CN117487685A - A strain of Lactobacillus crispatus and its application in preventing or treating female vaginitis - Google Patents

Abstract

The invention relates to the technical field of functional microorganism screening and application, in particular to a lactobacillus crispatus strain which is prepared from lactobacillus crispatusLactobacillus crispatus) And applications thereof. The lactobacillus crispatus is obtained by screening vaginal secretion of healthy women, belongs to normal vaginal strains, and can effectively inhibit various vaginal pathogenic bacteria such as gardnerella, candida albicans, staphylococcus aureus and the like. The Lactobacillus crispatus has been deposited at university of Wuhan, china at 2023, 4 and 24 daysThe preservation number of the China center for type culture collection is CCTCC NO: m2023608 can be widely applied to the prevention or auxiliary treatment of female bacterial vaginitis and mycotic vaginitis.

Description

A strain of Lactobacillus crispatus and its application in preventing or treating female vaginitis

Technical field

The present invention relates to the technical field of probiotic screening and application, and specifically relates to a strain of Lactobacillus crispatus, which has the effect of preventing or treating female vaginitis.

Background technique

Vaginal flora play an irreplaceable role in maintaining the stability and regulation of the vaginal environment. The vaginal flora is very complex. In addition to protozoa and fungi, it also includes many aerobic and anaerobic bacteria, such as Streptococcus lactis, Enterobacter, Proteus, Gavageococcus, Veillonella and other bacteria. These microorganisms can be divided into Both commensal and pathological microorganisms grow in this common environment, and there may be antagonistic effects between microorganisms. At pH 3.8 to 4.2, it is conducive to the reproduction of commensal probiotics, especially Lactobacillus, which is the main probiotic species in a healthy vagina. The density in vaginal fluid can reach 10 ⁵ to 10 ⁸ mL. When the vagina is infected by pathogens After microbial infection, even if Lactobacillus is dominant, it can still maintain a weakly acidic environment of pH 3.8 to 4.2 in the vagina, and some Lactobacilli can also produce H ₂ 0 ₂ , which has toxic effects on other microorganisms and inhibits their reproduction. No disease will occur. Once the reproductive capacity of pathogenic microorganisms exceeds that of probiotics, it will destroy the growth environment of lactic acid bacteria, thereby destroying the vaginal environment, eventually leading to the occurrence of gynecological diseases. After gynecological diseases occur, effective treatment is often not possible, and stubborn inflammation recurs, seriously affecting women's reproductive health. Among them, bacterial vaginosis (BV) is one of the diseases caused by imbalance of vaginal flora. It is mostly characterized by a decrease in Lactobacilli and an increase in anaerobic bacteria (including Gardnerella vaginalis and Atropobacter vaginalis). feature. The incidence rate of BV is between 5% and 50%. It can cause itching, odor and abnormal leucorrhea. It has become an important issue affecting women's daily life, sexual life quality and public health safety worldwide. Currently, bacterial vaginosis is mainly treated with antibiotics (including metronidazole and clindamycin). However, antibiotic therapy has a low cure rate and high recurrence rate, making it difficult to meet patients' expectations. Therefore, we urgently need to find a safe and effective treatment method to reduce the physical and psychological burden of bacterial vaginosis patients.

Probiotics were first discovered by Russian microbiologists in the early 20th century and have been developed for more than a century. For more than a century, the concept and efficacy of probiotics have been recognized by scientists and consumers. Probiotics can not only form dominant flora in the human intestine or skin, but can also be tightly combined with the intestinal mucosa or skin to form a natural biological barrier, keeping the entire human intestine or skin in a "healthy" state. Probiotics mainly regulate intestinal health and physical health by antagonizing bacterial adhesion or producing antimicrobial peptides. The types of lactobacilli in the vagina are relatively complex. There are about 25 species of vaginal lactobacilli reported so far, with Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jansoni being the dominant bacteria. In vitro studies have shown that Lactobacilli isolated from the vagina of healthy women can inhibit the adhesion of Gardnerella vaginalis and can even replace Gardnerella that has already adhered to the vaginal epithelium. Research by KremLeva and others found that H ₂ O ₂ -producing Lactobacilli can stimulate vaginal epithelial cells to secrete antibacterial substances and increase the activity of already synthesized protective factors, such as lysozyme, lactoferrin, etc. A severe lactobacilli deficiency is found in women with bacterial vaginosis, especially pregnant women. Therefore, using dominant vaginal strains screened from healthy women's vaginal secretions to make pharmaceuticals or sanitary products is a safe and effective way to prevent and treat bacterial vaginosis.

Contents of the invention

The object of the present invention is to provide a strain of Lactobacillus crispatus and its application. The Lactobacillus crispatus is screened from vaginal secretions of healthy women, belongs to normal vaginal strains, can effectively inhibit various vaginal pathogenic bacteria such as Gardnerella, Candida albicans, and Staphylococcus aureus, and can be widely used in female bacterial infections. and prevention or adjuvant treatment of fungal vaginitis.

The present invention first provides a strain of Lactobacillus crispatus VHProbi E04, which has been deposited in the Chinese Typical Culture Collection Center of Wuhan University, Wuhan, China on April 24, 2023, and its deposit number is CCTCC NO: M2023608.

The 16s rDNA sequence of the Lactobacillus crispatus VHProbi E04 strain is SEQ ID NO: 1.

The MALDI-TOF-MS protein fingerprint of the Lactobacillus crispatus VHProbi E04 strain is shown in Figure 3; its rep-PCR fingerprint is shown in Figure 4; and its RAPD fingerprint is shown in Figure 5.

The present invention also provides the application of the Lactobacillus crispatus VHProbi E04 strain in preparing bacteriostatic agents.

The present invention also provides the use of the Lactobacillus crispatus VHProbi E04 strain in the preparation of medicines for preventing or treating vaginitis caused by bacteria or fungi.

The bacteria are Gardnerella or Staphylococcus aureus.

The mold is Candida albicans.

The present invention also provides a composition for vaginal inflammation, comprising live bacteria, inactivated bacteria or fermentation products of the Lactobacillus crispatus VHProbi E04 strain.

The composition is a non-therapeutic vaginal health product, including vaginal health care products, vaginal cleaning products, vaginal care products, vaginal cosmetics and vaginal hygiene products.

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

The composition is a medical device for vaginal use.

Lactobacillus crispatus VHProbi E04 provided by the invention can produce antibacterial substance H ₂ O ₂ and can effectively inhibit the growth of vaginal pathogenic bacteria Gardnerella and Staphylococcus aureus. The diameter of the inhibition zone reaches 1.38±0.05cm and 2.15± respectively. 0.21cm; this strain can effectively inhibit the growth of Gardnerella and Candida albicans under mixed culture conditions, with an inhibition rate of 94% and 100% respectively, and this strain has certain adhesion to human vaginal epithelial cells. It can be colonized in the vagina, so it can be widely used in the prevention and auxiliary treatment of bacterial vaginosis and fungal vaginitis.

Lactobacillus crispatus VHProbi E04 provided by the invention is sensitive to common antibiotics and has good biological safety; it can tolerate higher salinity, with a maximum tolerated salt concentration of 3%; it can grow at 45°C and has certain heat resistance. This strain has strong antioxidant function, with the scavenging rates of DPPH and HRS free radicals being 24.28%±1.03% and 24.19%±4.63% respectively; the anti-lipid content of its supernatant, bacterial cells and intracellular extracts The peroxidation inhibition rates were 39.18%±0.17%, 10.71%±0.19% and 15.40%±0.19% respectively, with significant effects.

Lactobacillus crispatus VHProbi E04 provided by the present invention can be made into bacteriostatic agents, medicines or female hygiene products, which will help relieve the uncomfortable symptoms of bacterial vaginosis and fungal vaginal and maintain the health of female vaginas.

Description of the drawings

Figure 1 is a diagram of hydrogen peroxide production by Lactobacillus crispatus VHProbi E04;

Figure 2 is a colony morphology diagram of Lactobacillus crispatus VHProbi E04;

Figure 3 shows the MALDI-TOF-MS protein fingerprint of Lactobacillus crispatus VHProbi E04;

Figure 4 shows the rep-PCR fingerprint of Lactobacillus crispatus VHProbi E04;

Figure 5 shows the RAPD fingerprint of Lactobacillus crispatus VHProbi E04;

Figure 6 is a diagram showing the adhesion of Lactobacillus crispatus VHProbi E04 to human vaginal epithelial cells.

Detailed ways

The examples are only illustrative of the present invention and do not limit the scope of protection of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the method, steps or conditions of the present invention shall fall within the scope of the present invention.

Example 1 Isolation and screening of Lactobacillus crispatus VHProbi E04

1. Preliminary screening of lactic acid bacteria

According to the 2019 version of the "Ethical Code of Human Genetic Resources Bank", after signing a project commitment letter and informed consent form with the sample provider, and in accordance with the standard operating procedures of the biobank, the vaginal secretions of healthy women who have not consumed probiotic preparations within six months will be collected; the secretions will be collected Carry out serial dilutions of the material, take 100 μL of each of the three dilution gradient dilutions of 10 ^-1 , 10 ^-2 , and 10 ^-3 , apply them on the MRS selective medium, and incubate anaerobically at 37°C for 48 hours; wait until the cells grow on the plate. After colonization, a total of 18 strains of lactic acid bacteria with the shape of bacilli were selected through microscopic examination, which were named E01, E02,..., E18.

2. Rescreening against Gardnerella vaginalis and hydrogen peroxide-producing strains

(1) Prepare Columbia blood agar plate

Prepare Columbia blood agar plates in advance and let them dry.

(2) Preparation of Gardnerella vaginalis bacterial solution

Streak activated Gardnerella vaginalis BNCC337545 and BNCC354890 on Columbia blood agar plates, then pick single colonies into modified BHI broth medium (BHI plus 5% serum), culture aerobically at 37°C for 24 hours, and then Transfer it to the new modified BHI broth medium at a ratio of 1%, and incubate aerobically at 37°C for 24 hours to obtain fresh bacterial liquid. Mix the fresh bacterial liquid in an equal volume of 1:1 to obtain Gardnerella vaginalis. Bacteria liquid.

The Gardnerella vaginalis bacteria liquid described in the following examples is the same as this example.

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

(3) Oxford cup antibacterial experiment

Prepare the Columbia blood plate in advance, then take 50 μL of vaginal Lactobacillus gasseri liquid and spread it on the blood plate, put an Oxford cup on it, add 100 μL of the fermentation liquid of the first screening Lactobacillus to the wells, and incubate at 37°C for 48 hours, and observe whether there is any Zone of inhibition.

The results show that among the 18 Lactobacillus strains obtained through the preliminary screening of the present invention, E01, E04 and E11 strains have obvious inhibitory effects on Gardnerella, and the diameters of the inhibition zones produced by their fermentation broth are 1.23±0.03cm and 1.38 respectively. ±0.05cm and 1.28±0.02cm.

3. Determination of hydrogen peroxide production ability

Tetramethylaniline was prepared with ethanol to a concentration of 0.1g/100mL, and then filtered with a 0.22μm filter; horseradish peroxidase was dissolved in water, prepared to a concentration of 0.01g/100mL, and filtered with a 0.22μm filter.

Add 1% (w/v) glucose to the MRS culture medium, and then autoclave it at 121°C. When it drops to 50°C, add tetramethylaniline and horseradish peroxidase to obtain a final concentration of tetramethylaniline of 25 mg. /100mL, solid culture medium with a final concentration of horseradish peroxidase 1mg/100mL.

Then dilute the fresh bacterial liquids of E01, E04 and E11 respectively, take 50 μL of each and spread it on the above solid culture medium plate, culture it at 37°C for 48 hours, and observe whether blue colonies are produced.

The results are shown in Figure 1. Blue colonies were produced around the E04 colony, that is, hydrogen peroxide was produced, while the other two strains did not produce blue colonies. This shows that the E04 strain can produce hydrogen peroxide.

Example 2 Identification of Lactobacillus crispatus VHProbi E04

2.1 Identification of colony morphology

The colony morphology of the E04 strain is shown in Figure 2. The single colony is translucent, matte, with irregular and uneven edges. The colony diameter is 2-3mm, and the bacteria are rod-shaped under the microscope.

2.2 Identification of physiological and biochemical characteristics

The preparation of the inoculation solution in this example is as follows: under sterile conditions, take an appropriate amount of fresh bacterial solution, centrifuge it at 5000 rpm/min for 5 minutes, wash it twice with PBS buffer, resuspend it with the same volume of PBS buffer and dilute it 50 times. as an inoculum.

1. Temperature growth range experiment

Under sterile conditions, inoculate 10% of the inoculum into 10 mL MRS liquid medium. The 10 mL MRS liquid medium without bacteria was used as a control and placed in a constant temperature shaking incubator at 15°C for 7 days and 45 days respectively. Cultivate in a constant temperature shaking incubator for 2 days and observe whether the culture medium becomes turbid.

The results showed that after 7 days of constant temperature cultivation at 15°C, the culture medium was still clear; after 2 days of constant temperature cultivation at 45°C, the culture medium became turbid. This shows that the E04 strain cannot grow at 15°C but can grow normally at 45°C.

2. Carbon source metabolism experiment

API 50CHL reagent strips were used to determine the metabolic effects of E04 strain on 49 carbon sources.

Under sterile conditions, take an appropriate amount of fresh bacterial liquid of the E04 strain, centrifuge at 5000 rpm for 5 minutes, wash twice with pH 7.0 phosphate buffer, and resuspend with the same volume of buffer to prepare a bacterial suspension. Add the bacterial suspension to the culture medium of the API kit at an amount of 10%, then follow the instructions of the kit, add the culture medium into the hole of the test strip, seal it with paraffin, and then put the test strip into a Inside the box, add about 10 mL of sterile deionized water to the box substrate, cover it, and place it in a 37°C incubator for 24-48 hours. Observe the color change. If the bacteria grow, the color will change from blue to yellow, indicating no growth. The color remains unchanged. Record the test results and upload them to the identification software API web.

The results showed that the E04 strain could utilize galactose, glucose, fructose, mannose, N-acetylglucosamine, cork, esculin, salicin, cellobiose, maltose, sucrose, raffinose, and starch; it could not utilize Glycerin, mannitol, sorbitol, trehalose and D-arabitol; erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, ribose, D-arabinose Calendol, β-methyl-D-xyloside, sorbose, α-methyl-D-glucoside, dulbitol, amygdalin, rhamnose, inositol, α-methyl-mannoside, Lactose, melibiose, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, inulin, melezitose, gentiobiose, tulose, D-tatose Glutose, gluconate, L-arabitol, 2-ketogluconate and 5-ketogluconate.

API identification results: E04 strain is Lactobacillus crispatus, ID=99.6%, T value=0.87.

3. Glucose acid and gas production test

The culture medium formula used in this example is as follows:

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.

Dispense the prepared medium into large test tubes containing inverted small test tubes, 3 mL/tube, and autoclave at 121°C for 15 minutes.

Ingredients of salt solution A: KH ₂ PO ₄ 10g, K ₂ HPO ₄ 1.0g, dissolve in distilled water, and adjust the volume to 100mL.

Ingredients of salt solution B: MgSO ₄ ·7H ₂ O 11.5g, MnSO ₄ ·2H ₂ O 2.4g, FeSO ₄ ·7H ₂ O 0.68g. Dissolve in distilled water and adjust the volume to 100mL.

Under sterile conditions, the bacterial suspension was inoculated into the culture medium at an inoculation volume of 10%. The culture medium without bacteria was used as a control. Then the top was sealed with 2 mL of sterile liquid paraffin and placed at 37°C for culture. Cultivate continuously for 6 days, and observe whether the color of the culture medium changes every day.

The results showed that after 6 days of cultivation at 37°C, the culture medium changed from green to yellow, and there was no gas in the small inverted tube, indicating that the E04 strain fermented glucose to produce acid but no gas.

In summary, the physiological and biochemical identification results of the E04 strain are as follows: it cannot grow at 15°C but can grow normally at 45°C; it ferments glucose to produce acid but not gas; the carbon source metabolism is identified as Lactobacillus crispatus.

2.3 Molecular biology identification

Pick a single colony of the E04 strain on the plate and place it in the MRS broth medium, culture it at 37°C for 24 hours, then take 500 μL of the fermentation broth, and refer to the Tiangen Bacteria Genomic DNA Extraction Kit (Cat. No.: DP302) to obtain the genome of the strain. , the genome was used for subsequent molecular biology identification.

2.3.1 Protein spectrum identification

Use a toothpick to dip a single colony of the E04 strain on the plate onto the protein spectrometry plate, then use a toothpick to spread the bacterial mud evenly on the disc on the mass spectrometry plate. The bacterial mud does not need to be too thick, and then follow the protein spectrometry kit The instructions require adding 1 μL of the matrix solution in the mass spectrometry sample pretreatment box to cover the sample and allow it to dry naturally at room temperature. The dried mass spectrometer plate was placed on the Motitof protein spectrometer for identification.

The MALDI-TOF-MS protein fingerprint is shown in Figure 3. The E04 strain was identified as Lactobacillus crispatus.

2.3.1 Identification of 16s rDNA gene sequence

(1) 16s rDNA gene amplification

Primer sequence:

27F:AGAGTTTTGATCCTGGCTCA;

1492R: GGTTACCTTGTTACGACTT.

Table 1 16s rDNA PCR amplification system (50 μL)

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

(2) Electrophoresis verified that the size of the PCR amplification product was about 1500 bp, which met the requirements.

(3) PCR product sequencing

The sequencing results showed that the 16s rDNA sequence of strain E04 was SEQ ID NO: 1. The sequence was compared by BALST in the NCBI database, and it showed the highest similarity to Lactobacillus crispatus . Therefore, the E04 strain was initially determined to be Lactobacillus crispatus .

SEQ ID NO:1 is as follows:

GACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCAT GATGACTTGACGTCATCCCCACCTTCCTCCGGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTGGCCACCGCTTGTGC GCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCT CCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGC GGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTTCCAACTCGGCTATGCATCATCGCCTTGGTAAGCCTTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTAAAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTC CCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTACCGAAGTAAATCTGTTAGTTCCGCTCGCTCGACTGC.

2.3.3 RAPD fingerprint identification

1. RAPD fingerprint identification

1) Primer sequence: M13 (5’- GAGGGTGGCGGTTCT-3’).

2) RAPD reaction system

Table 2 RAPD reaction system

Element reaction volume TaqDNA polymerase (5U/μL) 0.2 μL 10×Buffer (including 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 capacity 20 μL

3) Electrophoresis

Prepare a 1.5% agarose gel plate, use DL2000 DNA Marker as a result control, conduct electrophoresis at a constant voltage of 100V for 80 minutes, and finally use a gel imaging system to detect the electrophoresis pattern. The RAPD fingerprint of the E04 strain is shown in Figure 4.

After comparison, no RAPD fingerprint consistent with Figure 4 was found in existing public reports, indicating that strain E04 is a new strain of Lactobacillus crispatus.

2.3.4 rep-PCR fingerprint identification

1) rep-PCR primers

GTGGTGGTGGGTGGTG.

2) Rep-PCR reaction system

Table 3 Reaction system of rep-PCR

Reaction components volume r TaqDNA polymerase 0.2μL 10×Ex Taq DNA Buffer (contains Mg ²⁺ ) 2μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 2μL DNA template 2μL Sterile double distilled water 12.8 μL

3) Electrophoresis

DL2000 DNA Marker was used as a result control. The voltage was 100 V and the electrophoresis time was 80 minutes to detect the amplification results. The rep-PCR fingerprint of E04 strain is shown in Figure 5.

After comparison, no rep-PCR fingerprint consistent with Figure 5 was found in the existing public reports, thus indicating that the E04 strain screened in the present invention is a new Lactobacillus crispatus strain.

2.3.5 Whole genome sequencing

The bacterial liquid of E04 strain was inoculated into 500mL MRS broth medium at a volume ratio of 1%, cultured at 37°C for 22h, and then centrifuged at 8000rpm for 10min to collect the bacteria. The bacteria were sent to the sequencing center to obtain the full gene sequence of the bacteria. The gene sequence was uploaded to the NCBI gene database with the accession number CP128800. The entire genome is 2133556bp long, with a GC content of 36.59% and a total number of genes of 2055.

After comparing the colony morphology and physiological and biochemical characteristics of the E04 strain, and integrating the identification results of molecular biology, it was determined that the E04 strain was a new strain of Lactobacillus crispatus and named Lactobacillus crispatus VHProbi E04.

Example 3 Salinity tolerance test of Lactobacillus crispatus VHProbi E04

Under sterile conditions, inoculate 10% of the inoculum into 5 mL MRS liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%. 5mL of MRS liquid medium without bacteria was used as a control, and cultured at 37°C with constant temperature shaking to observe whether the medium became turbid.

The results showed that Lactobacillus crispatus VHProbi E04 grew at a salt concentration of 1% to 3%, but did not grow at a salt concentration of 4% to 8%. The optimal salt concentration of Lactobacillus crispatus VHProbi E04 was 3%.

Example 4 Antibiotic tolerance test of Lactobacillus crispatus VHProbi E04

1. Preparation of antibiotics: Ampicillin, gentamicin, clindamycin, erythromycin, streptomycin, and tetracycline are all prepared into a storage solution of 2048 μg/mL, and stored at -20°C for later use. When used, the storage solution is serially diluted 2-fold with MRS liquid culture medium to obtain a usage solution, with a total of 11 gradients at a gradient dilution concentration of 1 to 1024 μg/mL.

2. Preparation of inoculation solution: Preparation of inoculum solution: Take an appropriate amount of fresh bacterial solution (cultured at 40°C for 24-48 hours), centrifuge at 5000 rpm for 5 minutes, wash once with sterile saline, resuspend with the same volume of saline and dilute 50 times , as inoculum.

3. Determination of the minimum inhibitory concentration MIC of antibiotics against Lactobacillus crispatus VHProbi E04 by broth microdilution method.

(1) Add MRS liquid culture medium without antibiotics to the first column of the 96-well plate as a negative control. Add 190 μL of MRS liquid culture medium containing different concentrations of antibiotics to the second to 12th columns, and then inoculate 10 μL of the above inoculation solution. , make 3 parallel wells, and use 1 well without bacterial solution as a blank.

(2) Add 50 μL paraffin oil to cover to prevent water evaporation.

(3) Take out the 96-well plate after shaking culture at 40°C for 48 hours, measure the OD ₆₀₀ value, and use the results of 48 hours to calculate the MIC value of the antibiotics against the strains. The results are shown in Table 5.

Table 4 Antibiotic MIC values of Lactobacillus crispatus VHProbi E04

Note: MIC unit is μg/mL.

It can be seen from the results in Table 4 that Lactobacillus crispatus VHProbi E04 provided by the present invention is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline and clindamycin, and is slightly sensitive to streptomycin and gentamicin. Overall Biosafety is good.

Example 5 Hydrophobic cell surface test of Lactobacillus crispatus VHProbi E04

1. Preparation of bacterial solution to be tested: Pick the purified Lactobacillus crispatus VHProbi E04 colony and inoculate it into the newly prepared MRS liquid medium, and culture it with shaking at 40°C for 24 to 48 hours. Then add an inoculum volume of 1% (V/V) to MRS liquid culture medium and continue to culture with shaking at 37°C for 24 to 48 hours. Then centrifuge at 6000×g for 10 minutes. Collect the cells and rinse them twice with sterile physiological saline. Sterilize 0.1M KNO ₃ 1mL solution and resuspend the bacterial cells as the bacterial liquid to be tested.

2. Surface hydrophobicity measurement: Take 50 μL of the above bacterial suspension, add 2450 μL of 0.1M KNO ₃ , and record the OD ₆₀₀ as A ₀ . Mix 1.5 mL of the bacterial suspension with 500 μL of xylene and let it stand at room temperature for 10 min (at this time form a two-phase system). Vortex the two-phase system for 2 minutes and then let it stand for 20 minutes to re-form the aqueous phase and the organic phase. Carefully absorb the aqueous phase (do not absorb the organic phase) and measure the absorbance A ₁ at 600 nm. Cell hydrophobicity was calculated according to the formula hydrophobicity = (A ₀ -A ₁ )/A ₁ ×100%, and the average value was taken from three experiments.

The results show that the cell surface hydrophobicity of Lactobacillus crispatus VHProbi E04 provided by the invention is 50.61%.

Example 6 Measurement of antioxidant function of Lactobacillus crispatus VHProbi E04

1. Determination of the ability of strains to scavenge DPPH and hydroxyl radicals (HRS)

1) Preparation of PBS bacterial suspension

Inoculate a single colony with excellent growth status into 3 mL of MRS liquid medium, and culture it at 37°C for 18-20 hours. Use this culture medium as the inoculum, and inoculate it into 50 mL of MRS liquid medium at an inoculum volume of 2%. Leave to culture for 18 hours to obtain the culture medium of the strain. Aspirate 1 mL of bacterial liquid to collect the cells, wash the cells twice with 1 mL of PBS buffer, and then add 2 mL of PBS solution to resuspend the cells for later use.

2) Determination of the ability of strains to scavenge DPPH free radicals

Take 1 mL of the PBS bacterial suspension of the strain to be tested, add 1 mL of 0.4 mM freshly prepared DPPH free radical solution, mix evenly, and then place it at room temperature for 30 minutes in the dark, and then measure the absorbance of the sample at a wavelength of 517 nm. _Sample A, measure 3 parallels. The samples in the control group were zeroed using equal volumes of PBS solution and DPPH·ethanol mixture, and equal volumes of PBS bacterial suspension and ethanol mixture.

The clearance rate is calculated according to the following formula: clearance rate = [1-(A _sample -A _blank )/A _control ]×100%.

The results show that the DPPH free radical scavenging rate of Lactobacillus crispatus VHProbi E04 provided by the present invention is as high as 24.28%±1.03%.

3) Determination of the ability of strains to scavenge hydroxyl radical HRS

Mix 100μL 5mM sodium salicylate-ethanol solution, 100μL 5mM ferrous sulfate, 500μL deionized water and 200μL Lactobacillus crispatus VHProbi E04 bacterial suspension, add 100μL hydrogen peroxide solution (3mM), and keep in a water bath at 37°C for 15 minutes. Then measure the sample absorbance at a wavelength of 510 nm.

The hydroxyl radical scavenging rate is calculated according to the following formula: scavenging rate = (A _sample - A _control ) / (A _blank - A _control ) × 100%.

Among them: A _control is deionized water substitute sample, A _blank is deionized water substitute sample and H ₂ O ₂ .

The results show that the Lactobacillus crispatus VHProbi E04 provided by the present invention has a scavenging rate of HRS free radicals as high as 24.19% ± 4.63%.

2. Determination of the anti-lipid peroxidation ability of strains

1) Strain culture and preparation of fermentation supernatant, bacterial cells, and intracellular extracts:

The strain was cultured in MRS liquid medium at 37°C for 24 hours. After 3 generations, it was centrifuged at 6000 r/min and 4°C for 10 min. The supernatant was collected as the fermentation supernatant. The collected cells were centrifuged with PBS buffer (pH 7.4) at 6000 r/min for 10 min and washed three times. Resuspend the bacteria in PBS buffer, adjust the bacterial concentration to 1.0×10 ⁹ cells/mL, and obtain a bacterial suspension. The bacterial suspension was ultrasonically disrupted with an ultrasonic disruptor for 20 minutes to obtain the intracellular extract.

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

3) Add 1 mL of linoleic acid emulsion and 1 mL FeSO ₄ (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample, and wait in a 37°C water bath for 1.5 h. Add 0.2 mL of TCA (4%) to the mixture. ), 2 mL TBA (0.8%), 100°C water bath for 30 minutes, cool quickly, centrifuge at 4000r/min for 15 minutes, collect the supernatant and measure the absorbance at OD ₅₃₂ nm, which is A; in the control group, 0.5 mL distilled water is used instead of the sample, which is A ₀ .

Inhibition rate = (A ₀ -A)/A ₀ ×100%.

The results show that: the anti-lipid peroxidation inhibition rate of the supernatant of Lactobacillus crispatus VHProbi E04 provided by the invention is 39.18%±0.17%; the anti-lipid peroxidation inhibition rate of the bacteria is 10.71%±0.19%; the intracellular extract The lipid peroxidation inhibition rate is 15.40%±0.19%.

Example 8 Inhibitory effect of Lactobacillus crispatus VHProbi E04 on Gardnerella

Take 100 μL of Lactobacillus crispatus liquid (10 ⁹ CFU/mL) and 100 μL of Gardnerella bacteria liquid (10 ⁹ CFU/mL) and inoculate it into a test tube containing 5 mL of modified BHI liquid culture medium. Shake evenly and place the test tube at 37 Incubate in a ℃ incubator for 24 hours to obtain the culture medium of the experimental group.

Take 100 μL of MRS liquid culture medium and 100 μL of Gardnerella bacteria liquid and inoculate it into a test tube containing 5 mL of modified BHI culture medium. Shake evenly and incubate the test tube in a 37°C incubator for 24 hours to obtain the control culture medium.

Gradient dilute the culture solution of each group with sterilized PBS buffer, use a pipette to absorb 100 μL and apply it to Columbia blood agar solid medium. Incubate it upside down in a 37°C incubator for 48 hours and then perform plate counting to obtain the contents of each group's culture solution. Gardnerella colony count.

The results showed that the concentration of Gardnerella in the culture medium of the control group was 2.15×10 ⁹ CFU/mL, while the concentration of Gardnerella in the culture medium of the Lactobacillus crispatus group was 3.45×10 ^{7 CFU/mL, indicating that the concentration of Gardnerella in the culture medium of the Lactobacillus crispatus group was 2.15×10 9} CFU/mL. Lactobacillus VHProbi E04 can significantly inhibit the growth of Gardnerella under co-culture conditions, with an inhibition rate of 98%.

Example 9 Inhibitory effect of Lactobacillus crispatus VHProbi E04 on Candida albicans

Take 50 μL of Lactobacillus crispatus liquid (10 ⁸ CFU/mL) and 50 μL of Candida albicans liquid (10 ⁷ CFU/mL) and inoculate it into a test tube containing 5 mL of MRS liquid culture medium. Shake evenly to serve as the culture medium of the experimental group;

Take 50 μL MRS liquid medium and 50 μL Candida albicans bacterial liquid and inoculate it into a test tube containing 5 mL MRS liquid medium as the control culture medium.

The culture medium of the control group and the experimental group was cultured in a 37°C incubator for 24 hours; after gradient dilution with sterilized PBS buffer, use a pipette to absorb 100 μL and apply it to Sabouraud dextrose solid medium, and incubate it upside down in a 37°C incubator for 48 hours. Then perform plate counting to obtain the number of Candida albicans colonies in each group of culture fluids.

The results showed that the concentration of Candida albicans in the culture medium of the control group was 5.05×10 ⁶ CFU/mL, while the concentration of Candida albicans in the culture medium of the Lactobacillus crispatus group was only 430 CFU/mL. It shows that Lactobacillus crispatus VHProbi E04 has a strong inhibitory effect on Candida albicans, with an inhibition rate of almost 100%.

Example 10 Antibacterial effect of Lactobacillus crispatus VHProbi E04 on Staphylococcus aureus

Take out the glycerin tube of Staphylococcus aureus, use an inoculation loop to pick a little bacterial liquid and streak it on the nutrient agar plate. After the colonies grow, pick a single colony and culture it in nutrient broth for 24 hours. The bacterial liquid is set aside.

Referring to the Oxford cup antibacterial test method described in Example 1, the inhibitory effect of Lactobacillus crispatus VHProbi E04 fermentation broth on Staphylococcus aureus that causes bacterial vaginosis was detected.

The results showed that the diameter of the inhibition zone of this strain against Staphylococcus aureus reached 2.15±0.21cm. This shows that Lactobacillus crispatus VHProbi E04 can effectively inhibit Staphylococcus aureus.

Example 11 Adhesion of Lactobacillus crispatus VHProbi E04 to human vaginal epithelial cells

1. Cell pre-culture

Human vaginal epithelial cells were resuscitated in liquid nitrogen and cultured to the required amount. When the cell density grew to about 80%, they were digested with trypsin into a single cell suspension and counted with a hemocytometer. The number of cells was 5×10 ⁵ cells/mL. . Then take 500 μL of cell suspension and inoculate it into a 24-well plate with cell slides. The inoculation density is 2.5×10 ⁵ cells/well. The cells are cultured overnight until they are completely adherent. Discard the culture medium and rinse twice with fresh culture medium for later use. .

2. Preparation of bacterial suspension

Wash the fresh bacterial solution of Lactobacillus crispatus VHProbi E04 twice with pH 7.0 phosphate buffer, then resuspend it in the same volume of RPMI-1640 culture medium containing 10% calf serum, adjust the absorbance so that OD ₆₀₀ = 0.8-1.0 between.

3. Cell culture

Add 500 μL of bacterial suspension to the prepared 24-well plate containing human vaginal epithelial cells, and culture symbiotically in a carbon dioxide incubator for 2 hours; wash 3 times with pH 7.0 phosphate buffer to remove non-adherent bacteria.

4. Counting

The cell slides were fixed with methanol for 15 minutes, then stained with Giemsa stain for 5 minutes, washed and rinsed with pH 7.0 phosphate buffer, and then the cells were removed and placed on a glass slide. Observe under a microscope.

The results are shown in Figure 6. Lactobacillus crispatus VHProbi E04 has certain adhesion to human vaginal epithelial cells and therefore can effectively colonize the vagina.

Lactobacillus crispatus VHProbi E04 was screened from the vaginal secretions of healthy women. It is one of the normal vaginal strains and can adhere to the surface of vaginal epithelial cells to colonize. Moreover, this strain has a broad antibacterial spectrum and can not only inhibit Gardnerella and Candida albicans can also effectively inhibit Staphylococcus aureus, so it can be made into antibiotics, medicines or female hygiene products, which will help prevent or treat bacterial vaginosis and fungal vaginitis, and maintain the health of women's vagina.

Claims (10)

1. A strain of Lactobacillus crispatus ( Lactobacillus crispatus ), characterized in that the Lactobacillus crispatus has been deposited in the Chinese Typical Culture Collection Center of Wuhan University, Wuhan, China on April 24, 2023, and its deposit number is CCTCCNO: M2023608 . 2. Lactobacillus crispatus according to claim 1, characterized in that the 16s rDNA sequence of Lactobacillus crispatus is SEQ ID NO: 1. 3. The use of Lactobacillus crispatus according to claim 1 in the preparation of bacteriostatic agents. 4. Use of Lactobacillus crispatus according to claim 1 in the preparation of medicines for preventing or treating vaginitis caused by bacteria or mold. 5. The application according to claim 4, wherein the bacterium is Gardnerella or Staphylococcus aureus. 6. The application according to claim 4, characterized in that the mold is Candida albicans. 7. A composition for vaginal use, characterized by comprising the live Lactobacillus crispatus bacteria, inactivated bacteria or fermentation products thereof according to claim 1. 8. The composition according to claim 7, wherein the composition is a non-therapeutic vaginal health product, including vaginal health care products, vaginal cleaning products, vaginal care products, vaginal cosmetics and vaginal hygiene products. . 9. The composition of claim 7, wherein the composition is a therapeutic vaginal health product, including vaginal medicines. 10. The composition of claim 7, which is a medical device for vaginal use.