for 48 h. Plate counts were performed to count the number of Gardnerella vaginalis colonies in each group's culture medium.

[0162] The results showed that the concentration of Gardnerella vaginalis in the control group culture medium was 2.15 × 10⁻⁶. 9 CFU / mL, the concentration of Gardnerella vaginalis in the experimental group culture medium was 1.90 × 10⁻⁶ CFU / mL. 7 CFU / mL. This indicates that, under co-culture conditions, Lactobacillus gasseri VHProbiE09 can significantly inhibit the growth of Gardnerella vaginalis, with an inhibition rate of 99.12% ± 0.07%.

[0163] 2. Inhibitory effect of Lactobacillus gasseri VHProbi E09 on Candida albicans

[0164] The experiment was divided into two groups: the experimental group and the control group.

[0165] (1) Experimental group: Take 50 μL of Lactobacillus gasseri VHProbi E09 bacterial culture (10 8 CFU / mL) and 50 μL of Candida albicans culture (10 7 CFU / mL) was inoculated into a test tube containing 5 mL of MRS liquid medium, shaken well, and the test tube was incubated at 37°C for 24 h to obtain the experimental group culture medium;

[0166] (2) Control group: 50 μL of MRS liquid culture medium and 50 μL of Candida albicans bacterial suspension were inoculated into a test tube containing 5 mL of MRS liquid culture medium, shaken evenly, and the test tube was incubated in a 37℃ incubator for 24 h to obtain the control group culture medium.

[0167] After serially diluting each group of culture medium with sterile physiological saline, 100 μL of each medium was spread onto Sabouraud dextrose agar and incubated upside down in a 37°C incubator for 48 h. Plate counts were performed to count the number of Candida albicans colonies in the culture medium of the experimental and control groups.

[0168] The results showed that the concentration of Candida albicans in the culture medium of the control group was 7.40 × 10⁻⁶. 6 The concentration of Candida albicans in the experimental group culture medium was 7667 CFU / mL. This indicates that Lactobacillus gasseri VHProbi E09 has a strong inhibitory effect on Candida albicans, with an inhibition rate of 99.9% ± 0.01%.

[0169] 3. Inhibitory effect of Lactobacillus gasseri VHProbi E09 on other pathogens

[0170] The Oxford cup inhibition test was used to detect the inhibitory effects of Lactobacillus gasseri VHProbi E09 on Salmonella enteritidis, Staphylococcus aureus, and Escherichia coli. The diameters of the inhibition zones are shown in Table 7.

[0171] Table 7. Antibacterial effect of Lactobacillus gasseri VHProbi E09

[0172] E. coli Salmonella enteritidis Staphylococcus aureus Diameter of the inhibition zone (cm) 1.2±0.05 1.44±0.02 1.0±0.0

[0173] Example 10 Adhesion of Lactobacillus gasseri VHProbi E09 to Human Vaginal Epithelial Cells

[0174] 1. Cell pre-culture

[0175] Human vaginal epithelial cells were resuscitated and cultured. When the cell density reached approximately 80%, they were digested with trypsin into a single-cell suspension and counted using a hemocytometer, with a cell count of 5 × 10⁻⁶. 5 Cells / mL. Then, seed the cell suspension into 24-well plates at a seeding rate of 500 μL / well, incubate overnight until fully adhered, discard the culture medium, wash twice with fresh culture medium, and set aside.

[0176] 2. Preparation of bacterial suspension

[0177] Fresh Lactobacillus gasseri VHProbi E09 culture was washed twice with pH 7.0 phosphate buffer, then resuspended in an equal volume of epithelial cell culture medium containing 10% fetal bovine serum. The absorbance was adjusted to achieve the desired OD. 600 = Between 0.8 and 1.0.

[0178] 3. Cell Culture

[0179] Add 500 μL of Lactobacillus gasseri VHProbi E09 suspension to a prepared 24-well plate containing human vaginal epithelial cells and co-culture in a carbon dioxide incubator for 2 h; wash three times with pH 7.0 phosphate buffer to remove unadhered bacteria.

[0180] 4. Counting

[0181] Add 300 μL of trypsin for 3 minutes, then add 700 μL of cell culture medium to stop the digestion. Repeat the pipetting process and collect the resulting solution into sterile EP tubes. Perform serial dilutions of the collected solution at 10-fold, 100-fold, 1000-fold, and 10000-fold, and plate the cells for cell counting. Simultaneously, count the cells in the control group. Calculate the adhesion ability of the tested strain using the following formula:

[0182] Adhesion capacity (CFU / cells) = Total number of bacteria adhering in each culture well / Total number of cells in each culture well.

[0183] The results showed that the adhesion index of Lactobacillus gasseri VHProbi E09 was 7.0. This indicates that Lactobacillus gasseri VHProbi E09 has good adhesion properties and can effectively colonize the vagina.

[0184] In summary, the *Lactobacillus gasseri* VHProbi E09 provided by this invention possesses strong antioxidant and hydrophobic properties, exhibits high tolerance to artificial gastric and intestinal fluids, and effectively inhibits intestinal pathogens such as *Escherichia coli* and *Salmonella enteritidis*. Therefore, this strain can exert its potential probiotic effects through oral administration. *Lactobacillus gasseri* VHProbi E09 can stably secrete substances such as lactic acid and hydrogen peroxide, maintaining the acidic environment of the vagina. Under mixed culture conditions, it can efficiently inhibit the growth of *Candida albicans* and *Gardnerella vaginalis*, and its excellent adhesion properties allow it to colonize the vagina effectively. Therefore, it is suitable for the prevention or treatment of fungal and bacterial vaginosis. *Lactobacillus gasseri* VHProbi E09 and its metabolites can be formulated into bacterial agents or drugs, or added to feminine hygiene products for the prevention or treatment of vaginitis, showing broad application prospects.

Claims

1. A strain of Lactobacillus gasseri ( Lactobacillus gasseri ), characterized in that, The preservation number of the Lactobacillus gasseri is CCTCC NO: M2023609.

2. An antibacterial agent, characterized in that, The antibacterial agent comprises Lactobacillus gasseri as described in claim 1.

3. The use of Lactobacillus gasseri as described in claim 1 in the preparation of a medicine having the effect of preventing or treating vaginitis in women.

4. A medicine for use in the vagina, characterized in that, The drug contains live Lactobacillus gasseri as described in claim 1.

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