WO2021027742A1

WO2021027742A1 - Lactobacillus crispatus and application thereof

Info

Publication number: WO2021027742A1
Application number: PCT/CN2020/107919
Authority: WO
Inventors: 承磊; 刘瑶; 王琼; 曾婉秋
Original assignee: 四川厌氧生物科技有限责任公司
Priority date: 2019-08-09
Filing date: 2020-08-07
Publication date: 2021-02-18
Also published as: CN110982726A; CN110982726B

Abstract

Provided is a Lactobacillus crispatus Lcris-2, which was preserved in the China Center for Type Culture Collection in June 2019 with the preservation number: CCTCC No. M2019427. The strain belongs to the dominant strain of women's vaginas in China, and may be used in the preparation of vaginal microecological preparations and health care products.

Description

Lactobacillus crispatus and its application

Technical field

The invention relates to the field of microorganisms, in particular to Lactobacillus crispatus and its applications.

Background technique

From the publication of the journal "Research Progress and Clinical Significance of Vaginal Microecology" published in the journal Practical Gynecology, and the document "Research Progress of Lactobacillus in the Vaginal Microecology" published in the journal Chinese Journal of Microecology, we can see that There are more than 300 kinds of microorganisms symbiosis in the human vagina. They restrict and balance each other to form a dynamic balance. The occurrence of various vaginitis is related to the imbalance of the vaginal microecological environment.

A healthy female vagina of childbearing age is a microenvironment with Lactobacillus as the dominant flora. They can produce lactic acid, H ₂ O ₂ , and bacteriocins, and competitively adhere to the vaginal epithelium, occupy binding sites, and consume intravaginal nutrition. Its dominant position in the vagina inhibits the excessive proliferation of other bacteria. Research over the past ten years has found that human vaginal lactobacilli are very diverse. There are more than 20 types of lactobacilli isolated from the vaginal microenvironment, and the types of lactobacilli in different regions and populations are different. In North America, the female vaginal ecological flora can be divided into 5 types, 4 of which are Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus jensenii. (Lactobacillus gasseri) is the dominant bacteria, the fifth species is other lactic acid bacteria and anaerobic bacteria; in Europe, the female vaginal ecological flora is mainly Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jannaschii, Lactobacillus johnseri, etc. Dominant flora; in China, the most common dominant lactobacilli in the vagina of healthy women are Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jannaschii, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus reuteri, and acidophilus Lactobacillus, Lactobacillus inert, etc. It can be seen that although the diversity of lactobacilli in the human vagina is very rich, Lactobacillus crispatus is one of the most common vaginal dominant lactobacilli.

When environmental factors or external interventions break the vaginal microecological balance, the vaginal microecological environment will enter a fragile state, which is not easy to resist the reproduction and invasion of pathogenic bacteria, and various vaginal inflammations occur. Bacterial vaginosis (bacterial vaginosis, hereinafter referred to as "BV") is a common gynecological disease with an infection rate of 15%-52%. It is based on excessive reproduction of Gardnerella vaginalis and other anaerobic bacteria instead of milk. Bacillus, a vaginal infectious disease that causes the clinical symptoms of the vaginal flora to be imbalanced. According to data, BV is the cause of tissue choriodritis, amniotic fluid infection, post-cesarean endometritis, and other poor pregnancy and pregnancy complications Risk factors for disease.

For BV, the clinical treatment method is to use metronidazole or clindamycin. Metronidazole is a prodrug. In an anaerobic environment, bacterial intracellular enzymatic reduction reduces the nitro group of metronidazole to an amino group. Thereby converting the antibiotic into an active form, and then by covalently binding with DNA to destroy its helical structure and cause single-strand and double-strand breaks, thereby degrading DNA and death of pathogens; Clindamycin can interact with the 50S ribosome on the bacterial ribosome The combination of subunits prevents the extension of the peptide chain, thereby inhibiting the protein synthesis of bacterial cells and causing the death of bacteria. Although antibiotic treatment is effective, it also has great shortcomings. It has the following two aspects: (1) It has an inhibitory effect on all antibiotic-sensitive microorganisms in the vaginal microenvironment. Therefore, after treatment, the vaginal microecology has not recovered to a healthy, A balanced state that can resist the invasion of pathogenic bacteria. The inhibited or killed pathogenic microorganisms or foreign pathogenic microorganisms will reproduce or even cause disease and relapse or new vaginal inflammation; (2) The microorganisms develop drug resistance and antibiotics The inability to balance the vaginal microecological environment results in refractory BV. Therefore, although antibiotic treatment is effective, the recurrence rate is high, and the recurrence rate is as high as 30% within 3 months.

The treatment of vaginal microecological imbalance includes three steps: sterilization, mucosal repair, and restoration of vaginal microecological balance. Sterilization is the first step in the treatment of vaginal inflammation. It inhibits or kills pathogenic microorganisms, including over-proliferation of aerobic and anaerobes, spores or hyphae, trichomonas, etc. After the pathogenic microorganisms are inhibited or killed, the immune repair of the vaginal mucosa and the recovery of dominant lactobacilli are the ultimate goals of treating vaginal inflammation. During this period, if the repair of the vaginal mucosa and the recovery process of Lactobacillus are affected, and the physical and chemical environment in the vagina does not return to normal, the inhibited pathogenic microorganisms or foreign pathogenic microorganisms will reproduce and even cause disease and relapse or new ones. Inflammation of the vagina. Probiotics can quickly occupy the receptors of the vaginal epithelium in the vagina and produce a protective effect on the vagina, thereby promoting the vagina to return to the normal microenvironment and reducing the recurrence of vaginal inflammation. Therefore, the use of probiotic microecological preparations to treat BV is the most preferred way in view of current technical means.

At present, there are only two types of vaginal microecological preparations on the market in China. One is a commercial drug produced by Xi’an Zhenghao Biopharmaceutical Co., Ltd. under the trade name "Yanhua", which contains a type of Streptococcus faecalis. It is not a vaginal dominant strain, and the bacteria under this species are conditionally pathogenic; the other is a commercial drug produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. under the trade name "Wanze Shuangqi", which contains 1 milk Bacillus-Lactobacillus delbrueckii (Lactobacillus delbrueckii), this strain does not belong to the dominant vaginal strain of women in China.

Therefore, searching for the dominant bacterial strains of Chinese women's vagina with strong probiotics is beneficial to the treatment of bacterial vaginosis. Lactobacillus crispatus is one of the dominant strains of female vaginas in my country. There are many studies on Lactobacillus crispatus isolated and screened from healthy women. For example, Chinese patent document 201811488762.5 discloses a Lactobacillus crispatus preparation and its application, such as Chinese patent document 201310002837.5 discloses a Lactobacillus crispatus strain and its applications. For example, Chinese Patent Document 201710935239.1 discloses a Lactobacillus crispatus and its applications. Lactic acid is one of the main antibacterial substances for Lactobacillus to inhibit the growth of pathogenic bacteria. In the prior art, these Lactobacillus crispatus screened from the vagina of healthy women are not high enough to produce lactic acid.

Summary of the invention

The purpose of the present invention is to provide an application technology of Lactobacillus crispatus Lcris-2 (Lactobacillus crispatus Lcris-2), which utilizes and belongs to the dominant strain of the female vagina in my country, and can be used in vaginal microecological preparations by using the dominant strain. Lactobacillus crispatus Lcris-2 was deposited in the China Type Culture Collection on June 4, 2019. The collection number of this strain collection is: CCTCC No: M2019427, Address: Wuhan University, Wuhan City, Hubei Province, Zip Code: 430072, Tel: 027-68754052.

The inventors obtained a Lactobacillus strain from the vagina of healthy women in my country, which proved that the Lactobacillus strain has superior lactic acid production ability and cell adhesion ability, can inhibit the growth of various pathogenic bacteria and produce hydrogen peroxide. This strain constitutes the first aspect of the present invention.

Because the strain is a dominant strain of women's vagina in my country, it can be used in women's health care products or drugs for treating vaginitis, which constitutes the second aspect of the present invention. Women's health care products refer to health care products for external use.

The third aspect of the present invention is to provide an inoculum containing Lactobacillus crispatus Lcris-2 as an active ingredient. The inoculum can be a bacterial suspension or a freeze-dried bacterial powder.

Lactobacillus crispatus Lcris-2 can also be used in the preparation of external genital hygiene products, such as sanitary napkins, tampons or sanitary care solutions for external genitalia.

Lactobacillus crispatus Lcris-2 can also be used in the preparation of medicines or health care products that regulate the balance of vaginal flora.

Lactobacillus crispatus Lcris-2 can also be used in the preparation of drugs or health care products with vaginal epithelial cell adhesion function.

Lactobacillus crispatus Lcris-2 can also be used in the preparation of medicines or health care products for the prevention and treatment of vaginal pathogens. Pathogenic bacteria include, but are not limited to, any one or more of Gardnerella Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli ability, Salmonella paratyphi B ability, and Shigella dysenteriae.

Lactobacillus crispatus Lcris-2 can also be used in external care products for infants delivered by caesarean section. Because babies born by caesarean section do not pass through the female vagina, no exogenous probiotics are obtained during the production process. Therefore, the probiotics screened in the female vagina can be made into external care products for the baby's body to smear or bathe.

The sequence of Lactobacillus crispatus Lcris-216SrRNA gene is shown in the sequence table.

After uploading the whole genome sequence of Lactobacillus crispatus Lcris-2 to EzBiocloud, comparing it with the whole genome sequence that can be linked to all the same species, the homology ratio (ANI) obtained is shown in Table 1 below, which proves It is a new strain of Lactobacillus.

Table 1: Comparison of Lcris-2 and different Lactobacillus crispatus whole genome ANI

The beneficial effects of the present invention are:

The selected Lactobacillus crispatus is genetically stable and has excellent bacteriostatic ability against Staphylococcus aureus, Pseudomonas agrobacterium, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae, and has good lactic acid production. Ability and ability to adhere to Hela cells.

Description of the drawings

Figure 1 is a frontal photo of the colony morphology of Lactobacillus crispatus Lcris-2.

Fig. 2 is an observation picture of Lactobacillus crispatus Lcris-2 Gram stain.

Figure 3 is a scanning electron microscope observation picture of Lactobacillus crispatus Lcris-2.

detailed description

The specific embodiments of the present invention will be described in detail below. The embodiments of the present invention include but are not limited to the following examples. In order to avoid too many unnecessary details, the well-known structures or functions will not be described in detail in the following embodiments. Except for definitions, the technical and scientific terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified.

The bacterial culture medium components and preparation methods used in the following examples are as follows:

Preparation of MRS broth: Weigh 52.0g of MRS product culture medium powder and dissolve it in 1L of distilled water; heat to boil, cool to room temperature and add 0.55g of cysteine hydrochloride, stir to dissolve and adjust the pH to 6.5; load quantitatively Dispense the dispenser with N ₂ , heat it to boiling, boil for 20 minutes in a slightly boiling state, and dispense into 10 mL anaerobic tubes after cooling, sterilize at 118°C with high temperature and humidity for 20 minutes, store in the shade, avoid light, and reserve.

Preparation of MRS solid medium: weigh 52.0g of MRS finished medium powder and 15.0g of agar powder, dissolve in 1L of distilled water, heat to boil, add 0.55g of cysteine hydrochloride after boiling, adjust the pH to 6.5,118 Sterilize at high temperature, humidity and heat for 20 minutes, store in a cool, dark place for later use.

Hydrogen peroxide semi-quantitative medium preparation: Weigh 52.0g of MRS finished medium powder and 15.0g of agar powder, dissolve in 1L distilled water, adjust pH to 6.5, sterilize at 118℃ high temperature and humidity for 20min, put it in after sterilization Incubate the 50℃ water bath for 30 minutes, add TMB (to make the final concentration of TMB be 0.25mg/mL) and HRP (to make the final concentration of HRP be 0.01mg/mL) and mix well; after cooling and solidification, mark the medium name and preparation date, and put Keep in refrigerator at 4℃ for later use.

The preparation of anaerobic PBS: weigh 0.27g potassium dihydrogen phosphate, 1.42g disodium hydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, dissolve in 1L of distilled water, heat to boil, cool to room temperature and add 0.55g half Cystine hydrochloride, stir to dissolve, adjust the pH value to 6.5, install a quantitative dispenser and pass N ₂ , heat to boiling, boil for 30 minutes at a slight boiling state, and then dispensed into a 10 mL anaerobic tube after cooling. Sterilize at 121°C for 30min under high temperature, humidity and heat. Store in a cool, dark place for later use.

Anaerobic BHI liquid medium preparation: Weigh 37.0g of BHI finished medium powder, dissolve it in 1L of distilled water, heat to boil, cool to room temperature, add 0.55g of cysteine hydrochloride, stir to dissolve and adjust the pH to 6.5, Install a quantitative dispenser and pass N ₂ , heat to boiling, boil for 20 minutes in a slightly boiling state, pass in N ₂ and CO ₂ (1:1 ratio) during cooling and dispensing, and dispense to 10 mL anaerobic tubes Sterilize at high temperature and humidity at 118°C for 20 minutes, store in a cool, dark place for later use.

Anaerobic BHI semi-solid medium preparation: weigh 37.0g of BHI finished medium powder and dissolve in 1L distilled water; heat to boil, cool to room temperature, add 6g agar powder and 0.55g cysteine hydrochloride, stir to dissolve and adjust When the pH value reaches 6.5, install a quantitative dispenser and pass N ₂ , heat to boiling, boil for 20 minutes, cool slightly, and pass in N ₂ and CO ₂ (1:1 ratio) during the cooling and dispensing process , Timely aliquot into 10mL anaerobic tubes, sterilize at 118℃ high temperature and humidity for 20min, store in a cool, dark place.

Preparation of nutrient broth liquid medium: weigh 10g peptone, 3g beef powder, 5g sodium chloride and dissolve into 1L distilled water, adjust the pH to 7.2, heat and boil, cool to room temperature and then aliquot, each 10mL; 121℃ high temperature and humidity Sterilize for 15 minutes, store in a cool, dark place for later use.

Preparation of nutrient broth solid medium: weigh 10g peptone, 3g beef powder, 5g sodium chloride, 6g agar powder and dissolve it in 1L distilled water, adjust the pH to 7.2, cool it slightly, and timely aliquot it into a 10mL anaerobic tube; Sterilize at high temperature, humidity and heat for 15 minutes, store in a cool, dark place for later use.

Example 1

The method for separating Lactobacillus crispatus of the present invention includes the following steps:

Use vaginal cotton swabs to collect samples of vaginal secretions from Chinese women aged 20-40 who have passed the health examination; take 2 mL of sterile, oxygen-free PBS buffer in an anaerobic tube containing the cotton swabs, shake and mix thoroughly, And use it as the original solution for continuous 10-fold gradient dilution; take 100 μL of the 10,000-fold diluted liquid and spread it on the MRS solid medium and place it in an anaerobic incubator at 37°C. After culturing for 48 hours, pick a single colony of suspected Lactobacillus in MRS Cultivate in broth medium for 24 hours, and transfer a part of the bacterial solution obtained after culture to continue the culture, and the other part of the bacterial solution is subjected to bacterial DNA extraction; bacterial 16S rRNA amplification and sequencing, and BLAST comparison of the sequencing results, according to the comparison Results The strains were analyzed and stored separately, and a total of 1,336 strains of Lactobacillus were obtained. Through the low pH culture environment vitality test, 16S rRNA genotype screening, Gardnerella vaginalis test, lactic acid production test, hydrogen peroxide production test, and Hela cell adhesion test for the above-mentioned 1336 strains of Lactobacillus deposited above, we obtained The best-performing strain of Lactobacillus crispatus was named Lcris-2.

The screening method of Lactobacillus crispatus of the present invention includes the following steps:

1. Vitality test in low pH culture environment

1.1 Activation: The preservation of Lactobacillus is activated in MRS broth with pH 6.5 and cultured overnight at 37°C;

1.2 Transfer: transfer the activated bacterial solution to the MRS broth culture with pH 4-5, and measure the OD ₆₀₀ value every 2-3 h;

1.3 Compare the OD ₆₀₀ value and screen strains from different samples that can proliferate rapidly or have a higher OD ₆₀₀ value.

2. 16S rRNA genotype screening

Lactobacillus of the same species isolated from the same sample, if the 16S rRNA sequence is different, indicating that their genotypes are different, their physiological characteristics may also be different. Therefore, 16S rRNA was performed on the lactobacilli selected by the low pH culture vigor comparison Genotype screening.

3. Inhibition of Gardnerella vaginal test

After the Lactobacillus is activated, take 0.1 mL of the bacterial solution and mix it with the MRS solid medium, pour it into a 6cm plate, culture it at 37°C for 48h after complete solidification, take out the plate, and punch a hole on the agar medium with a hole punch with an inner diameter of 6mm , To obtain the bacterial cake; after the activation and transfer of Gardnerella vaginalis, use anaerobic sterile PBS buffer to dilute the Gardnerella bacterial solution to 100 times with a 10-fold gradient, and take 10 ^-1 and 10 ^{-2 respectively} Mix 0.5 mL of the diluted solution with 5.25 mL of BHI solid medium containing 5% horse serum, pour it into a 9cm plate, and after it has completely solidified, place the lactic acid bacteria cake lightly on the surface of the BHI agar, and place 4 bacterial cakes symmetrically on each plate. Two bacteria per strain are in parallel, and the plate is placed in an anaerobic sealed tank with an anaerobic gas generating bag, incubated at 37°C for 48 hours, and the size of the inhibition zone is measured with a vernier caliper.

4. Lactic acid production test

After activation, the Lactobacillus was transferred to the MRS broth medium, and the two were cultured in parallel at 37°C for 48 hours. The pH value of the Lactobacillus broth cultured for 48 hours was measured and recorded with a pH 0.5-5.0 test paper, according to the following two Condition selection of strains for liquid chromatography: Condition 1, the pH value of 2.5 for liquid chromatography; Condition 2, the same species of Lactobacillus, select the lower pH strain for liquid chromatography; determine the liquid chromatography sample Afterwards, the supernatant was diluted 5 times, concentrated sulfuric acid was added for pre-treatment, and filtered with a 0.22um syringe filter before loading. The relevant parameters of liquid chromatography are as follows:

Instrument model: Agilent, analytical liquid chromatography 1200

Column model: Bole, Aminex HPX-87H

Mobile phase: 0.005M H ₂ SO ₄ , speed 0.6mL/min

Detector and detection wavelength: DAD, 207nm; RID, refractive index signal

Injection volume: 20μL.

5. Ability to produce hydrogen peroxide

After the Lactobacillus has been activated, use a pipette to pipette 2μL of bacterial solution and spot it in a 0.25mg/mL 3,3',5,5'-tetramethylbenzidine solution and 0.01mg/mL horseradish peroxide In the enzyme MRS agar, set two parallels at 24h, 48h and 72h observation time points, and place the plates at the same observation time point in the same anaerobic sealed tank with anaerobic gas generating bags, and incubate at 37°C. After the corresponding time, take out the corresponding plate, expose it to the air, observe the color reaction after 30 minutes and take a photo to record: Take Lactobacillus delbrueckii as the positive control, the blue depth produced by Lactobacillus bideti is counted as 4 points. The blue color produced by Lactobacillus spp. is equivalent to 3 points, the light blue color produced by Lactobacillus Biederi is scored as 2 points, the blue color is very weak, 1 point (slight color reaction), and 0 points are not changed. .

6. Adhesion Hela cell test

After the activation of the Lactobacillus, the cells were washed twice by centrifugation, resuspended in PBS, and 100μL of the Lactobacillus suspension was sucked into a 96-well cell culture plate containing Hela cells, incubated at 37°C for 30 minutes, and washed twice with sterile PBS after 30 minutes To remove the adherent Lactobacillus, add 25μL of trypsin solution to each well of a 96-well cell culture plate containing Hela cells, and place the cells in a 37℃ incubator to digest the cells. After the Hela cells are digested and become spherical, add 75μL to each well for complete culture After complete digestion, draw 20 microliters of bacterial suspension, dilute with sterile PBS 10-fold gradient, select appropriate dilution gradient for pour counting experiment, culture at 37°C for 48h and count.

The probiotic properties of Lactobacillus crispatus Lcris-2 of the present invention are shown in Table 2 below after frequent screening.

Table 2 The probiotic properties of Lactobacillus crispatus Lcris-2

Example 2

The biochemical identification results of Lactobacillus crispatus Lcris-2 are as follows:

Passed the methyl red test (MR test), acetyl methyl methanol test (VP test), indigo matrix test, esculin hydrolysis test, triose iron test, Krebs disaccharide iron test, urease test, phenylalanine Deaminase test, amino acid decarboxylase test, gelatin liquefaction test, sodium malonate test, citrate test (citrate test), nitrate reduction test, litmus milk test, bacterial motility test, using French Mérieux The API 50 CHL Lactobacillus identification system produced by the company conducts biochemical identification of strains. The specific results are as follows:

Lactobacillus crispatus Lcris-2 can hydrolyze esculin to produce glucose and esculin. A negative MR test indicates that the organic acid produced by metabolizing glucose is not enough to change the color of the color reagent. A negative VP test indicates that metabolizing glucose does not produce pyruvate and indigo. The result of the matrix test is that the bacteria does not decompose tryptophan in peptone and produces indole and triose iron. The test shows that it metabolizes glucose, lactose, sucrose, and does not produce hydrogen sulfide. The Krishna disaccharide iron test shows that it metabolizes and fermented glucose, lactose, and does not produce hydrogen sulfide. , Urease test, phenylalanine deaminase test, amino acid decarboxylase test, gelatin liquefaction test are all negative, indicating that the bacteria does not produce urease, phenylalanine deaminase, amino acid decarboxylase, gelatinase, C The di-sodium test, citrate test (citrate test), and nitrate reduction test were all negative, indicating that the bacteria does not use sodium malonate as a carbon source and citrate as a nitrogen source and carbon source , Does not reduce nitrate to nitrite. The litmus milk test found that the bacteria can ferment milk but does not coagulate, indicating that the bacteria grows vigorously, does not produce rennet, and the bacterial motility test is negative; Lactobacillus crispatus can ferment half Lactose, salicin, glucose, fructose, N-acetyl-glucosamine, horse leaf spirit, cellobiose, maltose, sucrose, trehalose, starch, non-fermentable, glycerin, erythritol, D-arabinose, mannose Sugar, L-arabinose, α-methyl-D-glucoside, amygdalin, arbutin, glycogen, ribose, D-xylose, L-xylose, adonol, β-methyl-D -Xylose, sorbose, rhamnose, weidrol, inositol, mannitol, sorbitol, α-methyl-D-mannose, melibiose, inulin, melezitose, xylitol , D-turbiose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-keto-gluconate, 5-keto-gluconate, lactose, D-tagatose, raffinose, gentiobiose. .

Example 3

Antibiotic sensitivity test of Lactobacillus crispatus Lcris-2.

In accordance with the requirements of the antibiotic susceptibility test in the 2015 edition of the Pharmacopoeia, the third part of the general microecological living bacteria products, the agar diffusion disc method is used to determine the susceptibility of strains to antibiotics, and the sensitivity of the strains to antibiotics is judged according to the size of the inhibition zone.

Activation and streaking: Lactobacillus crispatus Lcris-2 is activated in MRS broth medium, E. coli is activated in nutrient broth and cultured at 37°C; after activation of lactic acid bacteria, pick a loop of bacteria liquid and streak on MRS solid medium Put the plate into an anaerobic sealed tank with an anaerobic gas production bag, and cultivate at 37°C; after the E. coli is activated, pick a ring of bacteria liquid and mark it on the nutrient broth solid medium, and place the plate Put it into a sealed tank and incubate at 37°C.

Pick a few single colonies from the cultured agar plate and directly inoculate them into physiological saline to make a bacterial suspension. Adjust the turbidity of the suspension to reach 0.5 McDonnell’s concentration. After adjusting the turbidity of the suspension, it’s best to be within 15 minutes Dip a sterile cotton swab into the suspension inside, and rotate the swab several times over the liquid against the inner wall of the test tube to remove excess liquid on the swab.

Streak the entire agar surface with a swab and inoculate it on a dry surface plate, inoculate the suspension of Lactobacillus crispatus Lcris-2 on the MRS agar plate, and inoculate the E. coli suspension on the MH agar plate and the MRS agar plate respectively; after the inoculation is completed , Use the drug sensitive paper dispenser to distribute and place the drug sensitive paper on the plates. Each plate is placed 6 drug sensitive papers in 3 parallel. There are a total of 12 drug sensitive papers (all OXOID) in the experiment. The information is as follows:

Table 3 Inhibitory ability of antibiotics on Lactobacillus crispatus Lcris-2

result:

(1) Lactobacillus crispatus Lcris-2 is sensitive to five antibiotics: ampicillin, erythromycin, imipenem, piperacillin/tazobactam and ceftizoxime;

(2) Lactobacillus crispatus Lcris-2 is resistant to four antibiotics: nystatin, metronidazole, fluconazole and gentamicin;

(3) Lactobacillus crispatus Lcris-2 is sensitive to vancomycin;

(4) Lactobacillus crispatus Lcris-2 is sensitive to clindamycin;

(5) Lactobacillus crispatus Lcris-2 is resistant to norfloxacin.

Example 4

Test of the ability of Lactobacillus crispatus Lcris-2 to inhibit Staphylococcus aureus, inhibit Pseudomonas aeruginosa, inhibit Escherichia coli, inhibit Salmonella paratyphi B and inhibit Shigella dysentery.

After the activation of Lactobacillus crispatus Lcris-2, take 0.1 mL of the bacterial solution and MRS solid medium and mix it, pour it into a 6 cm plate, culture it at 37°C for 48 hours after complete solidification, take out the plate, and use a hole punch with an inner diameter of 6 mm to culture on agar Punch holes on the base to obtain a bacterial cake; Staphylococcus aureus, Pseudomonas agrobacterium, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae are activated and transferred with 0.9% saline at a 10-fold gradient. Dilute the pathogenic bacteria liquid to 100 times, take 0.5 mL of the 10 ^-1 and 10 ^-2 dilutions and 5 mL of nutrient agar solid medium and mix them, pour them into a 9 cm plate, and place the lactic acid bacteria cake lightly on the nutrition after it has completely solidified. On the agar surface, place 4 bacterial cakes on each dish symmetrically, 2 bacteria per strain in parallel, put the dish upright in a sealed jar, incubate at 37°C for 24 hours, measure the size of the inhibition zone with a vernier caliper.

The results are shown in the following table:

Example 5

Bacterial genomic DNA was extracted from the 0th generation (T0) and 30th generation (T30) of Lactobacillus crispatus Lcris-2, and primer set 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACCTTGTTACGACTT-3') ) Perform PCR and sequence the PCR amplified products of T0 and T30. The sequencing results of T0 and T30 are consistent, indicating genetic stability.

Example 6 Toxicity test

Test product: Lactobacillus crispatus Lcris-2 live bacteria capsules: low dose: 2.0*10 ⁹ CFU/capsule; high dose: 1.0*10 ¹⁰ CFU/capsule.

Route of administration: vaginal administration

Dosing frequency and duration: 1 dose on DAY1

Administration method: the blank control group was given blank capsules, the low-dose and high-dose groups of the test product were given corresponding specifications of the test product capsules, each of which was given 1 capsule vaginally.

Reasons for choosing the route of administration: To simulate the planned route of administration in clinical practice, vaginal administration is used.

Observation frequency and time: After administration of animals in groups 1 to 3, observe the acute toxicity reaction at the cage side for at least 4 hours. For animals without obvious abnormal reactions, the observation shall be terminated after 4 hours; for animals with obvious abnormalities, detailed clinical practice should be performed Observation, the end time of the day's observation is determined by the person in charge of the topic.

Detailed clinical observation: When animals in groups 1 to 3 are received, they will be taken out of the cage once a week after the start of administration for detailed clinical observation. The frequency of observation can be increased as needed. The detailed clinical observation period may not be recorded for general clinical observation. Observations include but are not limited to behavioral activities, skin, coat, eyes, ears, nose, abdomen, external genitalia, anus, limbs, feet, and breathing.

Weight: After the animals are received, before grouping, and once a week after medicine, all animals are also weighed before they are found dead or dying.

Food intake: The food intake (additional amount/remaining amount) of animals in groups 1 to 3 is measured once a week after administration, and it is expressed in the form of "g/head/day". Before the animal is fasted overnight, the amount of feed remaining in the cage is measured.

As a result, there were no obvious abnormalities in clinical observation, body weight, and food intake, which proved non-toxic.

According to the above-mentioned embodiments, the present invention can be well realized. It is worth noting that, based on the above-mentioned structural design, in order to solve the same technical problem, even if some insubstantial changes or polishes are made in the present invention, the essence of the technical solution adopted is still the same as that of the present invention. Therefore, it should also fall within the protection scope of the present invention.

Claims

A Lactobacillus crispatus Lcris-2 is characterized in that its deposit number is: CCTCC No. M2019427.
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of a medicine for preventing or treating vaginitis.
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of a health care product for women for preventing vaginitis.
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of female external genital hygiene products.
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of medicines or health care products for regulating the balance of vaginal flora.
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of drugs or health care products with vaginal epithelial cell adhesion function
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of health care products and medicines for preventing or treating vaginal pathogens.
The use according to claim 7, wherein the pathogenic bacteria include but are not limited to Gardnerella vaginalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shiga dysentery Any one or more of bacteria.
The use of Lactobacillus crispatus Lcris-2 according to claim 1 in the preparation of external care products for infants delivered by caesarean section.