A Vaginal Lactobacillus with Broad-Spectrum Bacteriostatic Activity Derived from Pigs and Its Application

Abstract

The present invention relates to the technical field of vaginal lactobacilli, and specifically to a porcine-derived broad-spectrum antibacterial vaginal lactobacillus and its application. The vaginal lactobacillus provided by the present invention is Lactobacillus vagmalis G1903, which is screened from the feces of healthy pigs through bacterial isolation and identification, simulated gastrointestinal tolerance test, antibacterial spectrum determination, and intestinal cell adhesion and colonization test. It has antibacterial effects and a relatively broad antibacterial spectrum, and has a significant antibacterial effect on anaerobic bacteria after adjusting the pH of the supernatant. Its preservation number is CGMCC NO. 26728. The vaginal lactobacillus G1903 prepared by the present invention can be used to regulate the microecological balance of animal intestines, prevent diseases, and improve the growth and production performance of animals.

Description

Technical Field

[0001] The present invention relates to the technical field of vaginal lactobacilli, and specifically to a porcine-derived vaginal lactobacillus with broad-spectrum antibacterial activity and its application. Background Art

[0002] Currently, the main way to deal with piglet intestinal infections in the breeding environment is to use antibiotics for treatment. The long-term use of high-dose and multi-variety antibiotics will cause bacterial drug resistance, damage the intestinal barrier function and disrupt the stability of the piglet intestinal flora. At the same time, people's requirements for the quality of animal products are getting higher and higher. Using safe, environmentally friendly and efficient alternatives to replace antibiotics has become a trend. Therefore, the emergence of lactic acid bacteria preparations is expected to become a safe and effective prevention and treatment means. Vaginal lactobacillus (Lactobacillus vaginalis, L. vaginalis) is a Gram-positive bacillus, which is widely present in the gastrointestinal tract and vagina of animals. Microbiota analysis found that a high abundance of L. vaginalis is not only beneficial for maintaining the intestinal health of piglets; but also can play a certain protective role against other diseases caused by hormonal disorders. Exogenous addition of porcine-derived lactobacilli can be better used in piglet production to promote the growth of piglets. However, there must be certain criteria for the selection of lactobacillus strains, such as the tolerance of the strains to gastric acid and bile salts, the colonization ability in intestinal epithelium, and antibacterial characteristics, etc. Although lactobacilli have great probiotic potential, due to their strain specificity and classification complexity, not all strains have ideal abilities and potential for application in livestock production. Therefore, screening strains with good probiotic properties and suitable for animal production is of great significance for livestock production and environmental protection. Summary of the Invention

[0003] The purpose of the present invention is to provide a porcine-derived vaginal lactobacillus with broad-spectrum antibacterial activity and its application to solve the problems raised in the above background art.

[0004] To solve the above problems, the present invention provides the following technical solutions:

[0005] A porcine-derived vaginal lactobacillus with broad-spectrum antibacterial activity, characterized in that: the DNA sequence of the vaginal lactobacillus is as shown in SEQ ID NO.1.

[0006] Further, the vaginal lactobacillus is porcine vaginal lactobacillus G1903, and the preservation number is CGMCC NO.26728.

[0007] Further, the screening method of the porcine vaginal lactobacillus G1903 is:

[0008] Sterilely collect the fecal samples of healthy pigs. Put the pig feces into a sterile centrifuge tube, add sterile PBS and mix well with the fecal samples, inoculate and streak on the MRS solid medium, and culture anaerobically and statically at 37°C. Pick the single colonies suspected to be Lactobacillus and continue to inoculate and streak on the MRS plate, and culture anaerobically and statically at 37°C. Streak continuously in this way for 3 times until single colonies with uniform morphology grow on the plate, and the strain is isolated.

[0009] Furthermore, the application of the vaginal Lactobacillus or its fermentation broth or its metabolite or its bacterial suspension or its culture solution in the preparation of antibacterial and / or bacteriostatic products.

[0010] Furthermore, the preparation method of the fermentation broth is as follows:

[0011] S1: Activate and culture the porcine vaginal Lactobacillus to obtain an activated bacterial solution;

[0012] S2: Centrifuge the activated bacterial solution obtained in S1 at a speed of 6000 r / min for 6 min at 4°C, filter to obtain the supernatant;

[0013] Take the supernatant, add NaOH reagent, adjust the pH of the system to obtain a fermentation broth with a pH of 6.0.

[0014] Furthermore, the specific steps of the activation culture in step S1 are as follows: Inoculate and streak the porcine vaginal Lactobacillus on the MRS medium, and culture anaerobically and statically at 37°C for 48 h; Pick the single colonies and culture anaerobically and statically at 37°C for 12 - 24 h to obtain an activated bacterial solution.

[0015] Furthermore, when the fermentation broth is applied, the viable count of the porcine vaginal Lactobacillus is 1×10 8 ~

[0016] 1×10 10 CFU / mL.

[0017] Furthermore, the application of the vaginal Lactobacillus or its preparation or its fermentation broth or its metabolite or its bacterial suspension or its culture solution in the preparation of products for improving the growth performance of animals.

[0018] Furthermore, the product prepared from the porcine vaginal Lactobacillus has any one or more of the following functions:

[0019] (1) Antibacterial and / or bacteriostatic;

[0020] (2) Regulating the intestinal microecological balance of animals;

[0021] (3) Preventing animal diseases;

[0022] (4) Reducing the probability of animals getting sick;

[0023] (5) Improve the growth performance of animals.

[0024] Compared with the prior art, the beneficial effects of the invention are as follows:

[0025] From the fecal samples of healthy piglets, through bacterial isolation and identification, determination of the antibacterial spectrum, and animal regression tests, a Lactobacillus vaginalis G1903 with antibacterial effects and a relatively broad antibacterial spectrum, and a significant antibacterial effect on anaerobic bacteria after adjusting the supernatant pH, was screened out. Experiments have proved that Lactobacillus vaginalis G1903 has antibacterial effects and has inhibitory activities on aerobic bacteria such as Enterobacter, Enterococcus, Staphylococcus, Salmonella, Klebsiella pneumoniae, and anaerobic bacteria such as Clostridium perfringens and Clostridium difficile; and the antibacterial effect of the supernatant fermentation broth of Lactobacillus vaginalis G1903 with a pH of 6.0 on the anaerobic intestinal pathogen - Clostridium perfringens can reach 35 mm. Lactobacillus vaginalis G1903 can also be used to regulate the microecological balance of animals, prevent animal diseases, improve the growth performance of animals, etc., and has good application prospects. Description of the Drawings

[0026] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0027] Figure 1 is the morphological diagram of Lactobacillus vaginalis G1903 in the embodiment of the present invention observed under a fluorescence microscope;

[0028] Figure 2 is the antibacterial effect diagram of the supernatant fermentation broth of Lactobacillus vaginalis G1903 with a pH of 6.0 on Clostridium perfringens in the embodiment of the present invention (note: Lactobacillus reuteri F5, Lactobacillus vaginalis G1903, Lactobacillus salivarius M26903, and Lactobacillus johnsonii SLD3 are arranged in a clockwise direction; F5, M26903, and SLD3 are control strains of porcine Lactobacillus isolated in the same batch as G1903). Detailed Embodiments

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used in the embodiments can be obtained from commercial channels unless otherwise specified; for the quantitative tests in the embodiments, three repeated experiments are set, and the results are averaged.

[0031] The indicator strains in the following examples, ATCC13124 (a human gas gangrene isolate), Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Clostridium difficile ATCC43593, were purchased from the American Type Culture Collection (ATCC). Clostridium perfringens X27 was a clinical isolate from the feces of diarrheic piglets. Salmonella abortus equi CVCC514 was purchased from the China Institute of Veterinary Drug Control. Salmonella strains SL1344, SL7207, SP, SE, and other Enterococcus, Staphylococcus, Escherichia coli, and Klebsiella pneumoniae strains were clinical isolates and were all stored in this experiment.

[0032] Example

[0033] Isolation, Identification, Screening, and Characterization Analysis of Porcine Vaginal Lactobacilli

[0034] (1) Sterilely collect fecal samples from healthy pigs. Take about 0.1 g of pig feces into a sterile centrifuge tube, add 1 mL of sterile PBS, and mix well with the fecal sample. Use a sterilized inoculation loop to dip a small amount and streak it on MRS solid medium. After inoculation, anaerobically statically culture it in a 37 °C constant temperature incubator for 48 h. Pick suspected Lactobacillus single colonies and continue to streak them on MRS plates, and anaerobically statically culture them at 37 °C. Streak continuously in this way 3 times until uniform single colonies grow on the plate;

[0035] (2) For the purified suspected strains, perform Gram staining and microscopic examination. The isolated suspected strains were sent to Jilin Kumei Biotechnology Co., Ltd. for 16S rDNA sequencing identification.

[0036] (3) The sequencing results showed that the sequence of the PCR product was as shown in SEQ ID NO.1. This sequence was analyzed by alignment on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and was identified as Lactobacillus vaginalis. Based on the above identification results, the strain name of G1903 was determined to be Lactobacillus vaginalis, and its taxonomic name was Lactobacillus vaginalis.

[0037] The sequence of Lactobacillus vaginalis G1903 is as follows:

[0038] This sequence is a 16S rDNA sequence:

[0039]

[0040] Experiment:

[0041] Experiment 1: Tolerance test of vaginal Lactobacillus to acid-base and bile salts

[0042] Use NaOH and HCl solutions to adjust the pH value of MRS broth medium to 2.0, 4.0, 6.0, 8.0 and 10.0 respectively. The test strain was anaerobically cultured in normal MRS broth medium at 37 °C for 48 h. The bacterial liquid was centrifuged at 8000 r / min for 10 min at 4 °C to obtain bacterial cells, and then resuspended with PBS to 10 9 CFU / mL (excluding the influence of the original medium); then, the obtained resuspended liquid was inoculated into MRS test tubes with adjusted pH values at an inoculation ratio of 2%, and colony counting was performed after anaerobic culture at 37 °C for 48 h.

[0043] Take the same resuspended liquid mentioned above and inoculate it into the medium containing 0.3% (mg / mL) porcine bile salt and normal medium at an inoculation ratio of 2% respectively. Colony counting was performed after anaerobic culture at 37 °C for 12 h.

[0044] The results showed that the survival rates of vaginal Lactobacillus G1903 in acid, alkali and bile salts were 25.01% ± 0.005, 65.30% ± 0.03987 and 87.46% ± 0.016 respectively. Compared with pH 2.0, the strain showed stronger survival ability in the environment with pH 4.0.

[0045] Experiment 2: Evaluation of the antibacterial effect of porcine-derived vaginal Lactobacillus

[0046] 1. Cultivation of isolated strains and preparation of cell-free supernatant (CFS)

[0047] Streak inoculate the glycerol bacteria of vaginal Lactobacillus isolated strains stored at -80 °C on MRS solid medium and culture them at 37 °C under anaerobic conditions for 48 h. Pick single colonies into liquid medium and culture them statically at 37 °C under anaerobic conditions for 12 - 24 h. Take the activated bacterial liquid, centrifuge it at 6000 r / min for 6 min at 4 °C, discard the precipitate, take the supernatant, filter it with a 0.22 μm filter to obtain cell-free supernatant (CFS), and store it at 4 °C for later use. Add NaOH to the above supernatant to obtain a supernatant fermentation broth with a pH of 6.0 at the same time.

[0048] 2. Cultivation of indicator bacteria and determination of antibacterial effect

[0049] Take the strains stored at -80 °C and streak them twice on the solid medium plate. Pick single colonies and inoculate them into the liquid medium suitable for the growth of the strains, and culture them under the appropriate conditions of each strain to adjust the concentration of the indicator bacteria to 10 7 -10 8CFU / mL, OD600 = 0.75 - 0.78. After evenly spreading 100 μL of the indicator bacteria on the solid agar plate, use a sterile pipette tip to evenly punch holes with a diameter of 9 mm on the culture medium; add the prepared supernatant and the supernatant fermentation broth with a pH of 6.0 to the corresponding culture medium plate holes, and measure the diameter of the inhibition zone after culturing for 12 h under the corresponding conditions. Each test is set with three replicates.

[0050] The results showed that a total of 52 strains of indicator bacteria were used for the antibacterial experiment. The supernatant of Lactobacillus vaginalis G1903 showed antibacterial effects against 28 of the indicator bacteria, and the supernatant of Lactobacillus vaginalis G1903 with a pH of 6.0 showed strong antibacterial effects against Clostridium perfringens ATCC13124 and X27. No obvious antibacterial effects were shown by the other strains. The supernatant of Lactobacillus vaginalis G1903 had antibacterial effects against Enterococcus (2 strains), Staphylococcus (5 strains), Salmonella (4 strains), Escherichia coli (15 strains), Klebsiella pneumoniae (1 strain), and Clostridium perfringens (1 strain).

[0051] Experiment 3: Determination of the adhesion and colonization ability of porcine-derived Lactobacillus vaginalis to intestinal epithelial cells

[0052] 1. In vitro hemolytic assay

[0053] Streak the fresh glycerol bacteria stored at -80 °C onto the LB blood plate and observe whether there is a hemolytic zone after culturing anaerobically at 37 °C for 48 h. The results showed that Lactobacillus vaginalis G1903 had no hemolytic property.

[0054] 2. Adhesion of Lactobacillus vaginalis to mouse intestinal epithelial cells in vitro

[0055] The activated single colony was resuspended with PBS to prepare a bacterial suspension (10 9 CFU / mL) for standby. Sterilely collect the jejunum of healthy mice and cut it into 1 cm 2 intestinal segments, lay them flat in 2 ml sterile centrifuge tubes. The mouse intestinal segments were previously soaked in an antibiotic (2 mg / mL metronidazole) solution for 30 min. Add 0.5 mL of the prepared bacterial suspension, incubate anaerobically at 37 °C for 3 h, then rinse with PBS and transfer to a new centrifuge tube containing grinding beads for grinding. After serial dilution of the grinding solution, plate count was performed on MRS medium.

[0056] The results showed that the adhesion rate of Lactobacillus vaginalis G1903 reached 85.29% ± 0.084.

[0057] Experiment 4: Safety determination after intragastric administration of Lactobacillus vaginalis to mice

[0058] Six-week-old (20 - 22 g) SPF-grade BALB / c female mice were selected as experimental animals and randomly divided into two groups. A control group (normal saline) and an experimental group (fluorescently labeled G1903) with 4 mice in each group were set up. All mice were deprived of water and weighed 2 hours before the experiment. The mice in the experimental group were administered the drug by gavage at a dose of 100 mg / Kg. The control group was given the same volume of the solvent, normal saline, by gavage. At 6 hours and 12 hours after gavage, the mice were weighed and fluorescence imaging of the mouse intestine was performed using a laser confocal microscope.

[0059] The results showed that there was no significant change in the body weight of the mice after gavage with G1903, and the mice were in good condition; there was no obvious damage to the mouse intestine observed by laser confocal microscopy, and the fluorescently labeled G1903 appeared at the terminal of the cecum 12 hours after gavage.

[0060] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0061] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Lactobacillus vaginalis with broad-spectrum antibacterial activity from pigs, characterized in that: The Lactobacillus vaginalis is Lactobacillus vaginalis of porcine origin G1903, with the preservation number of CGMCC NO.26728.

2. Use of the vaginal Lactobacillus or its supernatant fermentation broth or its bacterial suspension or its culture solution described in any one of claims 1 in the preparation of an antibacterial and / or bacteriostatic product, characterized in that: The bacteria strains with antibacterial properties are Enterococcus, Staphylococcus, Salmonella, Escherichia coli, Klebsiella pneumoniae, and Clostridium perfringens.

3. The application according to claim 2, wherein: The preparation method of the supernatant fermentation broth is as follows: S1: Activate and culture Lactobacillus vaginalis of porcine origin to obtain an activated bacterial solution; S2: Centrifuge the activated bacterial solution obtained in S1 at a temperature of 4°C and a rotation speed of 6000 r / min for 6 min, and filter to obtain a supernatant; Take the supernatant, add NaOH reagent, and adjust the pH of the system to obtain a supernatant fermentation broth with a pH of 6.

0.

4. The application according to claim 2, characterized in that: The specific steps of the activation culture in step S1 are: inoculate and streak Lactobacillus vaginalis of porcine origin onto an MRS medium, and anaerobically statically culture at 37°C for 48 h; pick a single colony and anaerobically statically culture at 37°C for 12 - 24 h to obtain an activated bacterial solution.

5. The application according to claim 2, wherein: When the supernatant fermentation broth is applied, the viable count of porcine vaginal Lactobacillus is 1×10 8 ~1×10 10 CFU / mL.

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