Lactobacillus crispatus MYT-LC01 and application

Abstract

The invention discloses a lactobacillus crispatus MYT-LC01 and an application of the lactobacillus crispatus MYT-LC01. The preservation number of the Lactobacillus crispatus MYT-LC01 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) NO: 32209, and the preservation number of the Lactobacillus crispatus MYT-LC01 provided by the invention is CGMCC NO: 32209. The 16S rDNA (ribosomal deoxyribonucleic acid) sequence of the lactobacillus crispatus MYT-LC01 is as shown in SEQ ID NO: 1. The lactobacillus crispatus MYT-LC01 has an antagonistic effect on vagina-related harmful bacteria (candida albicans, staphylococcus aureus and escherichia coli), and can be used for inhibiting the occurrence of vulvovaginal candidiasis, vulvofolliculitis, bacterial vaginitis, cervicitis and urinary tract infection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Lactobacillus crispatus MYT-LC01 and an application thereof. Background Art

[0002] The vaginal microbiota of healthy women is dominated by lactobacilli, which play a key role in maintaining the balance of vaginal microecology. Among Chinese women, common dominant lactobacilli include Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii. Lactobacillus protects vaginal health through multiple mechanisms:

[0003] (1) Producing lactic acid: Lactobacillus can use glycogen in the vagina to produce lactic acid, maintaining a low pH environment in the vagina (usually between 3.8 and 4.5); this low pH environment is not conducive to the growth of most pathogenic bacteria, thereby effectively preventing the invasion and reproduction of pathogenic bacteria;

[0004] (2) Produce antibacterial substances: Lactobacillus can also produce antibacterial substances such as hydrogen peroxide and bacteriocins, which can directly inhibit or kill pathogens and further enhance the defense ability of the vagina;

[0005] (3) Competitive exclusion: Lactobacillus competitively adsorbs epithelial cells, preventing the adhesion and colonization of other microorganisms, thereby reducing the chance of infection by pathogens; this competitive exclusion mechanism helps maintain the stability of the vaginal microbiome;

[0006] (4) Immune regulation: Lactobacillus can also regulate the host's immune response, enhance local immunity, and resist the invasion of pathogens.

[0007] However, due to various factors (such as antibiotic use, hormone changes, bad living habits, etc.), the balance of vaginal microecology may be disrupted, resulting in a decrease in the number of lactobacilli, thereby increasing the risk of gynecological diseases such as vulvovaginal candidiasis, bacterial vaginosis, candidal infection, etc. Therefore, it is of great clinical significance to develop a product that can effectively supplement and restore the balance of vaginal lactobacilli. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art, and a lactobacillus crispatus MYT-LC01 and application thereof are provided. The present invention finds that vaginal lactobacillus crispatus MYT-LC01 has the best antibacterial effect through probiotic separation and antagonism detection experiments, and further finds that vaginal lactobacillus crispatus MYT-LC01 has an antagonistic effect on vaginal-related harmful bacteria (Candida albicans, Staphylococcus aureus, Escherichia coli) through strain identification, so that gynecological diseases caused by the above-mentioned strains can be treated or prevented, thereby inhibiting the occurrence of vulvovaginal candidiasis, vulvar folliculitis, bacterial vaginitis, cervicitis and urinary tract infection.

[0009] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0010] The invention provides a Lactobacillus crispatus MYT-LC01, whose preservation number is CGMCC NO:32209.

[0011] The above-mentioned Lactobacillus crispatus was identified and named: It was named Lactobacillus crispatus MYT-LC01, deposited on October 14, 2024, with the deposit number CGMCC NO: 32209, and the deposit unit was the General Microbiology Center of China National Culture Collection Administration; the deposit address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101.

[0012] The 16S rDNA sequence of the Lactobacillus crispatus MYT-LC01 is shown in SEQ ID NO:1.

[0013] The present invention also provides a use of the Lactobacillus crispatus MYT-LC01 in inhibiting the growth of harmful vaginal bacteria.

[0014] The present invention also provides a use of the Lactobacillus crispatus MYT-LC01 in preparing an antibacterial agent for inhibiting the growth of harmful vaginal bacteria.

[0015] Furthermore, the above-mentioned vaginal harmful bacteria is any one of Candida albicans, Staphylococcus aureus and Escherichia coli.

[0016] The present invention also provides a use of the Lactobacillus crispatus MYT-LC01 in preparing a medicine for treating or preventing gynecological diseases.

[0017] Furthermore, the gynecological disease is any one of vulvovaginal candidiasis, candidiasis, bacterial vaginitis and urinary tract infection.

[0018] The above-mentioned vulvovaginal candidiasis is a gynecological disease caused by Candida albicans;

[0019] The above-mentioned vulvar folliculitis, bacterial vaginitis, and cervicitis are gynecological diseases caused by Staphylococcus aureus;

[0020] The above-mentioned urinary tract infection is a gynecological disease caused by Escherichia coli.

[0021] The present invention also provides a Lactobacillus crispatus fermentation agent, wherein the fermentation agent comprises Lactobacillus crispatus MYT-LC01 at a concentration of 10 8 ~10 12 CFU / mL.

[0022] The present invention also provides a method for preparing the Lactobacillus crispatus fermentation agent, which specifically comprises the following steps: picking a single colony of Lactobacillus crispatus MYT-LC01 in an MRS liquid culture medium, and culturing the colony at 37° C. for 48 hours to obtain the Lactobacillus crispatus fermentation agent.

[0023] The present invention also provides a use of the Lactobacillus crispatus fermentation agent in preparing an antibacterial agent for inhibiting the growth of harmful vaginal bacteria.

[0024] Among the above antibacterial agents, the concentration of Lactobacillus crispatus MYT-LC01 is 10 8 ~10 12 CFU / mL.

[0025] The present invention also provides an application of the Lactobacillus crispatus fermentation agent in preparing a drug for treating or preventing gynecological diseases, wherein the gynecological disease is any one of vulvovaginal candidiasis, vulvar folliculitis, bacterial vaginitis, cervicitis and urinary tract infection.

[0026] The present invention also provides a drug for treating or preventing gynecological diseases, wherein the drug comprises the above-mentioned Lactobacillus crispatus MYT-LC01 or the above-mentioned Lactobacillus crispatus fermentation agent, and the concentration of Lactobacillus crispatus MYT-LC01 in the drug is 10 8 ~10 12 CFU / mL.

[0027] Beneficial effects of the present invention:

[0028] 1. The present invention screened and identified a specific vaginal Lactobacillus crispatus MYT-LC01 from the vagina of healthy women. The strain has antagonistic effects on Candida albicans, Staphylococcus aureus, and Escherichia coli, and can effectively prevent and treat common gynecological diseases such as vulvovaginal candidiasis, vulvar folliculitis, bacterial vaginitis, cervicitis, and urinary tract infection.

[0029] 2. The present invention has developed an efficient and stable culture method, which can be used to culture and prepare Lactobacillus crispatus MYT-LC01 on a large scale in the laboratory and on an industrial scale, ensuring the high vitality and high yield of the strain while ensuring the stability and safety of the strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of the inhibition of Lactobacillus crispatus MYT-LC01 on Candida albicans;

[0031] Figure 2 This is the result of the inhibition of Lactobacillus crispatus MYT-LC01 on Escherichia coli;

[0032] Figure 3 This is the result of the inhibition of Lactobacillus crispatus MYT-LC01 on Staphylococcus aureus;

[0033] Figure 4 This is a plate diagram showing the antibacterial effect of Lactobacillus crispatus MYT-LC01 on harmful vaginal bacteria. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0035] Example 1

[0036] Isolation and identification of Lactobacillus crispatus MYT-LC01

[0037] 1. Sample collection

[0038] Insert the cotton swab about 5 cm (2 inches) into the vaginal opening of a healthy female. Gently rotate the cotton swab for about 30 seconds to ensure that the cotton swab is in full contact with the vaginal wall. Pull out the cotton swab without touching the skin. Place the cotton swab head at the mouth of the sampling tube, forming an angle of about 45 degrees with the tube wall. Gently break the stem of the cotton swab to allow the cotton head to fall into the tube.

[0039] 2. Strain Isolation

[0040] The strains attached to the cotton head were separated by anaerobic lactic acid bacteria culture medium (MRS), pH 6.2-6.6, 37°C, anaerobic static culture for 2-3 days, and 4 single colonies were isolated, one of which was named MYT-LC01 and identified.

[0041] 3. Culture of strain MYT-LC01

[0042] (1) Preparation of slant: Prepare anaerobic MRS solid culture medium, quantitatively dispense it into test tubes, and solidify it into a slant. The volume of culture medium poured into the test tube should be about 1 / 4, and the length of the slant should not exceed half of the test tube. Generally, a 15×150 mm test tube with a capacity of 5 ml is used.

[0043] (2) Inoculation: The prepared slant was subjected to a sterility test. The strain MYT-LC01 was inoculated only after it was confirmed to be sterile. The slant was cultured anaerobically at 37°C for 2 to 3 days.

[0044] 4. Identification of strain MYT-LC01

[0045] (1) Determination of 16S rDNA sequence (bacterial species identification)

[0046] DNA extraction: Use the full-form gold plant DNA extraction kit (universal type), the specific steps are as follows:

[0047] ① Place the Spin Column in the Collection Tube, add 250 μl Buffer BL, and centrifuge at 12000 rpm / min for 1 min to activate the silica membrane;

[0048] ②Take the bacterial cells of strain MYT-LC01 (no more than 20 mg), add liquid nitrogen and grind thoroughly. After grinding, place in a 1.5 ml centrifuge tube, add 400 μl Buffer gP1, vortex for 1 min, and place in a 65°C water bath for 10 to 30 min. During this period, you can take out and mix by inversion to fully lyse;

[0049] ③ Add 150 μl Buffer gP2, vortex for 1 min, and place on ice for 5 min;

[0050] ④ Centrifuge at 12000rpm / min for 5min and transfer the supernatant to a new centrifuge tube;

[0051] ⑤ Add an equal volume of anhydrous ethanol to the supernatant, immediately shake and mix thoroughly, transfer all the liquid into the Spin Column, centrifuge at 12,000 rpm / min for 30 seconds, and discard the waste liquid;

[0052] ⑥ Add 500 μl Buffer Pw (anhydrous ethanol has been added before use) to the Spin Column, centrifuge at 12000 rpm / min for 30 seconds, and discard the waste liquid;

[0053] ⑦ Add 500 μl Wash Buffer (anhydrous ethanol has been added before use) to the Spin Column, centrifuge at 12000 rpm / min for 30 seconds, and discard the waste liquid;

[0054] ⑧Repeat step 7;

[0055] ⑨Put the Spin Column back into the Collection Tube, centrifuge at 12,000 rpm / min for 2 min, and open the lid to dry for 1 min;

[0056] ⑩Take out the Spin Column and place it in a clean centrifuge tube. Add 50-100 μl TE Buffer (preheated TE Buffer at 65°C) to the center of the adsorption membrane. Incubate at 20-25°C for 2 minutes. Centrifuge at 12,000 rpm / min for 2 minutes to obtain bacterial DNA.

[0057] (2) PCR amplification

[0058] The universal primers for bacterial species identification are shown in Table 1. The extracted bacterial DNA samples were appropriately diluted and used as PCR templates for amplification with full-form gold 1×mix. The components of the amplification system are shown in Table 2, and the amplification program is shown in Table 3.

[0059] Table 1 Universal primers

[0060]

[0061]

[0062] Table 2 Amplification system

[0063] Full gold 1×mix 45μl 27F(10P) 2μl 1492R(10P) 2μl DNA template 1μl

[0064] Table 3 Amplification procedures

[0065]

[0066] (3) Electrophoresis detection

[0067] The amplified PCR product was subjected to agarose gel electrophoresis (2 μl sample + 6 μl bromophenol blue) at 300 V for 12 min to obtain an identification gel image, and first-generation sequencing (sequencing primers were 27F / 1492R) was performed to obtain the sequencing results.

[0068] (4) Identification

[0069] ① The sequencing results were spliced ​​using ContigExpress and the inaccurate parts at both ends were removed. The 16SrDNA sequence of strain MYT-LC01 is shown in SEQ ID NO: 1.

[0070] ② The spliced ​​sequences were aligned in the NCBI database (blast.ncbi.nlm.nih.gov), and the alignment results are shown in Table 4. The strain MYT-LC01 has a high match with Lactobacillus crispatus, and is named Lactobacillus crispatus MYT-LC01, which was deposited on October 14, 2024, with the deposit number CGMCC NO: 32209, and the deposit unit is the General Microbiology Center of the China Microbiological Culture Collection Administration; the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101.

[0071] Table 4 Comparison results

[0072]

[0073] Example 2

[0074] Antibacterial effect detection of Lactobacillus crispatus MYT-LC01

[0075] 1. Methods:

[0076] Lactobacillus crispatus MYT-LC01 antagonistic detection: The double-layer plate method detects the antagonistic effect of Lactobacillus crispatus MYT-LC01 on vaginal harmful strains (Candida albicans, Staphylococcus aureus, Escherichia coli). The lower layer of the double-layer plate is an agar nutrient medium with an agar content of 1.5%, and the upper layer is a soft agar medium with an agar content of 0.5%. The vaginal harmful bacteria and the soft agar medium are mixed according to a volume ratio of 1:100 and cultured overnight. After the plate is solidified and the surface is dried, a 1ml gun tip is used to punch holes, and Lactobacillus crispatus MYT-LC01 is inoculated. According to the size of the inhibition zone, the inhibitory effect of the strain on the vaginal harmful bacteria is observed. At the same time, the remaining 3 single colonies obtained by separation in Example 1 are selected as controls for antagonistic detection, and are named strain No. 1, strain No. 2 and strain No. 3 respectively.

[0077] (2) Results:

[0078] The results are shown in Table 5 and Figure 1 to Figure 4 As shown, the larger the inhibition zone, the more obvious the antibacterial effect. The results showed that all four strains of bacteria had a significant inhibitory effect on the growth of harmful vaginal bacteria, among which Lactobacillus crispatus MYT-LC01 had the most obvious antibacterial effect.

[0079] Table 5 The size of the inhibition zone of the strains against vaginal harmful bacteria

[0080]

[0081]

[0082] Note: strains 1 to 3 are strains obtained simultaneously when screening Lactobacillus crispatus MYT-LC01.

[0083] Lactobacillus crispatus MYT-LC01 has an antagonistic effect on vaginal-related harmful bacteria (Candida albicans, Staphylococcus aureus, and Escherichia coli). It can be seen that Lactobacillus crispatus MYT-LC01 and its related products can treat or prevent gynecological diseases (vulvovaginal candidiasis, vulvar folliculitis, bacterial vaginitis, cervicitis, and urinary tract infection) caused by the above-mentioned strains.

[0084] Example 3

[0085] Preparation of Lactobacillus crispatus fermentation agent

[0086] A single colony of Lactobacillus crispatus MYT-LC01 was selected and placed in MRS liquid culture medium. After culturing at 37°C for 48 hours, a fermentation agent of Lactobacillus crispatus MYT-LC01 was obtained. The concentration of Lactobacillus crispatus MYT-LC01 was 10 8 ~10 12 CFU / mL.

[0087] According to actual conditions, Lactobacillus crispatus MYT-LC01 or Lactobacillus crispatus fermented bacteria can be used to prepare antibacterial agents for inhibiting the growth of harmful vaginal bacteria, and further prepared drugs for treating or preventing gynecological diseases, which are used to treat or prevent gynecological diseases caused by the above strains.

[0088] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A Lactobacillus crispatus MYT-LC01, whose deposit number is CGMCC NO: 32209.

2. A use of the Lactobacillus crispatus MYT-LC01 according to claim 1 in inhibiting the growth of harmful vaginal bacteria.

3. A use of the Lactobacillus crispatus MYT-LC01 according to claim 1 in the preparation of an antibacterial agent for inhibiting the growth of harmful vaginal bacteria.

4. The use according to claim 2 or 3, characterized in that: The vaginal harmful bacteria is any one of Candida albicans, Staphylococcus aureus and Escherichia coli.

5. Use of the Lactobacillus crispatus MYT-LC01 according to claim 1 in preparing a drug for treating or preventing gynecological diseases.

6. The application according to claim 5, characterized in that: The gynecological disease is any one of vulvovaginal candidiasis, vulvar folliculitis, bacterial vaginitis, cervicitis and urinary tract infection.

7. A Lactobacillus crispatus fermentation agent, characterized in that: The fermentation agent comprises the Lactobacillus crispatus MYT-LC01 of claim 1, and its concentration is 10 8 ~10 12 CFU / mL.

8. Use of the Lactobacillus crispatus fermentation agent according to claim 7 in the preparation of an antibacterial agent for inhibiting the growth of harmful vaginal bacteria.

9. Use of the Lactobacillus crispatus fermentation agent according to claim 7 in the preparation of a drug for treating or preventing gynecological diseases, characterized in that: The gynecological disease is any one of vulvovaginal candidiasis, vulvar folliculitis, bacterial vaginitis, cervicitis and urinary tract infection.

10. A drug for treating or preventing gynecological diseases, characterized in that: The drug comprises the Lactobacillus crispatus MYT-LC01 of claim 1 or the Lactobacillus crispatus fermentation agent of claim 7, and the concentration of Lactobacillus crispatus MYT-LC01 in the drug is 10 8 ~10 12 CFU / mL.

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