CN116814474A - Lactobacillus crispatus and application thereof - Google Patents

Abstract

The invention discloses a strain of Lactobacillus crispatus and its application. The Lactobacillus crispatus is deposited in the General Microorganism Center of China Microbial Culture Collection Committee, and the deposit number is CGMCC No. 24781. The Lactobacillus crispatus LSA-573 of the present invention is used to develop a live lactobacillus preparation, which has the advantages of safety, reliability, good effect, etc., and can be used to treat and/or prevent female lower urinary tract infections and vaginitis-related diseases, and regulate women's Reproductive tract microecological balance, preventing women's reproductive tract diseases, and reducing the probability of women suffering from female reproductive tract diseases are of great significance to female reproductive health, population eugenics, and family and social harmony and stability.

Description

A strain of Lactobacillus crispatus and its application

Technical field

The invention belongs to the field of microorganisms, and specifically relates to a strain of Lactobacillus crispatus and its application.

Background technique

Urogenital tract infections are common diseases that seriously affect the physical and mental health of women, including vaginal infections, lower urinary tract infections, etc. The female lower reproductive tract is an open cavity, home to a large number of different types of microorganisms. These microorganisms grow and reproduce in the vagina and restrict each other, forming the vaginal micro-ecosystem, which is closely related to female reproductive health and is one of the important factors in the human body. Microecological zone. Affected by hormone levels, behavioral habits, ethnic genes, and the environment, the composition of microorganisms in the reproductive tract of women continues to change throughout their lives. Before puberty, Escherichia coli and anaerobic bacteria dominate. The diversity of vaginal flora is high, and the pH is neutral to alkaline. During puberty, Lactobacilli begin to colonize, and the pH becomes acidic (3.8-4.5). The vagina of healthy women of childbearing age is a microenvironment in which Lactobacillus is the dominant flora. The most common ones are Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jansoni, Lactobacillus iners, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus rhamnosus, etc. The normal vaginal flora during pregnancy is very similar to that during the childbearing period. During the puerperium, the vaginal flora is affected by hormones. The decrease in estrogen makes the vaginal flora of women in the puerperium not very similar to that of the childbearing period, but more like menopausal women. . In postmenopausal women, as estrogen decreases, vaginal pH increases, and anaerobic bacteria and mycoplasma dominate.

Statistics released by the World Health Organization (WHO) show that 40% of women suffer from reproductive tract infections of varying degrees. According to this estimate, at least 200 million women suffer from reproductive tract infections and related diseases every year. From a clinical perspective, women Reproductive infections are mostly related to microecological imbalance. The reproductive tract flora is disordered, lactobacilli are reduced, and other pathogenic bacteria increase, thus causing various gynecological inflammations. Bacterial vaginosis (BV) is the most common vaginal disease in women of childbearing age. It is caused by the reduction or disappearance of lactobacilli that normally produce hydrogen peroxide in the vagina, facultative anaerobic bacteria and anaerobic bacteria (such as Gardnerella vaginalis). Vaginal infections are mainly caused by an increase. The detection rate among obstetrics and gynecology outpatients is as high as 18.8%, and 50% of BV patients are asymptomatic. Fungal vaginitis (VVC), also known as vulvovaginal candidiasis, is caused by yeasts mainly Candida albicans. About 70%-75% of women experience VVC at least once in their lives, and 50% of them experience VVC at least once in their lives. Women will experience multiple attacks of the disease throughout their lives, and 5%-10% of them are likely to develop recurrent VVC. Urinary tract infection (UTI) is an inflammatory response caused by bacterial invasion of urothelial cells. The pathogens include uropathogenic Escherichia coli, Klebsiella, Staphylococcus and Enterococcus species and other pathogens. Studies have shown that approximately 50% of women will suffer from a urinary tract infection once in their lifetime, and 25% of patients will have a recurrence within 6 months. Female vaginitis is not only stubborn and harmful, it seriously affects women's quality of life and can also cause a variety of complications. UTI can affect the risk of kidney infection, and UTI infection 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 delivery, premature rupture of membranes, and neonatal infection. Left untreated, VVC may lead to some serious complications of the upper reproductive tract, such as endometritis, salpingitis, and pelvic inflammatory disease, ultimately leading to fallopian tube scarring, infertility, or ectopic pregnancy.

For female vaginitis, the existing treatment methods are mainly antibiotics. Although they 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 resistance of pathogenic bacteria, which has aggravated the prevalence and stubbornness of female vaginitis. Lactobacilli account for 70% to 95% of female vaginal flora and play an important protective role in the vagina. At present, lactobacillus live bacteria preparations and strains related to the treatment and improvement of vaginitis/lower urinary tract infection in China are still in their infancy, and a large amount of research data and lactobacillus strains are needed as technical support and candidate research objects.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a strain, the strain is Lactobacillus crispatus LSA-573, which can be used to develop live lactobacillus preparations, has the advantages of safety, reliability, good effect, etc., and can be used for treatment and/or prevention Diseases related to female lower urinary tract infection and vaginitis can also help restore the vaginal microecological environment, which is beneficial to women's health.

The present invention also proposes a product containing the above-mentioned bacterial strain.

The present invention also proposes the application of the above-mentioned bacterial strain or products containing the above-mentioned bacterial strain.

In one aspect of the present invention, a strain is proposed. The strain is Lactobacillus crispatus LSA-573, which is deposited in the General Microorganism Center of the China Committee for the Collection of Microorganisms. The deposit number is CGMCC No. 24781, and the deposit date is It is April 26, 2022.

In some embodiments of the invention, the Lactobacillus crispatus LSA-573 is isolated from the posterior vaginal fornix of healthy women.

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

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

(1) The above strains;

(2) Inoculants containing the above strains;

(3) Live bacterial liquid containing the above strains;

(4) Dead bacterial liquid containing the above strains;

(5) Contains metabolites of the above strains;

(6) Extracts containing the above strains.

In some embodiments of the invention, the product is a pharmaceutical, medical device, food or hygiene product.

In some embodiments of the present invention, the medicine further includes pharmaceutical carriers and/or pharmaceutical excipients.

In some embodiments of the invention, the pharmaceutical carrier includes diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, sweeteners, and At least one of the fragrances.

In some embodiments of the invention, the excipient includes water.

In some embodiments of the invention, the filler includes at least one of starch and sucrose.

In some embodiments of the invention, the binder includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

In some embodiments of the invention, the humectant includes glycerin.

In some embodiments of the invention, the disintegrant includes at least one of agar, calcium carbonate and sodium bicarbonate.

In some embodiments of the invention, the absorption enhancer includes a quaternary ammonium compound.

In some embodiments of the invention, the surfactant includes cetyl alcohol.

In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

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

In some embodiments of the invention, the medical device is a gel.

In some embodiments of the present invention, the food is dairy products, soy products, or fruit and vegetable products produced using a starter culture containing the above bacterial strain; or the food is a beverage or snack containing the above bacterial strain.

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

In some embodiments of the invention, the sanitary napkin includes a tampon, a safety panty, a menstrual cup or a panty liner.

In some embodiments of the present invention, the product is in the form of one of powder, ointment, gel, drop, suppository, lozenge, granule, capsule, spray, tablet, pill and solution; preferably, the The capsules are capsules.

In a third aspect of the present invention, the application of the above-mentioned Lactobacillus crispatus strain or the above-mentioned product is proposed, and the application is in the preparation of antibacterial products.

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 .

In some embodiments of the present invention, the application is in the preparation of medicaments for treating or preventing genitourinary tract infections, or vaginal care products.

In some embodiments of the invention, the genitourinary tract infections include vaginal infections and lower urinary tract infections.

In some embodiments of the present invention, the vaginal infection includes bacterial vaginitis, candida vaginitis, trichomonal vaginitis, childhood vaginitis, menstrual vaginitis, senile vaginitis, mixed infectious vaginal infection inflammation and at least one of the following: HPV infection, infection that threatens the fetus in pregnant women, or vaginal infection that causes premature labor or miscarriage.

In some embodiments of the invention, the lower urinary tract infection includes urethritis and cystitis.

In some embodiments of the invention, the application is in the preparation of drug delivery vehicles.

In some embodiments of the invention, the use is in the preparation of a product for adhesion to cervical cancer cells or bladder transitional cell cancer cells.

In some embodiments of the invention, the application is in the production of lactic acid or in the preparation of products for the production of lactic acid.

In some embodiments of the invention, the lactic acid is D-lactic acid and L-lactic acid.

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 ability to produce lactic acid and H ₂ O ₂ and is effective against urogenital tract pathogenic bacteria such as urine It has good antibacterial effect against pathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa and Candida albicans, and has good antibacterial effect on human cervical epithelial cells HeLa cells and human bladder transitional cell carcinoma T24 cells. High adhesion ability. The Lactobacillus crispatus LSA-573 of the present invention is used to develop a live Lactobacillus preparation, which has the advantages of safety, reliability, good effect, etc., and can be used to treat and/or prevent female lower urinary tract infections and vaginitis-related diseases, and regulate women's Reproductive tract microecological balance, preventing women's reproductive tract diseases, and reducing the probability of women suffering from female reproductive tract diseases are of great significance to female reproductive health, population eugenics, and family and social harmony and stability.

Description of the drawings

The present invention will be further described below in conjunction with the accompanying drawings and examples, wherein:

Figure 1 is a colony morphology diagram of the LSA-573 strain in Example 1 of the present invention;

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

Figure 3 is an experimental flow chart for the establishment of Gardnerella vaginalis model mice in Example 6 of the present invention.

Detailed ways

The concept of the present invention and the technical effects produced will be clearly and completely described below with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without exerting creative efforts are all protection scope of the present invention.

The culture medium involved in the following examples and its preparation method are as follows:

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

MRS agar medium: Weigh 24.05g of MRS broth powder and 10.0g of agar powder, add 500mL of deionized water, stir and dissolve, adjust the pH to 6.2±0.2, autoclave at 121°C for 15 minutes, and cool the medium to 50 to After reaching 60°C, pour into Petri dishes. Pour about 15-20mL of culture medium into each Petri dish. Cool and solidify and place in a 4°C refrigerator for later use.

Hydrogen peroxide semi-quantitative assay medium (TMB-HRP medium):

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

(2) Weigh 0.05g of 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 1mL of dimethyl sulfoxide solution to obtain a 50mg/mL TMB solution, and then add it to the culture medium 0.5mL of TMB solution, mix well;

(3) Paste the sterilization instruction tape and mark the culture medium name and preparation date. Place the culture medium in an autoclave and sterilize it at 121°C for 15 minutes;

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

(5) Cool the sterilized MRS culture medium to 50 to 60°C, add 0.5 mL of HRP solution, mix well, and pour the plate onto the plate to prepare TMB-HRP culture medium. After drying, store at 4°C for later use.

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

Isolation, purification and enrichment culture of Lactobacillus delbrueckii DM8909: I purchased the drug "Dingjunsheng" from a very large pharmacy store. Open the capsule shell of "Dingjunsheng" tablets in a clean work area, use an inoculating loop to dip the bacterial powder into the MRS broth culture medium, mix well, and place it in a 37°C incubator for anaerobic cultivation for 24 hours. Then use an inoculation loop to dip the cultured bacterial liquid into streaks on MRS agar medium to isolate, and then culture it anaerobically in a 37°C incubator for 48 hours. After the culture, a single colony on the plate was picked and inoculated into MRS broth medium, and cultured anaerobically in a 37°C incubator for 24 hours. Finally, the bacterial liquid was taken for 16s rDNA sequence identification. The identified 16s rDNA sequence of Lactobacillus delbrueckii DM8909 is shown in SEQ ID NO: 4. The bacterial liquid was preserved and frozen in a -80°C refrigerator to obtain the reference strain German milk. Bacillus DM8909.

Isolation, purification and enrichment culture of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1: I purchased "Kelitun Women's Probiotic Capsules" from the online store of Kelitun Overseas Store. Dissolve the capsule in 10 mL of physiological saline in a clean work area, shake well and carry out gradient dilution, then draw the bacterial suspension onto the MRS plate, shake the glass beads and spread them, and culture them anaerobically in a 37°C incubator until they grow on the MRS agar. When obvious colonies appear, single colonies of different shapes are picked and inoculated into MRS broth, and cultured anaerobically at 37°C for 24 hours. The bacterial liquids were taken separately for strain identification. The identified 16s rDNA sequence of Lactobacillus reuteri RC-14 is shown in SEQ ID NO: 5, and the 16s rDNA sequence of Lactobacillus rhamnosus GR-1 is shown in SEQ ID NO: 6. As shown, the bacterial solution was preserved and frozen in a -80°C refrigerator to obtain the reference strains Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1.

Uropathogenic Escherichia coli (purchased from Ningbo Mingzhou Biotechnology Co., Ltd.); Gardnerella vaginalis (purchased from Guangdong Microorganism Collection Center); Pseudomonas aeruginosa (isolated from a patient with bacterial vaginosis BV and identified The 16s rDNA sequence of Pseudomonas aeruginosa is shown in SEQ ID NO: 7); Candida albicans (purchased from China General Microbial Culture Collection and Management Center).

Example 1

1. Isolation of LSA-573 strain

LSA-573 strain was isolated from the posterior vaginal fornix of healthy women. The specific separation method is as follows: use vaginal swabs to collect vaginal secretion samples from women of childbearing age who have passed the health examination, place the vaginal swabs in ESwab transportation preservation solution (Copan Company), shake and mix thoroughly to obtain a mixed solution, and put the mixed solution Add sterile PBS solution for ten-fold gradient dilution, apply the dilutions of different concentrations on MRS agar medium, and place it in an incubator at 37°C for anaerobic cultivation for 48 hours. After obvious colonies grow on the culture medium, transfer different colonies to culture, pick out strains with typical characteristics of lactic acid bacteria such as white round edges, neat edges, and shiny surfaces, and streak them on the MRS plate one by one. Culture isolation. Through screening, we obtained products with strong ability to produce lactic acid and H ₂ O ₂ , which are resistant to genitourinary tract pathogenic bacteria such as uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa and Candida albicans. Lactobacillus crispatus, which has good antibacterial effect and has high adhesion ability to human cervical epithelial cells HeLa cells and human bladder transitional cell carcinoma T24 cells, is named LSA-573. At the same time, the bacterial solution is preserved, marked with the corresponding strain number, and frozen in a -80°C refrigerator.

2. Identification of LSA-573 strain

(1) Culture characteristics, microscopic examination and morphological characteristics

The isolated LSA-573 strain was cultured anaerobically on MRS agar medium. The results are shown in Figure 1. The colonies were about 2-3 mm in diameter, gray-white round colonies, opaque, with a protrusion in the middle, and The edges are neat and the size and shape are stable. Gram staining was performed on the smear of this bacterium. The results are shown in Figure 2. The bacterium is Gram-positive, has no flagella, is short rod-shaped, can be connected into long chains, and has no spores.

(2)16s rDNA gene sequence identification

The DNA of Lactobacillus was extracted using the bacterial genomic DNA rapid extraction kit and used as a template, using primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) and 1492R: 5'-TACGGTTACCTTGTTACGACTT-3'( SEQ ID NO: 3) Amplify the conserved region of the 16S rDNA gene to obtain a PCR product. and sequence the PCR products.

PCR system (25 μL): 12.5 μL Taq PCR Master Mix buffer (including Taq DNA polymerase, dNTP, MgCl ₂ , PCR buffer, PCR reaction stabilizer, loading buffer and bromophenol blue dye), 1 μL each 10 μmol/ L of upstream and downstream primers, 1-2 μL of DNA template, and the remainder to 25 μL with nuclease-free water.

PCR conditions: pre-denaturation at 94°C for 5 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 90 seconds; extension at 72°C for 10 minutes.

The obtained PCR product is detected by 1% gel electrophoresis. If the result is positive, the product is sent to a sequencing company for bidirectional sequencing. The obtained bacterial sequence was then subjected to Blast sequence analysis on the NCBI database, and LSA-573 was finally determined to be Lactobacillus crispatus, with a homology score of 99.8%.

After the above identification results, the name of the LSA-573 strain was determined to be Lactobacillus crispatus, and its classification was named Lactobacillus crispatus. The strain was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on April 26, 2022. (abbreviated as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), and the collection number is CGMCC No. 24781.

The sequence of 16s rDNA of Lactobacillus crispatus LSA-573 is as follows:

TTGTTACGACTTCACCCCAGTCATCTGCCCTGCCTTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATCCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGT ACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGG TTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACT GGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTA TCCTTCTTCACCAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCTTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTA AAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTGACCGAAGTAAATCTGTTAGTTCCGCTCGCTCGACTGCATGTATAGC (SEQ ID NO: 1).

Example 2. Content analysis of metabolites of LSA-573 strain

1. Determination of D-type and L-type lactic acid production of Lactobacillus crispatus LSA-573

Use activated Lactobacillus crispatus LSA-573 as the experimental group, Lactobacillus germansii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 as the control group. Centrifuge respectively and discard the supernatant. Resuspend the bacterial cells and adjust the turbidity of the liquid to a suitable turbidity (~10 ⁸ CFU/mL). Take the bacterial suspension and inoculate it into MRS liquid medium (10% v/v), and incubate anaerobically at 37°C for 24 hours. The cultured Lactobacillus bacteria liquid was centrifuged, and the supernatant was taken for determination of lactic acid content. High-performance liquid chromatography was used 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. The measurement results of the lactic acid content in the fermentation broth are shown in Table 2.

Table 1 Chromatographic conditions for determination of lactic acid content in Lactobacillus fermentation broth using HPLC method

Table 2 Lactic acid content in fermentation broth of Lactobacillus crispatus LSA-573 and control strains

Note: "ND" means that this type of lactic acid is not produced.

The measurement 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 D-lactic acid and L-lactic acid at the same time, and the lactic acid output is mainly D-lactic acid. Accounting for 67.55% of the total lactic acid; the total lactic acid content is higher than Lactobacillus germansii DM8909 and Lactobacillus rhamnosus GR-1, and much higher than Lactobacillus reuteri RC-14.

2. Determination of H ₂ O ₂ production ability of Lactobacillus crispatus LSA-573

Activated Lactobacillus crispatus LSA-573 was used as the experimental group, Lactobacillus germansii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 were used as the control group, respectively, in TMB-HRP medium. Underline the line and incubate anaerobically for 48 hours at 37°C in a three-gas incubator. Take out the plate, expose the bacteria to the air, and observe the color change of the colonies. Finally, according to the time of color change, the ability of the strain to produce hydrogen peroxide is judged as strong (change time <10 min), medium (change time 10 to 20 min), weak (change time 20 to 30 min) or none (change time >30min or no discoloration).

The measurement results are as follows:

Table 3 Results of semi-quantitative hydrogen peroxide production capacity determination of Lactobacillus crispatus LSA-573 and control strains

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

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 minutes, Lactobacillus crispatus LSA-573 began to change color, and the colonies were slightly blue. At 3 to 6 minutes, the blue color of the colonies continued to deepen, and at 9 minutes, the colonies became obviously dark blue; Reuteri The colonies of Lactobacillus RC-14 appeared blue when exposed to air for 10 minutes, and the colonies showed obvious dark blue after 20 minutes; the colonies of Lactobacillus rhamnosus GR-1 were still milky white when exposed to air for 30 minutes; Lactobacillus germansii DM8909 Obvious dark blue colonies appeared after being exposed to air for 1 minute. Therefore, according to the judgment criteria of hydrogen peroxide production capacity, Lactobacillus crispatus LSA-573 has a strong hydrogen peroxide production capacity.

Example 3. Analysis of the antibacterial effect of Lactobacillus crispatus LSA-573 on pathogenic bacteria

Activated Lactobacillus crispatus LSA-573 was used as the experimental group, Lactobacillus germansii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 were used as the control group, and equal amounts of bacterial liquid were centrifuged. , discard the supernatant, resuspend the bacteria and adjust the turbidity of the bacterial solution to the appropriate turbidity (~10 ⁸ CFU/mL), take 0.5mL of Lactobacillus resuspension and culture it with 20mL of MRS agar cooled to 50~60℃ Mix the base, cool and solidify, and then culture anaerobically at 37°C for 24 hours, then punch holes to prepare bacterial cakes; centrifuge the activated uropathogenic Escherichia coli/Gardnerella vaginalis/Pseudomonas aeruginosa and Candida albicans, and discard Clear, resuspend the bacterial cells, adjust the turbidity to the appropriate turbidity (~10 ⁸ CFU/mL), take 100 μL of bacterial suspension and resuspend it in nutrient agar solid culture medium, use glass beads to evenly spread; The cake is placed on an agar plate coated with pathogenic bacteria and incubated anaerobically at 37°C for 48 hours. If an inhibition zone appears, use a vernier caliper to measure the diameter of the inhibition zone.

Table 4 Inhibitory zone diameter (mm) of Lactobacillus crispatus LSA-573 and control bacteria against pathogenic bacteria

"-" means it has no bacteriostatic effect on pathogenic bacteria.

The antibacterial results of Lactobacillus crispatus LSA-573 and control bacteria against pathogenic bacteria are shown in Table 4. From the table, it can be seen that Lactobacillus crispatus LSA-573 is effective against uropathogenic Escherichia coli, Gardnerella vaginalis and aeruginosa. The antibacterial ability of Pseudomonas is higher than that of Lactobacillus germansii DM8909 and Lactobacillus reuteri RC-14, and it can significantly inhibit Candida albicans that cannot be inhibited by other lactobacilli.

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

1. Analysis of the adhesion effect of LSA-573 strain on cervical cancer cell Hela

HeLa cells are derived from women's cervical cancer cells, proliferate rapidly, and are often used as surrogate cells for vaginal epithelial cells for in vitro research. The stronger the adhesion of probiotics to epithelial cells, the more likely they are to colonize the human body, compete with pathogenic bacteria for space, prevent the invasion of pathogenic bacteria, and are more conducive to their probiotic functions.

Cultivate Hela cells (purchased from the Cell Bank of the Chinese Academy of Sciences) in T75 cell culture flasks until the cell coverage rate is about 80%. Digest with 3 mL of 0.25% trypsin-EDTA solution for 2 min and 30 s. Add 3 mL of 10% FBS and 89% DMEM. Use sugar culture medium and a mixture containing 1% penicillin and streptomycin. Gently pipet to remove the cells from the culture bottle. The cells are seeded into 2 mL of a 6-well plate at a density of 4×10 ⁴ cells/mL. Single cells will be formed after 48 hours of culture. layer.

Activated Lactobacillus crispatus LSA-573 was used as the experimental group, Lactobacillus germansii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 were used as the control group, and the lactobacilli in each group were cultured. After centrifuging the liquid to remove the supernatant, resuspend the precipitated bacterial cells in PBS solution, adjust the turbidity of the bacterial liquid to 1.0MCF, centrifuge to remove the supernatant, and resuspend the bacterial cells in an equal volume of DMEM high-sugar medium to obtain a bacterial suspension. The bacterial suspension was diluted and plated on MRS agar. After anaerobic culture for 48 hours, the number of colonies on each plate was counted.

Before the adhesion experiment, aspirate the culture medium in the cell culture plate, add 2 mL of PBS solution to each well to wash the cells 3 times, then add 2 mL of bacterial suspension, gently shake the cell culture plate to fully disperse the bacteria, and place the cell culture plate and The remaining bacterial suspension was placed in a 37°C, 5% _CO2 incubator for 2 hours, with 2 cells in each group.

After adhesion is completed, aspirate the culture medium from the cell culture plate, add 2 mL of PBS solution to each well, wash the cells 3 times, add 0.5 mL of 0.25% trypsin-EDTA solution for digestion for 2 min and 30 s, aspirate the digestion liquid, and then add 1 mL of PBS solution. Gently pipet the cells to detach them and add 1 mL of 0.05% TritonX-100 to lyse the cells to make a bacterial suspension. Gradually dilute it. Take 100 μL of bacterial liquid and spread it evenly on MRS agar; after 48 hours of anaerobic culture, count each plate. number of clones.

2. Analysis of the adhesion of LSA-573 strain to human bladder transitional cell carcinoma T24 cells

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%, digested with 3 mL of 0.25% trypsin-EDTA solution for 2 min and 30 s, and added 3 mL of 10% FBS, Use a mixture of 89% McCoy's 5a culture medium and 1% penicillin and streptomycin. Gently pipet to remove the cells from the culture bottle. The cells are seeded into 2 mL of a 6-well plate at a density of 4×10 ⁴ cells/mL. After culturing for 48 hours, Form a single cell layer.

Activated Lactobacillus crispatus LSA-573 was used as the experimental group, Lactobacillus germansii DM8909, Lactobacillus reuteri RC-14, and Lactobacillus rhamnosus GR-1 were used as the control group. The culture fluids of each group were centrifuged. After removing the supernatant, resuspend the precipitated bacterial cells in PBS solution, adjust the turbidity of the bacterial solution to 1.0MCF, centrifuge to remove the supernatant, and resuspend the bacterial cells with an equal volume of McCoy's 5a culture medium to obtain a bacterial suspension. Take the bacterial suspension. The solution was diluted and plated on MRS agar. After anaerobic incubation for 48 hours, the initial number of bacteria on each plate was counted.

Before the adhesion experiment, aspirate the culture medium in the cell culture plate, add 2 mL of PBS solution to each well to wash the cells 3 times, then add 2 mL of bacterial suspension, gently shake the cell culture plate to fully disperse the bacteria, and place the cell culture plate and The remaining bacterial suspension was placed in a 37°C, 5% CO ₂ incubator for 2 hours, and 2 parallel cells were made.

After adhesion, the culture medium in the cell culture plate was aspirated, and 2 mL of PBS solution was added to each well to wash the cells three times. Add 0.5 mL of 0.25% trypsin-EDTA solution to each well for digestion for 2 min and 30 s, aspirate the digestion liquid, add 1 mL of PBS solution and gently pipet the cells to detach them, add 1 mL of 0.05% TritonX-100 to lyse the cells, and prepare a bacterial suspension. After gradient dilution, take 100 μL of bacterial solution and spread it evenly on MRS agar; after 48 hours of anaerobic culture, count the number of viable bacteria after bacterial adhesion treatment on each plate.

The adhesion rate is calculated as follows:

Adhesion rate = strain survival rate (%) = N _l /N ₀ ×100%;

In the formula, N ₁ -the number of viable bacteria after strain adhesion treatment (CFU/mL); N ₀ -the number of viable bacteria before strain adhesion (CFU/mL).

Table 5 Cell adhesion results

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

The calculation results of the adhesion rate of LSA-573 strain to cervical cancer cells HeLa and human bladder transitional cell carcinoma T24 cells are shown in Table 5. From the table, it can be seen that Lactobacillus crispatus LSA-573 has a strong adhesion rate to human cervical epithelial cells. Hela cells and human bladder transitional cell carcinoma T24 cells have higher adhesion ability and better colonization ability.

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

Forty SPF grade female mice weighing 14-18g and 6-8 weeks old were selected and randomly divided into low-dose group, high-dose group and control group, with 10 mice in each group. Centrifuge the activated Lactobacillus crispatus LSA-573, discard the supernatant, and resuspend the bacteria in PBS solution to adjust to low turbidity (~10 ⁸ CFU/mL) and high turbidity (~10 ¹⁰ CFU/mL). All mice were given standard chow ad libitum. The mice in the low-dose group were perfused into the vagina with 20 μL of low-turbidity LSA-573 bacterial solution every day, the high-dose group were perfused with the same volume of high-turbidity LSA-573 bacterial solution every day, and the control group was perfused with the same volume of normal saline every day, for a total of After 14 days, the body weight and toxic reactions of the mice were observed.

One day before infusion and on the 14th day of infusion, use a micropipette to draw 50 μL of normal saline, repeatedly flush the vagina of each group of mice 5-6 times, and take 30 μL of the above-mentioned perfusion solution to form a suitable gradient (~10 ^-4 ) Lactobacilli were then counted on MRS agar.

Fourteen days after vaginal infusion of bacterial solution, mice in each group showed no abnormality. Compared with the control group, there was no abnormal weight, no signs of tremors, abnormal posture, exophthalmos, or death, and urination and breathing were normal. The results indicate that Lactobacillus crispatus LSA-573 is non-toxic.

Table 6 The number of Lactobacilli (CFU/mL) in mice in different experimental groups before and after vaginal infusion of Lactobacillus

The changes in the number of lactobacilli before and after vaginal infusion of bacterial solution in mice are shown in Table 6. The lactobacilli in the vagina of mice infused with low turbidity LSA-573 every day increased approximately 8-9 times, while the number of lactobacilli in the vagina of mice infused with high turbidity LSA-573 increased by about 8-9 times. Lactobacillus vaginalis increased approximately 60-fold in mice. This result indicates that Lactobacillus crispatus LSA-573 has the ability to colonize the vagina.

Example 6 Therapeutic ability of Lactobacillus crispatus LSA-573 on Gardnerella vaginalis model mice

Forty SPF-level female mice weighing 14-18g and 6-8 weeks old were selected and randomly divided into healthy group, infection group, metronidazole group and LSA-573 group, with 10 mice in each group. The definition of vaginal garde was established. The day of the Gardnerella vaginalis model mice is Day 1. The experimental plan for establishing the Gardnerella vaginalis model mice is shown in Figure 3. From Day 1 to Day 3, each mouse was injected with 0.5 mg of estradiol every day for estrogenization, and then 20 μL of Gardnerella vaginalis with an appropriate concentration (~10 ⁸ CFU/mL) was injected into the vagina of the mouse continuously. Four days after inoculation (Day 4 to Day 8), mice in the healthy group were injected with 20 μL of physiological saline instead of bacterial solution. On Day 8, use a sterile swab to dip a small amount of mucus from the mouse vagina, and stain it under a microscope to observe the presence of Gardnerella vaginalis to ensure the colonization of the mouse vagina with Gardnerella vaginalis and the Gardnerella vaginalis model. Establishment of mice.

Centrifuge the activated Lactobacillus crispatus LSA-573, discard the supernatant, and resuspend the bacteria in PBS solution to adjust to appropriate turbidity (~10 ⁹ CFU/mL). From the first 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 injected vaginally into the vagina of the mice in the LSA-573 group; metronidazole The mice in the group were injected vaginally with 20 μL of metronidazole solution; the mice in the healthy group and the infected group were given the same volume of normal saline vaginally every day.

After modeling, before administration, on the 1st day after administration, and on the 7th day after administration, use a microsampler to take 50 μL of normal saline, rinse the mouse vagina 5-6 times repeatedly, and take 30 μL of the above-mentioned lavage solution and release it into The appropriate gradient was then spread on agar media for enumeration of Lactobacilli and Gardnerella vaginalis. Lactobacillus agar was counted using MRS agar, and Gardnerella vaginalis was counted using Colombian agar supplemented with gentamicin sulfate (4 mg/L), nalidixic acid (30 mg/L), and amphotericin B (2 mg/L). Blood plate.

Table 7 The number of Lactobacilli (CFU/mL) in different experimental groups of mice before and after vaginal modeling administration

The results are shown in Table 7. It can be seen from Table 7 that on the first day after administration, compared with the infection group, the number of Gardnerella bacteria in the vagina of mice infused with Lactobacillus crispatus LSA-573 was significantly reduced by about 1. orders of magnitude, 2 orders of magnitude lower than that of the metronidazole group. 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 administration, the number of Gardnerella vaginalis in the vagina of the mice in the LSA-573 group was similar to that in the metronidazole group, but the number of Lactobacilli was about 3 orders of magnitude higher than that in the metronidazole group, and was in the healthy group. 10-15 times. The above results show that Lactobacillus crispatus LSA-573 has similar inhibitory ability to Gardnerella vaginalis as metronidazole, and can colonize the vagina of mice and restore the number of Lactobacilli in the vagina to normal levels and above. It shows that Lactobacillus crispatus LSA-573 has preventive and therapeutic effects on bacterial vaginosis.

The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various modifications can be made without departing from the purpose of the present invention. kind of change. In addition, the embodiments of the present invention and the features in the embodiments may be combined with each other without conflict.

Claims (10)

1. A bacterial strain, characterized in that the bacterial strain is Lactobacillus crispatus (Lactobacillus crispatus) LSA-573, which is deposited in the General Microbiology Center of the Chinese Microbial Culture Collection Committee. The deposit number is CGMCC No. 24781, and the deposit date is 2022. April 26th. 2. The strain according to claim 1, characterized in that the strain has the 16SrDNA sequence shown in SEQ ID NO: 1. 3. A product, characterized in that it includes one of (1) to (6): (1) The strain according to claim 1 or 2; (2) A bacterial agent containing the strain described in claim 1 or 2; (3) A viable bacterial solution containing the bacterial strain described in claim 1 or 2; (4) Dead bacterial liquid containing the bacterial strain according to claim 1 or 2; (5) Metabolites containing the strain described in claim 1 or 2; (6) An extract containing the bacterial strain according to claim 1 or 2. 4. The product according to claim 3, characterized in that the product is medicine, medical equipment, food or hygiene products; preferably, the hygiene products are tampons, sanitary napkins, menstrual pads, diapers, soap and At least one type of condom. 5. The product according to claim 4, wherein the medicine further includes a pharmaceutical carrier and/or pharmaceutical excipients; preferably, the pharmaceutical carrier includes diluents, excipients, fillers, adhesives, At least one of an agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent. 6. The product according to claim 3, characterized in that the form of the product is one of powder, ointment, gel, drop, suppository, lozenge, granule, capsule, spray, tablet, pill and solution. kind; Preferably, the capsule is a capsule. 7. Use of the bacterial strain described in claim 1 or 2, or the product described in any one of claims 3-6, in the preparation of antibacterial products. 8. Application according to claim 7, characterized in that the pathogenic bacteria inhibited by the antibacterial product are uropathogenic Escherichia coli, Gardnerella vaginalis, Pseudomonas aeruginosa and Candida albicans. at least one species of bacteria. 9. The use of the bacterial strain according to claim 1 or 2, or the product according to any one of claims 3 to 6, in the preparation of medicines for treating or preventing genitourinary tract infections, or vaginal care products. 10. Use of the strain described in claim 1 or 2, or the product described in any one of claims 3-6, in the preparation of drug delivery carriers.