A Lactobacillus crispatus and its application

Abstract

The invention discloses a strain of Lactobacillus crispatus and an application thereof. The Lactobacillus crispatus is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, and the deposit number is CGMCC No. 24781. The Lactobacillus crispatus LSA-573 of the scheme of the invention is used to prepare a live lactobacillus preparation, which has the advantages of safety, reliability, good effect, etc., can be used to treat and / or prevent female lower urinary tract infection and vaginitis-related diseases, regulate the microecological balance of female reproductive tract, prevent female reproductive tract diseases, and reduce the probability of female reproductive tract diseases, which is of great significance to female reproductive health, population eugenics, and family and social harmony and stability.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Lactobacillus crispatus and its application. Background Art

[0002] Urinary and reproductive tract infections are common diseases that seriously affect the physical and mental health of women, including vaginal infections, lower urinary tract infections, etc. The lower female genital tract is an open cavity, inhabited by a large number of different types of microorganisms. These microorganisms grow and reproduce in the vagina and restrict each other, constituting the vaginal microecosystem, which is closely related to female reproductive health and is one of the important microecological regions in the human body. Affected by hormone levels, behavior habits, ethnic genes, and the environment, the composition of reproductive tract microorganisms in women changes continuously throughout their lives. Before puberty, Escherichia coli and anaerobic bacteria are dominant, the vaginal flora diversity is relatively high, and the pH is neutral to alkaline. During puberty, Lactobacillus begins to colonize, and the pH becomes acidic (3.8 - 4.5). The vagina of healthy women of childbearing age is a microenvironment with Lactobacillus as the dominant flora. The most common ones include Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus rhamnosus, etc. The normal vaginal flora during pregnancy is very similar to that of women of childbearing age. During the puerperium, the vaginal flora is affected by hormones. The decrease in estrogen makes the vaginal flora of puerperal women not very similar to that of women of childbearing age, but more like that of postmenopausal women. After menopause, with the decrease in estrogen and the increase in vaginal pH, anaerobic bacteria and mycoplasma are dominant.

[0003] Statistics released by the World Health Organization (WHO) show that 40% of women suffer from reproductive tract infections of varying degrees. Based on this estimate, at least 200 million women suffer from reproductive tract infections and related diseases every year. From a clinical perspective, most female reproductive tract infections are related to microecological imbalance, with disorders in the reproductive tract flora, a decrease in lactobacilli, and an increase in other pathogenic bacteria, leading to various gynecological inflammations. Bacterial vaginitis (BV) is the most common vaginal disorder in women of childbearing age. It is a vaginal infection mainly caused by a decrease or disappearance of normal hydrogen peroxide-producing lactobacilli in the vagina and an increase in facultative anaerobes and anaerobes (such as Gardnerella vaginalis). The detection rate in obstetrics and gynecology outpatient patients is as high as 18.8%, and 50% of BV patients have no clinical symptoms. Vulvovaginal candidiasis (VVC), also known as vulvovaginal candidiasis, is caused by yeasts mainly Candida albicans. Approximately 70%-75% of women experience VVC at least once in their lives, 50% of women will experience multiple attacks of the disease in their lives, and 5%-10% of women are likely to develop recurrent VVC. Urinary tract infection (UTI) is an inflammatory reaction caused by bacteria invading uroepithelial cells. Pathogens include uropathogenic Escherichia coli, Klebsiella, Staphylococcus, Enterococcus species, and other pathogens. Research shows that approximately 50% of women will suffer from UTI once in their lives, and 25% of patients will relapse within 6 months. Female vaginitis is not only stubborn and harmful in itself, seriously affecting the quality of life of women, but also causes various complications. UTI can affect the probability of kidney infection, and UTI during pregnancy can also cause symptoms such as acute cystitis and acute pyelonephritis. BV can cause pelvic inflammatory disease, infection after gynecological surgery, and infertility. BV during pregnancy can cause adverse pregnancy outcomes such as miscarriage, premature birth, premature rupture of membranes, and neonatal infection. If VVC is not treated in time, it may lead to some serious complications in the upper reproductive tract, such as endometritis, salpingitis, and pelvic inflammatory disease, ultimately resulting in tubal scarring, infertility, or ectopic pregnancy.

[0004] For female vaginitis, the existing treatment methods mainly rely on antibiotic treatment. Although antibiotics can kill pathogens, they cannot restore the vaginal microecological balance, mainly because they cannot restore the quality and quantity of lactobacilli, and cannot effectively prevent recurrent / refractory vaginitis. Moreover, the extensive use of antibiotics has led to an increase in the drug resistance of pathogenic bacteria, further exacerbating the prevalence and stubbornness of female vaginitis. Lactobacilli account for 70%-95% of the female vaginal flora and play an important protective role in the vagina. At present, the relevant live lactobacilli preparations and strains for the treatment and improvement of vaginitis / lower urinary tract infections in China are still in their infancy and require a large amount of research data and lactobacilli strains as technical support and candidate research objects. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a strain, namely Lactobacillus crispatus LSA-573, which can be used for the development of live lactobacillus preparations, and has the advantages of safety, reliability, good effect, etc. It can be used for the treatment and / or prevention of female lower urinary tract infections and vaginitis-related diseases, and can also help restore the vaginal microecological environment, which is beneficial to women's health.

[0006] The present invention also provides a product containing the above-mentioned strain.

[0007] The present invention also provides the use of the above-mentioned strain or the product containing the above-mentioned strain.

[0008] In one aspect of the present invention, a strain is provided, namely Lactobacillus crispatus LSA-573, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 24781 and the deposit date of April 26, 2022.

[0009] In some embodiments of the present invention, the Lactobacillus crispatus LSA-573 is isolated from the posterior fornix of the vagina of healthy women.

[0010] In some embodiments of the present invention, the strain has the 16S rDNA sequence shown in SEQ ID NO: 1.

[0011] In the second aspect of the present invention, a product is provided, including at least one of (1) to (6):

[0012] (1) The above-mentioned strain;

[0013] (2) A bacterial agent containing the above-mentioned strain;

[0014] (3) A live bacterial liquid containing the above-mentioned strain;

[0015] (4) A dead bacterial liquid containing the above-mentioned strain;

[0016] (5) A metabolite containing the above-mentioned strain;

[0017] (6) An extract containing the above-mentioned strain.

[0018] In some embodiments of the present invention, the product is a drug, a medical device, a food or a hygiene product.

[0019] In some embodiments of the present invention, the drug further includes a pharmaceutical carrier and / or pharmaceutical excipients.

[0020] In some embodiments of the present invention, the pharmaceutical carrier comprises at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetening agent, and a flavoring agent.

[0021] In some embodiments of the present invention, the excipient comprises water.

[0022] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0023] In some embodiments of the present invention, the binder comprises at least one of a cellulose derivative, an alginate, gelatin, and polyvinylpyrrolidone.

[0024] In some embodiments of the present invention, the wetting agent comprises glycerol.

[0025] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.

[0026] In some embodiments of the present invention, the absorption enhancer comprises a quaternary ammonium compound.

[0027] In some embodiments of the present invention, the surfactant comprises cetyl alcohol.

[0028] In some embodiments of the present invention, the adsorption carrier comprises at least one of kaolin and saponite.

[0029] In some embodiments of the present invention, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0030] In some embodiments of the present invention, the medical device is a gel.

[0031] In some embodiments of the present invention, the food is a dairy product, a soy product, or a fruit and vegetable product produced using a starter containing the above-mentioned strain; or the food is a beverage or snack containing the above-mentioned strain.

[0032] In some embodiments of the present invention, the hygiene product is at least one of a tampon, a sanitary napkin, a menstrual pad, a diaper, a soap, and a condom.

[0033] In some embodiments of the present invention, the sanitary napkin comprises a tampon, a pull-up panty liner, a menstrual cup, or a panty liner.

[0034] In some embodiments of the present invention, the form of the product is one of a powder, an ointment, a gel, a drop, a suppository, a lozenge, a granule, a capsule, a spray, a tablet, a pill, and a solution; preferably, the capsule is a soft capsule.

[0035] In the third aspect of the present invention, there is provided an application of the above-mentioned Lactobacillus crispatus strain or the above-mentioned product, and the application is for the preparation of an antibacterial product.

[0036] In some embodiments of the present invention, the pathogenic bacteria inhibited by the antibacterial product are at least one of uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa, and Candida albicans.

[0037] In some embodiments of the present invention, the application is for the preparation of a drug for treating or preventing urogenital tract infections, or for the preparation of a vaginal care product.

[0038] In some embodiments of the present invention, the urogenital tract infections include vaginal infections and lower urinary tract infections.

[0039] In some embodiments of the present invention, the vaginal infections include at least one of bacterial vaginitis, candidal vaginitis, trichomonal vaginitis, infantile vaginitis, menstrual vaginitis, senile vaginitis, mixed infective vaginitis, and vaginal infections caused by HPV infection, infections endangering the fetus in pregnant women, and vaginal infections causing premature birth or miscarriage.

[0040] In some embodiments of the present invention, the lower urinary tract infections include urethritis and cystitis.

[0041] In some embodiments of the present invention, the application is for the preparation of a drug delivery carrier.

[0042] In some embodiments of the present invention, the application is for the preparation of a product for adhering to cervical cancer cells or bladder transitional cell carcinoma cells.

[0043] In some embodiments of the present invention, the application is for the production of lactic acid or for the preparation of a product for the production of lactic acid.

[0044] In some embodiments of the present invention, the lactic acid is D-lactic acid and L-lactic acid.

[0045] According to some embodiments of the present invention, it has at least the following beneficial effects: The present invention provides a strain of Lactobacillus crispatus LSA-573, which has strong lactic acid-producing and H2O2-producing abilities, has good antibacterial effects against urogenital pathogenic bacteria such as uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa, and Candida albicans, and has a high adhesion ability to human cervical epithelial cells Hela cells and human bladder transitional cell carcinoma cells T24. Using the Lactobacillus crispatus LSA-573 of the present invention's solution to develop a live lactobacillus preparation has the advantages of safety, reliability, and good effect, and can be used for the treatment and / or prevention of female lower urinary tract infections and vaginitis-related diseases, regulating the microecological balance of the female reproductive tract, preventing female reproductive tract diseases, reducing the probability of female reproductive tract diseases, and has very important significance for female reproductive health, population eugenics, and family and social harmony and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The following further describes the present invention in conjunction with the drawings and embodiments, wherein:

[0047] Figure 1 It is the colony morphology diagram of the LSA-573 strain in Example 1 of the present invention;

[0048] Figure 2 It is the morphological diagram of the LSA-573 strain after Gram staining in Example 1 of the present invention;

[0049] Figure 3 It is the experimental flow chart for the establishment of a Gardnerella vaginalis model mouse in Example 6 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0051] The culture media involved in the following embodiments and their preparation methods are as follows:

[0052] MRS broth medium: Weigh 24.05 g of MRS broth powder, add 500 mL of deionized water, stir to dissolve, and adjust the pH to 6.2 ± 0.2; autoclave at 121 °C for 15 min to obtain it.

[0053] MRS agar medium: Weigh 24.05 g of MRS broth powder and 10.0 g of agar powder, add 500 mL of deionized water, stir to dissolve, adjust the pH to 6.2 ± 0.2, autoclave at 121 °C for 15 min. After cooling the medium to 50 - 60 °C, pour it into petri dishes, about 15 - 20 mL of the medium is poured into each petri dish. After cooling and solidifying, store it in a 4 °C refrigerator for later use.

[0054] Hydrogen peroxide semi - quantitative determination medium (TMB - HRP medium):

[0055] (1) Weigh 4.81 g of MRS broth powder and 2.0 g of agar powder, place them in a conical flask, then add 100 mL of deionized water, stir to dissolve, and adjust the pH to 6.2 ± 0.2;

[0056] (2) Weigh 0.05 g of 3,3’,5,5’ - tetramethylbenzidine (TMB) and dissolve it in 1 mL of dimethyl sulfoxide solution to obtain a 50 mg / mL TMB solution. Then add 0.5 mL of the TMB solution to the medium and mix well;

[0057] (3) Paste the sterilization indicator tape, mark the name of the medium and the preparation date, place the medium in an autoclave, and sterilize at 121 °C for 15 min;

[0058] (4) Weigh 0.02 g of horseradish peroxidase aniline (HRP) and dissolve it in 2 mL of deionized water, and filter - sterilize to obtain a 10 mg / mL HRP solution;

[0059] (5) After cooling the sterilized MRS medium to 50 - 60 °C, add 0.5 mL of the HRP solution, mix well, pour the plates to prepare the TMB - HRP medium, let it dry and store at 4 °C for later use.

[0060] The sources or preparation methods of the microorganisms involved in the following examples are as follows:

[0061] Isolation, purification and enrichment culture of Lactobacillus delbrueckii DM8909: The drug "Ding Junsheng" was purchased from a very large pharmacy store. In a clean working area, the capsule shell of the "Ding Junsheng" tablet was opened, and a inoculation loop was used to dip the bacterial powder into MRS broth medium, mixed well, and anaerobically cultured in an incubator at 37°C for 24 h. Subsequently, a inoculation loop was used to dip the cultured bacterial liquid and streak it on MRS agar medium, and anaerobically cultured in an incubator at 37°C for 48 h. After the culture was completed, a single colony on the plate was picked and inoculated into MRS broth medium, and anaerobically cultured in an incubator at 37°C for 24 h. Finally, the bacterial liquid was taken for 16s rDNA sequence identification. The 16s rDNA sequence of Lactobacillus delbrueckii DM8909 obtained by identification was as shown in SEQ ID NO:4. The bacterial liquid was preserved and frozen in a -80°C refrigerator, and the reference strain Lactobacillus delbrueckii DM8909 was obtained.

[0062] Isolation, purification and enrichment culture of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1: The "Colibactin Women's Probiotic Capsules" were purchased from the online store of Kelichun Overseas Exclusive Store. In a clean working area, the capsules were dissolved in 10 mL of physiological saline, shaken well and then serially diluted. Subsequently, the bacterial suspension was aspirated and spread on MRS plates with glass beads, and anaerobically cultured in an incubator at 37°C until obvious colonies grew on MRS agar. Single colonies with different morphologies were picked and inoculated into MRS broth, and anaerobically cultured at 37°C for 24 h. The bacterial liquid was taken for species identification respectively. The 16s rDNA sequence of Lactobacillus reuteri RC-14 obtained by identification was as shown in SEQID NO:5, and the 16s rDNA sequence of Lactobacillus rhamnosus GR-1 was as shown in SEQ ID NO:6. The bacterial liquid was preserved and frozen in a -80°C refrigerator, and the reference strains Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 were obtained.

[0063] Uropathogenic Escherichia coli (purchased from Ningbo Mingzhou Biotechnology Co., Ltd.); Gardnerella vaginalis (purchased from Guangdong Microbial Preservation Center); Pseudomonas aeruginosa (isolated from a patient with bacterial vaginitis BV, and the 16s rDNA sequence of Pseudomonas aeruginosa obtained by identification was as shown in SEQ ID NO:7); Candida albicans (purchased from China General Microbiological Culture Collection Center).

[0064] Example 1

[0065] 1. Isolation of LSA-573 strain

[0066] The LSA-573 strain was isolated from the posterior fornix of the vagina of a healthy woman. The specific isolation method is as follows: vaginal secretions of women of childbearing age who passed a health examination were collected with a vaginal swab, and the vaginal swab was placed in ESwab transport and preservation solution (Copan), shaken well to obtain a mixed solution. The mixed solution was added to sterile PBS solution for serial ten-fold gradient dilution. Dilutions of different concentrations were taken and spread on MRS agar medium, and anaerobically cultured in an incubator at 37°C for 48 h. After obvious colonies grew on the medium, different colonies were subcultured, and the strains with typical characteristics of lactic acid bacteria such as white round edges, neat edges, and shiny surfaces were selected and streaked on MRS plates for isolation one by one. By screening, a Lactobacillus crispatus strain with strong lactic acid production and H2O2 production abilities, good antibacterial effects against urogenital pathogenic bacteria such as uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa, and Candida albicans, and high adhesion ability to human cervical epithelial cells Hela cells and human bladder transitional cell carcinoma cells T24 was obtained, and it was named LSA-573. At the same time, the bacterial liquid was preserved, the corresponding strain number was marked, and it was stored in a -80°C refrigerator.

[0067] 2. Identification of the LSA-573 strain

[0068] (1) Culture characteristics, microscopic examination and morphological characteristics

[0069] The isolated LSA-573 strain was streaked on MRS agar medium and anaerobically cultured. The results are as Figure 1 shown. The colonies showed a diameter of about 2-3 mm, were grayish-white circular colonies, opaque, with a raised middle, and had neat colony edges and stable size and shape. The smear of this bacterium was Gram-stained. The results are as Figure 2 shown. This bacterium was Gram-positive, without flagella, was short rod-shaped, could form long chains, and had no spores.

[0070] (2) 16s rDNA gene sequence identification

[0071] The DNA of Lactobacillus was extracted by a rapid bacterial genomic DNA extraction kit and used as a template. Primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:2) and 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO:3) were used to amplify the conserved region of the 16S rDNA gene to obtain a PCR product. And the PCR product was sequenced.

[0072] PCR system (25 μL): 12.5 μL Taq PCR Master Mix buffer (containing Taq DNA polymerase, dNTP, MgCl2, PCR buffer, PCR reaction stabilizer, loading buffer and bromophenol blue dye), 1 μL each of 10 μmol / L upstream and downstream primers, 1 - 2 μL DNA template, and the rest is supplemented with nuclease-free water to 25 μL.

[0073] PCR conditions: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, for a total of 35 cycles; extension at 72 °C for 10 min.

[0074] The obtained PCR products were detected by 1% gel electrophoresis. If the result was positive, the products were sent to a sequencing company for bidirectional sequencing. Subsequently, Blast sequence analysis was performed on the obtained bacterial sequences in the NCBI database, and finally, LSA-573 was determined to be Lactobacillus crispatus, with a homology score of 99.8%.

[0075] Based on the above identification results, the strain name of LSA-573 was determined to be Lactobacillus crispatus, and its taxonomic name was Lactobacillus crispatus. This strain was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on April 26, 2022, with the deposit number CGMCC No. 24781.

[0076] The sequence of the 16S rDNA of Lactobacillus crispatus LSA-573 is as follows:

[0077]

[0078] Example 2: Content Analysis of the Metabolites of Lactobacillus crispatus LSA-573

[0079] 1. Determination of the Yields of D- and L-Lactic Acids by Lactobacillus crispatus LSA-573

[0080] Using the activated Lactobacillus crispatus LSA-573 as the experimental group, and Lactobacillus delbrueckii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 as the control groups respectively, centrifuge them, discard the supernatant, resuspend the cells and adjust the turbidity of the liquid to an appropriate turbidity (~10 8 CFU / mL), take the bacterial suspension and inoculate it into MRS liquid medium (10% v / v), and culture anaerobically at 37 °C for 24 h. Centrifuge the cultured Lactobacillus bacterial liquid, and take the supernatant for the determination of lactic acid content. Use high performance liquid chromatography to detect the contents of D-lactic acid and L-lactic acid in the fermentation broth. The specific chromatographic conditions are shown in Table 1, and the determination results of the lactic acid content in the fermentation broth are shown in Table 2.

[0081] Table 1 Chromatographic Conditions for the Determination of Lactic Acid Content in Lactobacillus Fermentation Broth by High Performance Liquid Chromatography

[0082]

[0083]

[0084] Table 2 Lactic Acid Contents in the Fermentation Broths of Lactobacillus crispatus LSA-573 and Control Strains

[0085]

[0086] Note: "ND" indicates that this type of lactic acid is not produced.

[0087] The determination results of lactic acid content are shown in Table 2. It can be seen from the table that Lactobacillus crispatus LSA-573 of the present invention can produce both D-lactic acid and L-lactic acid, and the lactic acid production is mainly D-lactic acid, accounting for 67.55% of the total lactic acid; the total lactic acid content is higher than that of Lactobacillus delbrueckii DM8909 and Lactobacillus rhamnosus GR-1, and much higher than that of Lactobacillus reuteri RC-14.

[0088] 2. Determination of the Ability of Lactobacillus crispatus LSA-573 to Produce H2O2

[0089] Using the activated Lactobacillus crispatus LSA-573 as the experimental group and Lactobacillus delbrueckii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 as the control groups respectively, streak them on TMB-HRP medium and anaerobically culture them at 37 °C in a triple-gas incubator for 48 h. Take out the plates, expose the bacteria to the air, and observe the change in colony color. Finally, based on the time of color change, judge the ability of the strains to produce hydrogen peroxide, which are strong (change time < 10 min), medium (change time is 10 to 20 min), weak (change time is 20 to 30 min), or none (change time > 30 min or no color change).

[0090] The measurement results are as follows:

[0091] Table 3 Semi-quantitative determination results of hydrogen peroxide production capacity of Lactobacillus crispatus LSA-573 and control strains

[0092] Strain Discoloration time after exposure to air Hydrogen peroxide production capacity Lactobacillus crispatus LSA-573 1 - 2 min +++ Lactobacillus delbrueckii DM8909 <1min ++++ Lactobacillus rhamnosus GR-1 No discoloration － Lactobacillus reuteri RC-14 10 min ++

[0093] The experimental results are shown in Table 3. It can be seen from the table that the hydrogen peroxide production capacity of Lactobacillus crispatus LSA-573 is better than that of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1. When exposed to air for 1 to 2 min, the colonies of Lactobacillus crispatus LSA-573 began to change color and showed a slightly blue state. At 3 to 6 min, the blue color of the colonies continued to deepen, and at 9 min, the colonies showed an obvious dark blue color; the colonies of Lactobacillus reuteri RC-14 showed a sky blue color when exposed to air for 10 min and an obvious dark blue color at 20 min; the colonies of Lactobacillus rhamnosus GR-1 remained milky white when exposed to air for 30 min; the colonies of Lactobacillus delbrueckii DM8909 showed an obvious dark blue color when exposed to air for 1 min. Therefore, according to the judgment standard of hydrogen peroxide production capacity, Lactobacillus crispatus LSA-573 has a strong hydrogen peroxide production capacity.

[0094] Example 3 Analysis of the antibacterial effect of Lactobacillus crispatus LSA-573 against pathogenic bacteria

[0095] Using the activated Lactobacillus crispatus LSA-573 as the experimental group and Lactobacillus delbrueckii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 as the control groups respectively, take equal amounts of bacterial liquid and centrifuge, discard the supernatant, resuspend the bacteria, and adjust the turbidity of the bacterial liquid to an appropriate turbidity (~10 8 CFU / mL). Take 0.5 mL of the resuspended Lactobacillus liquid and mix it with 20 mL of MRS agar medium cooled to 50 - 60 °C, anaerobically culture it at 37 °C for 24 h after cooling and solidifying, and then punch to prepare bacterial cakes; centrifuge the activated uropathogenic Escherichia coli / Gardnerella vaginalis / Pseudomonas aeruginosa, and Candida albicans, discard the supernatant, resuspend the bacteria, and adjust the turbidity to an appropriate turbidity (~10 8(CFU / mL), take 100 μL of the bacterial suspension and resuspend it in a nutrient agar solid medium, and evenly coat it with glass beads; place the prepared bacterial cake on the agar plate coated with the pathogenic bacterium, and anaerobically culture it at 37 °C for 48 h. If an inhibition zone appears, use a vernier caliper to measure the diameter of the inhibition zone.

[0096] Table 4 Inhibition zone diameters (mm) of Lactobacillus crispatus LSA-573 and control bacteria against pathogenic bacteria

[0097]

[0098]

[0099] "-" indicates no inhibitory effect on pathogenic bacteria.

[0100] The inhibitory results of Lactobacillus crispatus LSA-573 and control bacteria against pathogenic bacteria are shown in Table 4. It can be seen from the table that Lactobacillus crispatus LSA-573 has a higher inhibitory ability against uropathogenic Escherichia coli, Gardnerella vaginalis, and Pseudomonas aeruginosa than Lactobacillus delbrueckii DM8909 and Lactobacillus reuteri RC-14, and can significantly inhibit Candida albicans, which cannot be inhibited by other lactobacilli.

[0101] Example 4 Analysis of the adhesion of LSA-573 strain to cervical cancer cells Hela and human bladder transitional cell carcinoma cells T24

[0102] 1. Analysis of the adhesion of LSA-573 strain to cervical cancer cells Hela

[0103] HeLa cells are derived from cervical cancer cells of women and have rapid proliferation. They are commonly used as substitute cells for vaginal epithelial cells in in vitro studies. The stronger the adhesion of probiotics to epithelial cells, the more beneficial it is for colonization in the human body, competing for occupancy with pathogenic bacteria to prevent the invasion of pathogenic bacteria, and it is also more conducive to the exertion of its probiotic functions.

[0104] Culture Hela cells (purchased from the Cell Bank of the Chinese Academy of Sciences) in a T75 cell culture flask until the cell coverage rate is about 80%. Digest with 3 mL of 0.25% trypsin-EDTA solution for 2 min 30 s, add 3 mL of a mixed solution containing 10% FBS, 89% high-glucose DMEM medium, and 1% penicillin-streptomycin, and gently pipette to detach the cells from the culture flask. The cells are inoculated at a density of 4×10 4 cells / mL and 2 mL are inoculated into a 6-well plate, and a single cell layer is formed after culturing for 48 h.

[0105] Using the activated Lactobacillus crispatus LSA-573 as the experimental group and Lactobacillus delbrueckii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 as the control groups respectively. After centrifuging the culture broth of each group of lactobacilli to remove the supernatant, the precipitated bacteria were resuspended in PBS solution, and the turbidity of the bacterial suspension was adjusted to 1.0 MCF. Then, the supernatant was removed by centrifugation, and the bacteria were resuspended in an equal volume of high-glucose DMEM medium to obtain a bacterial suspension. The bacterial suspension was diluted and spread on MRS agar plates. After anaerobic culture for 48 h, the number of colonies on each plate was counted.

[0106] Before the adhesion experiment, the culture broth in the cell culture plate was aspirated, and 2 mL of PBS solution was added to each well to wash the cells 3 times. Then, 2 mL of the bacterial suspension was added, and the cell culture plate was gently shaken to evenly disperse the bacteria. The cell culture plate and the remaining bacterial suspension were placed in an incubator at 37 °C and 5% CO2 for adhesion for 2 h, with 2 parallels in each group.

[0107] After the adhesion, the culture broth in the cell culture plate was aspirated, and 2 mL of PBS solution was added to each well to wash the cells 3 times. Then, 0.5 mL of 0.25% trypsin-EDTA solution was added to digest for 2 min 30 s. The digestion solution was aspirated, and then 1 mL of PBS solution was added to gently pipette the cells to detach them, and 1 mL of 0.05% TritonX-100 was added to lyse the cells to make a bacterial suspension. The suspension was serially diluted, and 100 μL of the bacterial suspension was evenly spread on MRS agar; after anaerobic culture for 48 h, the number of colonies on each plate was counted.

[0108] 2. Analysis of the adhesion effect of LSA-573 strain on human bladder transitional cell carcinoma T24 cells

[0109] Human bladder transitional cell carcinoma T24 cells (purchased from Beina Biotechnology) were cultured in a T75 cell culture flask until the coverage rate was about 80%. The cells were digested with 3 mL of 0.25% trypsin-EDTA solution for 2 min 30 s, and 3 mL of a mixed solution containing 10% FBS, 89% McCoy’s 5a medium, and 1% penicillin-streptomycin was added. The cells were gently pipetted to detach them from the culture flask. The cells were inoculated at a density of 4×10 4 cells / mL at 2 mL per well in a 6-well plate and cultured for 48 h to form a single cell layer.

[0110] Using the activated Lactobacillus crispatus LSA-573 as the experimental group, and Lactobacillus delbrueckii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 as the control groups respectively. After centrifuging the culture solutions of each group to remove the supernatant, the precipitated bacteria were resuspended in PBS solution, and the turbidity of the bacterial suspension was adjusted to 1.0 MCF. Then, the supernatant was removed by centrifugation, and the bacteria were resuspended in an equal volume of McCoy’s 5a medium to obtain a bacterial suspension. The bacterial suspension was taken and diluted and spread on MRS agar. After anaerobic culture for 48 h, the initial number of bacteria on each plate was counted.

[0111] Before the adhesion experiment, the culture solution in the cell culture plate was aspirated, and 2 mL of PBS solution was added to each well to wash the cells 3 times, and then 2 mL of the bacterial suspension was added. The cell culture plate was gently shaken to fully disperse the bacteria evenly. The cell culture plate and the remaining bacterial suspension were placed in an incubator at 37 °C and 5% CO2 for adhesion for 2 h, and 2 parallels were made.

[0112] After the adhesion was completed, the culture solution in the cell culture plate was aspirated, and 2 mL of PBS solution was added to each well to wash the cells 3 times. 0.5 mL of 0.25% trypsin-EDTA solution was added to each well to digest for 2 min and 30 s. The digestion solution was aspirated, 1 mL of PBS solution was added, and the cells were gently pipetted to make them detached. 1 mL of 0.05% TritonX-100 was added to lyse the cells to make a bacterial suspension, which was serially diluted. 100 μL of the bacterial suspension was evenly spread on MRS agar; after anaerobic culture for 48 h, the number of viable bacteria after the strain adhesion treatment on each plate was counted.

[0113] The calculation formula for the adhesion rate is as follows:

[0114] Adhesion rate = Viability rate of the strain (%) = N l / N0×100%;

[0115] In the formula, N1 - Number of viable bacteria after the strain adhesion treatment (CFU / mL); N0 - Number of viable bacteria before the strain adhesion (CFU / mL).

[0116] Table 5 Results of cell adhesion

[0117] Strain Hela cells Human bladder transitional cell carcinoma T24 cells Lactobacillus crispatus LSA-573 20％ 18％ Lactobacillus delbrueckii DM8909 13％ 8％ Lactobacillus rhamnosus GR-1 15％ 10％ Lactobacillus reuteri RC-14 8％ 6％

[0118] The calculation results of the adhesion rates of the LSA-573 strain to the cervical cancer cells Hela and human bladder transitional cell carcinoma cells T24 are shown in Table 5. As can be seen from the table, Lactobacillus crispatus LSA-573 has a high adhesion ability to human cervical epithelial cells Hela and human bladder transitional cell carcinoma cells T24, and has better colonization ability.

[0119] Example 5. Toxicity test and colonization ability of Lactobacillus crispatus LSA-573

[0120] Forty SPF-grade female mice weighing 14 - 18 g and 6 - 8 weeks old were selected and randomly divided into a low-dose group, a high-dose group, and a control group, with 10 mice in each group. The activated Lactobacillus crispatus LSA-573 was centrifuged, the supernatant was discarded, and the bacterial pellet was resuspended in PBS solution to a low turbidity (~10 8 CFU / mL) and a high turbidity (~10 10 CFU / mL). All mice were given standard feed for free intake. The vaginas of the mice in the low-dose group were perfused with 20 μL of the low-turbidity LSA-573 bacterial solution every day, the high-dose group was perfused with an equal volume of the high-turbidity LSA-573 bacterial solution every day, and the control group was perfused with an equal volume of normal saline every day for a total of 14 days. The body weight and toxic reactions of the mice were observed.

[0121] One day before perfusion and on the 14th day of perfusion, 50 μL of normal saline was aspirated with a micropipette and used to repeatedly rinse the vaginas of the mice in each group 5 - 6 times. Then, 30 μL of the above lavage fluid was diluted to an appropriate gradient (~10 -4 ) and then used for Lactobacillus counting on MRS agar.

[0122] Fourteen days after vaginal perfusion with the bacterial solution, there were no abnormal conditions in the mice of each group. Compared with the control group, there was no abnormal body weight, and no tremors, abnormal postures, exophthalmos, or deaths were found. Urination and breathing were normal. This result indicates that Lactobacillus crispatus LSA-573 is non-toxic.

[0123] Table 6 Lactobacillus counts (CFU / mL) in the vaginas of mice in different experimental groups before and after vaginal perfusion with Lactobacillus

[0124]

[0125] The changes in the Lactobacillus counts in the vaginas of the mice before and after vaginal perfusion with the bacterial solution are shown in Table 6. The Lactobacillus in the vaginas of the mice perfused with the low-turbidity LSA-573 increased by about 8 - 9 times per day, while the Lactobacillus in the vaginas of the mice perfused with the high-turbidity LSA-573 increased by about 60 times. This result indicates that Lactobacillus crispatus LSA-573 has the ability to colonize in the vagina.

[0126] Example 6 Therapeutic ability of Lactobacillus crispatus LSA-573 on mice with Gardnerella vaginalis model

[0127] Forty SPF-grade female mice weighing 14 - 18 g and 6 - 8 weeks old were selected and randomly divided into a healthy group, an infection group, a metronidazole group, and an LSA-573 group, with 10 mice in each group. The day when the Gardnerella vaginalis model mice began to be established was defined as Day1. The experimental protocol for establishing the Gardnerella vaginalis model mice is as Figure 3 shown. From Day1 to Day3, each mouse was injected with 0.5 mg of estradiol every day for estrogenization, and then 20 μL of an appropriate concentration of Gardnerella vaginalis solution (~108 (CFU / mL), and inoculated continuously for 4 days (Day4 - Day8). Mice in the healthy group were injected with 20 μL of normal saline instead of the bacterial solution. On Day8, a small amount of mucus was swabbed from the vagina of the mice with a sterile swab, and whether Gardnerella vaginalis was present was observed by staining under a microscope to ensure the colonization of Gardnerella vaginalis in the mouse vagina and the establishment of a mouse model of Gardnerella vaginalis.

[0128] Centrifuge the activated Lactobacillus crispatus LSA - 573, discard the supernatant, and resuspend the bacterial cells with PBS solution to an appropriate turbidity (~10 9 (CFU / mL). From the first day to the seventh day after modeling (Day8 - Day15), for 7 consecutive days, once a day, 20 μL of freshly prepared Lactobacillus crispatus LSA - 573 bacterial solution was perfused into the vagina of mice in the LSA - 573 group; 20 μL of metronidazole solution was injected into the vagina of mice in the metronidazole group; mice in the healthy group and the infection group were given the same volume of normal saline vaginally every day.

[0129] Before drug administration, on the 1st day after drug administration, and on the 7th day after drug administration, 50 μL of normal saline was taken with a micropipette, and the vagina of the mice was rinsed repeatedly 5 - 6 times. After diluting 30 μL of the above lavage fluid to an appropriate gradient, it was spread on an agar medium for counting Lactobacillus and Gardnerella vaginalis. MRS agar was used for counting Lactobacillus, and Columbia blood agar plates supplemented with gentamicin sulfate (4 mg / L), nalidixic acid (30 mg / L), and amphotericin B (2 mg / L) were used for counting Gardnerella vaginalis.

[0130] Table 7 Lactobacillus counts (CFU / mL) in the vagina of mice in different experimental groups before and after vaginal modeling and drug administration

[0131]

[0132] As shown in Table 7, it can be seen from Table 7 that on the 1st day after drug administration, compared with the infection group, the Gardnerella vaginalis in the vagina of mice perfused with Lactobacillus crispatus LSA - 573 decreased significantly by about 1 order of magnitude, and was lower than that of the metronidazole group by 2 orders of magnitude. However, the number of Lactobacillus in the LSA - 573 group of mice was about 8 - 12 times that of the metronidazole group and about 3 - 6 times that of the infection group. On the 7th day after drug administration, the number of Gardnerella vaginalis in the vagina of mice in the LSA - 573 group was similar to that of the metronidazole group, but the number of Lactobacillus was higher than that of the metronidazole group by about 3 orders of magnitude and was 10 - 15 times that of the healthy group. The above results indicate that Lactobacillus crispatus LSA - 573 has an inhibitory ability against Gardnerella vaginalis similar to that of metronidazole, and at the same time can colonize in the mouse vagina and restore the number of Lactobacillus in the vagina to normal levels and above, indicating that Lactobacillus crispatus LSA - 573 has a preventive and therapeutic effect on bacterial vaginitis.

[0133] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A strain, characterized in that, The strain is Lactobacillus crispatus ( Lactobacillus crispatus ), LSA-573, which is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 24781 and the deposit date of April 26, 2022.

2. The strain according to claim 1, characterized in that, The strain has a 16S rDNA sequence shown in SEQ ID NO:

1.

3. A product, characterized in that, It includes one of (1) to (3): (1) The strain described in claim 1 or 2; (2) A bacterial agent containing the strain described in claim 1 or 2; (3) A viable bacterial liquid containing the strain described in claim 1 or 2; The product is a drug or a hygienic product.

4. The product according to claim 3, characterized in that, The hygienic product is at least one of a tampon, a sanitary napkin, a menstrual pad, a diaper, and a condom.

5. The product according to claim 3, characterized in that, The drug further includes a pharmaceutical carrier and / or pharmaceutical excipients.

6. The product according to claim 5, wherein The pharmaceutical carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetening agent, and a flavoring agent.

7. The product according to claim 3, wherein The form of the product is one of a powder, an ointment, a gel, a drop, a suppository, a lozenge, a granule, a capsule, a spray, and a solution.

8. Use of the strain according to claim 1 or 2, or the product according to any one of claims 3-7, in the preparation of an antibacterial product, characterized in that, The pathogenic bacteria inhibited by the antibacterial product are at least one of uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa, and Candida albicans.

9. Use of the strain described in claim 1 or 2, or the product described in any one of claims 3 - 7 in the preparation of a drug for treating or preventing urogenital tract infections, or a vaginal care product.

Images