Bacillus velez and its production method and application in preparing anti-Enterobacterial tridecapeptide

Abstract

A strain of Bacillus velez, the strain is deposited in the China Center for Type Culture Collection, the deposit date is September 7, 2023, the address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No. M20231632. The strain can be used to prepare a 13-peptide with a unique structure that can strongly inhibit Gram-negative Enterobacteriaceae. The preparation method provided by the present invention can prepare a high-purity, high-activity, stable and safe anti-Enterobacteriaceae 13-peptide for the development and utilization of antibacterial drugs, lead veterinary drugs and feed additives.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to Bacillus velez, application thereof, and a method for preparing anti-Enterobacterium tridecapeptide. Background Art

[0002] Bacillus Velez Bacillus velezensis ) is a type of Gram-positive bacillus that can produce a variety of novel peptides and lipopeptides with antibacterial and other active compounds. It is one of the common strains for the actual industrial production of antimicrobial peptides. Lipocyclic antimicrobial peptides, also known as cyclolipopeptides, are secondary metabolites produced by microorganisms (such as certain Bacillus subtilis). They are an important class of antimicrobial peptides formed by the cyclization of amino acids and fatty acids, and are mainly synthesized through the non-ribosomal peptide and polyketide hybrid pathways of bacteria. The structure of lipocyclic peptides contains both hydrophilic amino acid residues and hydrophobic fatty acid chains, so the entire lipocyclic peptide has significant amphipathicity. Lipocyclic peptides interact with the phospholipid bilayer in the biological membrane, destroy the lipid membrane structure, form ion channels, promote the outflow of intracellular substances, and thus cause cell death, so that lipocyclic peptides can exhibit strong antibacterial activity. Therefore, lipocyclic antimicrobial peptides can be added as feed additives to partially replace the role of antibiotics and as candidates for the development of new antimicrobial drugs.

[0003] The emergence of multi-drug resistant pathogenic Enterobacteriaceae in actual clinical practice and aquaculture has made people realize the insufficiency of existing antimicrobial drugs. Although there are many studies on antimicrobial peptides, there are not many new antibacterial products that have been developed. This makes the discovery of more new antimicrobial peptides and their industrial development particularly urgent. The development of new antimicrobial peptide drugs or feed additives plays an important role in the treatment of diseases caused by or in the replacement of antibiotics in the feed of the aquaculture industry, and has important application prospects. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a strain capable of preparing specific, stable, highly active and highly pure anti-Enterobacterial tridecapeptide, as well as a preparation method and application of the tridecapeptide.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A strain of Bacillus velez ( Bacillus velezensis a5546), deposited in China Center for Type Culture Collection, deposit date: September 7, 2023, address: Wuhan University, Wuhan, China, deposit number: CCTCC No.M20231632.

[0007] The use of the Bacillus Velezii in the preparation of antimicrobial peptides. The antimicrobial peptides are preferably anti-Enterobacterial tridecapeptides.

[0008] The present invention also provides an anti-Enterobacterial tridecapeptide, the structural formula of which is as follows:

[0009]

[0010] The present invention also provides a method for preparing the anti-Enterobacterial tridecapeptide by the above-mentioned Bacillus Velezii, comprising the following steps:

[0011] Step 1: Bacillus Velezii (CCTCC No. M20231632) Bacillus velezensis a5546) was inoculated in LB slant medium for activation, cultured at 37°C for 12 h, and then inoculated into a sterilized LB medium shake flask, cultured at 37°C, 200 rpm, for 12 h to obtain seed liquid, and the seed liquid was inoculated into the optimized liquid medium at a volume ratio of 1% inoculation amount, and cultured at 37°C for 16 h to obtain the 13-peptide fermentation product. The fermentation liquid was centrifuged to obtain the supernatant, which was freeze-dried to obtain lyophilized powder.

[0012] Step 2: The obtained lyophilized powder was dissolved in ultrapure water and extracted with an equal volume of n-butanol. Ether was added to the n-butanol extract and the 13 peptide was precipitated at 4°C overnight.

[0013] Step 3: The obtained precipitate is dissolved with the initial mobile phase and then separated and purified by semi-preparative reverse phase high performance liquid chromatography. The obtained fraction is subjected to rotary evaporation to remove acetonitrile and then freeze-dried to obtain a high-purity sample of the tridecapeptide.

[0014] Preferably, the optimized liquid culture medium described in step 1 is prepared as follows: 3.3 g soluble starch, 10 g yeast extract powder, 10 g ammonium sulfate, and 3 g anhydrous magnesium sulfate are dissolved in 1 L ultrapure water, and the pH is adjusted to 6.5 with hydrochloric acid after dissolution. The culture medium is sterilized at 121°C for 20 min, the inoculation amount is 1%, the fermentation temperature is 37°C, the fermentation time is 16 h, and the fermentation broth is centrifuged to obtain the supernatant, which is freeze-dried to obtain freeze-dried powder.

[0015] Preferably, the extraction process in step 2 is as follows: the lyophilized powder obtained in step 1 is dissolved with ultrapure water, the ratio of lyophilized powder to ultrapure water is 1:1~1:5 (W / V), after dissolution, an equal volume of n-butanol is added and extracted 1~3 times, the n-butanol extract is transferred to a new conical flask, and an equal volume of ether is added to precipitate the tridecapeptide.

[0016] Preferably, the reverse phase high performance liquid chromatography preparation process in step 3 is: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phase contains 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B);

[0017] The elution mode is shown in Table 1:

[0018] Table 1 Elution gradient for preparation of thirteen peptides

[0019]

[0020] The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

[0021] The present invention also provides the use of the above-mentioned Bacillus Velezii and the above-mentioned anti-Enterobacterial tridecapeptide in the preparation of a drug or feed additive for resisting Gram-negative Enterobacteriaceae infection. The Gram-negative Enterobacteriaceae include but are not limited to Escherichia coli, Salmonella, Shigella dysenteriae, and Pseudomonas aeruginosa.

[0022] Beneficial effects:

[0023] The present invention provides a strain of Bacillus velezine for preparing anti-Enterobacterial tridecapeptide ( Bacillus velezensis ), can prepare thirteen peptides with anti-Gram-negative pathogenic Enterobacteriaceae activity, the peptides have a unique molecular structure, high activity, high purity and stable performance.

[0024] The invention provides an anti-Enterobacterial tridecapeptide, which can stably exist in a neutral and acidic environment, can maintain the stability of its structure and antibacterial activity in artificial gastric juice, can be degraded into other substances with certain antibacterial activity in an artificial intestinal fluid environment, and has no obvious hemolytic activity; through screening of antibacterial activity, it is found that the tridecapeptide has strong activity in inhibiting pathogenic Enterobacteriaceae such as Escherichia coli, Salmonella, Shigella dysenteriae, Pseudomonas aeruginosa, and can be used for the development and utilization of antibacterial drugs, leading veterinary drugs and feed additives for inhibiting related diseases and infections caused by various Gram-negative pathogenic Enterobacteriaceae.

[0025] 3. The present invention provides a method for preparing an anti-Enterobacterial tridecapeptide, which prepares a high-purity tridecapeptide product through liquid seed preparation, fermentation liquid preparation, fermentation liquid pretreatment, tridecapeptide extraction and purification, thereby greatly reducing the preparation and purification steps.

[0026] 4. The present invention utilizes extraction precipitation combined with preparative reverse phase high performance liquid chromatography to improve the preparation efficiency and sample purity of the 13 peptides. The purity of the 13 peptides prepared by this method is as high as 91.76%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the Bacillus Velez in Example 1 of the present invention ( Bacillus velezensis a5546) strain identification phylogenetic tree;

[0028] Figure 2Comparison diagram of PCR amplification results in identification of Bacillus velez strains in Example 1 of the present invention;

[0029] Figure 3 This is the HPLC preparation diagram of the 13 peptide in Example 2 of the present invention;

[0030] Figure 4 This is a HPLC chart of the purity analysis of the 13 peptides in Example 2 of the present invention;

[0031] Figure 5 The primary and secondary mass spectra of the thirteen peptides in Example 2 of the present invention;

[0032] Figure 6 The stability results of the tridecapeptide in Example 4 of the present invention in aqueous solutions of different pH values;

[0033] Figure 7 The stability results of the tridecapeptide in Example 5 of the present invention in artificial gastric juice;

[0034] Figure 8 The stability results of the 13 peptide in Example 6 of the present invention in artificial intestinal fluid;

[0035] Fig. 9 The hemolytic results of the thirteen peptides in Example 7 of the present invention. DETAILED DESCRIPTION

[0036] The strain used in the present invention is Bacillus Velezii isolated from the soil of Huanghe Village, Guangshun Town, Rongchang District, Chongqing (N 29°22′56″, E 105°29′46″) Bacillus velezensis a5546), which was deposited in the China Center for Type Culture Collection on September 7, 2023. The deposit number is CCTCC No. M20231632.

[0037] The strain isolated by the present invention has been identified, and its morphological characteristics are: the colony is white, the surface is shiny, and there is no wrinkled edge; the bacteria are rod-shaped, 1.0-3.5 μm long, 0.4-0.8 μm wide, the bacteria have flagella, can form spores, and are Gram-positive. The 16S rRNA gene of the strain was amplified by PCR, sequenced, and analyzed by phylogeny, and it was identified as Bacillus Velezii, and named Bacillus Velezii ( Bacillus velezensis a5546).

[0038] The fermentation and compound separation and purification parameters involved in the present invention are well known to those skilled in the art.

[0039] Example 1

[0040] An identification of a Bacillus velez strain for preparing anti-Enterobacterial tridecapeptide comprises the following steps:

[0041] (1) DNA extraction

[0042] Use TSINGKE Plant DNA Extraction Kit (Universal), the specific steps are as follows:

[0043] 1. Place the Spin Column in the Collection Tube, add 250 μL Buffer BL, and centrifuge at 12,000 rpm for 1 min to activate the silica membrane;

[0044] 2. Take the dried tissue of the sample (no more than 20 mg), add liquid nitrogen and grind it thoroughly. After grinding, place it in a 1.5 mL centrifuge tube, add 400 μL Buffer gP1, vortex for 1 min, and place it in a 65℃ water bath for 10-30 min. During this period, you can take it out and mix it upside down to fully lyse it;

[0045] 3. Add 150 μL Buffer gP2, vortex for 1 min, and place on ice for 5 min;

[0046] 4. Centrifuge at 12000 rpm for 5 min and transfer the supernatant to a new centrifuge tube;

[0047] 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 12000 rpm for 30 s, and discard the waste liquid;

[0048] 6. Add 500 μL Buffer Pw (anhydrous ethanol has been added before use) to the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0049] 7. Add 500 μL Wash Buffer (anhydrous ethanol has been added before use) to the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0050] 8. Repeat step 7;

[0051] 9. Place the Spin Column back into the Collection Tube, centrifuge at 12,000 rpm for 2 min, and open the cover to dry for 1 min;

[0052] 10. Take out the Spin Column and put it into a clean centrifuge tube. Add 50-100 μL TE Buffer (preheat TE Buffer at 65℃) to the center of the adsorption membrane. Incubate at 20-25℃ for 2 min and centrifuge at 12,000 rpm for 2 min.

[0053] (2) PCR amplification

[0054] 1. Universal primers for bacterial species identification

[0055]

[0056] 2. The extracted DNA sample was diluted appropriately and used as a PCR template. Amplification was performed using Qingke 1×TSE101 Gold Mix. The components of the amplification system are as follows:

[0057]

[0058] The above amplification system is amplified according to the following amplification program:

[0059]

[0060] (3) Electrophoresis detection

[0061] The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 minutes to obtain the identification gel image. The prepared PCR product was subjected to first-generation sequencing (sequencing primers were 722F / 907R).

[0062] (4) Identification

[0063] 1. Use ContigExpress to assemble the sequencing results and remove the inaccurate parts at both ends.

[0064] 2. Compare the assembled sequences in the NCBI database (blast.ncbi.nlm.nih.gov). The screenshot of the comparison result is as follows: Figure 2 .

[0065] 3. In the comparison results, the species with a homology of more than 97% and the highest homology, the highest ranking, and clear species information obtained from NCBI are generally used as reference species for identification. Figure 1 As shown, the 16S sequence comparison results of the identified strain samples are Bacillus velezensis (or the same genus), so it was named Bacillus Velezii ( Bacillus velezensis a5546).

[0066] Example 2

[0067] A method for preparing an anti-Enterobacterial tridecapeptide comprises the following steps:

[0068] (1) Fermentation

[0069] The Bacillus Velezii with the deposit number of CCTCC No. M20231632 ( Bacillus velezensis a5546) was inoculated in LB slant medium (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) for activation and culture. After culturing at 37°C for 12 hours, it was inoculated into a sterilized LB medium shake flask and cultured at 37°C, 200 rpm, for 12 hours to obtain seed liquid. The seed liquid was inoculated into the optimized liquid medium at a volume ratio of 1% inoculation amount. After culturing at a fermentation temperature of 37°C for 16 hours, the 13-peptide fermentation product was obtained. The fermentation liquid was centrifuged to obtain the supernatant, which was freeze-dried to obtain lyophilized powder.

[0070] The liquid culture medium was prepared as follows: 3.3 g soluble starch, 10 g yeast extract powder, 10 g ammonium sulfate, and 3 g anhydrous magnesium sulfate were dissolved in 1 L ultrapure water, and the pH was adjusted to 6.5 with hydrochloric acid after dissolution. The culture medium was sterilized at 121°C for 20 min before use.

[0071] (2) Extraction, separation and purification

[0072] The obtained lyophilized powder was dissolved in ultrapure water and extracted with an equal volume of n-butanol. Ether was added to the n-butanol extract and the 13 peptide was precipitated overnight at 4°C. The precipitate was dissolved with the initial mobile phase and then separated and purified by semi-preparative reversed-phase high performance liquid chromatography, such as Figure 3 As shown, the fraction corresponding to chromatographic peak No. 1 is the target 13-peptide. The chromatographic column used in the preparation process is an Agilent Zorbax 300SB-C18 PrepHT preparative liquid chromatography column, and the mobile phase is water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B);

[0073] The elution modes are shown in the table below:

[0074] Table 1 Elution gradient for preparation of tridecapeptides

[0075]

[0076] The flow rate was 5 mL / min, the column temperature was 30 °C, and the detection wavelength was 220 nm.

[0077] The obtained fractions were subjected to rotary evaporation to remove acetonitrile and then freeze-dried to obtain the pure tridecapeptide. The purity was tested by analytical reverse phase high performance liquid chromatography. The results were as follows: Figure 4 The purity of the 13 peptide sample prepared in the present invention can be as high as 91.76%. The molecular structure was identified by mass spectrometry, and the results are as follows: Figure 5 As shown. High-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gives its molecular ion peak as m / z 802.9321[M+2H] 2+ and 535.6245[M+3H] 3+The primary and secondary mass spectrometry information proves that the prepared sample of the present invention is a tridecapeptide represented by the following structural formula.

[0078]

[0079] Example 3 Antibacterial activity of anti-Enterobacterial tridecapeptide

[0080] Referring to the National Committee for Clinical Laboratory Standards, MH broth medium (Mueller Hinton Broth medium, Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) was used to serially dilute the 13 peptide to 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 μg / mL. 50 μL of the diluted 13 peptide was added to each well of the 96-well plate, and 50 μL of the peptide was resuspended in 2-7×10 5 CFU / mL indicator bacteria solution. Shake and culture for at least 18 h, read the OD value at 600 nm with a microplate reader to obtain the minimum inhibitory concentration of the 13 peptides. The indicator bacteria selected were: Escherichia coli ATCC 25922, Escherichia coli CVCC1556, Shigella dysenteriae CGMCC 1.1869, Salmonella CVCC534, Escherichia coli CCTCC AB 2013345, Pseudomonas aeruginosa ATCC 27853, Streptococcus dysgalactiae ATCC 35666, Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 29213, Listeria monocytogenes CVCC 3746, Bacillus cereus CVCC4101, and the results are shown in Table 2.

[0081] Table 2 MIC values ​​of the 13 peptides against 11 indicator bacteria

[0082]

[0083] Among them, the 13 peptides have good anti-Gram-negative Enterobacteriaceae activity, with MIC values ​​ranging from 4 to 8 μg / mL. The anti-Gram-positive bacteria activity is weak, with MIC values ​​ranging from 32 to 256 μg / mL. The results show that the 13 peptides have the research and development value of lead drugs and antimicrobial peptides for Gram-negative Enterobacteriaceae.

[0084] Example 4 Stability of anti-Enterobacterial tridecapeptide in aqueous solutions at different pH values

[0085] The pure 13 peptide in Example 2 was prepared into a solution with a concentration of 1.0 mg / mL using ultrapure water, and the pH was adjusted to 1.0, 3.0, 5.0, 7.0, 9.0 and 11.0 using hydrochloric acid and sodium hydroxide solutions, respectively. Samples were taken at 0, 0.5, 1, 2, 3, 4, 5 and 6 h for HPLC and inhibition zone detection. The stability of the 13 peptide in aqueous solutions at different pH values ​​was evaluated by the degradation retention rate obtained by liquid chromatography detection. The results are shown in Figure 2. Figure 6 shown.

[0086] As shown in the figure, the degradation residual rate of the 13 peptide in the pH = 11 aqueous solution dropped to 34.72% at 0.5 h, and the degradation residual rate was 8.21% at 2 h, and maintained at 8.21~10.88% within 6 h; the degradation residual rate of the 13 peptide in the pH = 9 aqueous solution decreased from 100.00% at 0 h to 95.06% at 6 h; the degradation residual rate of the 13 peptide in the pH = 7 aqueous solution was 96.05~100% within 0~6 h; the degradation residual rate of the 13 peptide in the pH = 5 aqueous solution was 99.19~100% within 0~6 h; the degradation residual rate of the 13 peptide in the pH = 3 aqueous solution was 100~100.95% within 0~6 h. The results show that the alkaline environment will cause the denaturation of the 13 peptide, while it can exist stably in the neutral and acidic environment. The results provide guidance for the selection of sample processing, excipients and storage conditions in the future product development process.

[0087] Example 5 Evaluation of the stability of anti-Enterobacterial tridecapeptide in artificial gastric juice

[0088] The stability of the anti-Enterobacterial tridecapeptide in the gastric protein solution was determined by HPLC and inhibition zone method. According to the "Pharmacopoeia of the People's Republic of China" (2020 edition), 16.4 mL of dilute hydrochloric acid was taken, about 800 mL of water and 10 g of pepsin were added, and then shaken well. Then, the mixture was transferred to a 1000 mL volumetric flask and made up to the mark with ultrapure water to obtain artificial gastric juice. The tridecapeptide was dissolved and diluted with artificial gastric juice to a concentration of 200 μg / mL, placed in a 37 ℃ water bath, and samples were taken at 0, 0.5, 1, 2, 4 and 6 h. After the sample was inactivated by 85 ℃ water bath for 5 min, it was centrifuged at 9500 rpm and 4 ℃ for 15 min, the chromatographic peak area of ​​the tridecapeptide at different time points was detected, and the remaining percentage was calculated. Take 100 μL of the solution and use the inhibition zone method to detect the antibacterial activity of the tridecapeptide. The results are as follows: Figure 7 As shown in the figure, the degradation retention rate of the 13 peptide in artificial gastric juice was 94.56-100% after 0-6 h in a 37℃ water bath, and the size of the inhibition zone for the indicator bacteria Escherichia coli ATCC 25922 was in the range of 1.97-1.83 cm. This shows that the 13 peptide can maintain the stability of its structure and antibacterial activity in artificial gastric juice.

[0089] Example 6 Evaluation of the stability of anti-Enterobacterial tridecapeptide in artificial intestinal fluid

[0090] The stability of the thirteen peptides in artificial intestinal fluid was determined by high performance liquid chromatography and the inhibition zone method. According to the "Pharmacopoeia of the People's Republic of China" (2020 edition), 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution was taken, 118 mL of 0.2 mol / L sodium hydroxide solution was added, and 10 g of pancreatic enzyme was taken. An appropriate amount of water was added to dissolve it. After the two liquids were mixed, they were transferred to a 1000 mL volumetric flask and diluted to the scale with water to obtain a trypsin solution. The thirteen peptides were dissolved and diluted with trypsin solution to a concentration of 200 μg / mL, placed in a 37 ℃ water bath, and samples were taken at 0, 0.5, 1, 2, 4 and 6 h. After the sample was inactivated by 85 ℃ water bath for 5 min, it was centrifuged at 9500 rpm and 4 ℃ for 15 min. The chromatographic peak area of ​​the thirteen peptides at different time points was detected, and the remaining percentage was calculated. Take 100 μL of the solution and use the inhibition zone method to detect the antibacterial activity of the thirteen peptides. The results are as follows Figure 8 As shown in the figure, the degradation retention rate of the 13 peptides in artificial intestinal fluid was 33.36-100% after 0-6 h in a 37 ℃ water bath, and the size of the inhibition zone for the indicator bacteria Escherichia coli ATCC 25922 was in the range of 1.97-2.07 cm. The 13 peptides were easily degraded into other substances with certain antibacterial activity in the artificial intestinal fluid environment.

[0091] Example 7 Evaluation of the hemolytic activity of the anti-Enterobacterial tridecapeptide

[0092] Whole blood from pigs was collected intravenously using a blood collection tube containing sodium heparin, and red blood cells were obtained after centrifugation. The blood cells were washed with 10 mM PBS (pH7.3) until the supernatant had no obvious red color. The blood cells were prepared into a 4% suspension with PBS, and 100 μL of 4% blood cells and 100 μL of a dilution of 13-peptide solution with a concentration range of 0-512 μg / mL were added to the wells of a 96-well plate, incubated in a 37°C incubator for 1 h, centrifuged at 1500 rpm for 5 min, and the supernatant was transferred to a new 96-well plate. The OD value was detected at 540 nm using an enzyme reader. 10 mM PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively. The hemolyticity of the 13-peptide was calculated. The calculation formula is as follows:

[0093]

[0094] Among them, A 十三肽 A is the OD value of different concentrations of 13 peptides added to the blood cell well; PBS A is the OD value of adding PBS into the blood cell well; 0.1% Triton X-100 The OD value of 0.1% Triton X-100 added to the blood cell well. Fig. 9As shown in the figure, the hemolytic rate of the 13 peptides was 0.303~1.924% in the concentration range of 256~0.5 μg / mL, and its variation was within 5%. The results showed that the 13 peptides had no obvious hemolytic activity.

[0095] In summary, in the present invention, based on Bacillus Velezii ( Bacillus velezensis The anti-Enterobacterial thirteen peptides produced by a5546 have the advantage of being able to resist a variety of Gram-negative pathogenic Enterobacteriaceae in various applications and have a very broad application prospect.

[0096] The above are only embodiments of the present invention. The invention is not limited to the fields involved in this implementation case. The common sense such as the known specific structures and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field to which the invention belongs before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the present invention. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to explain the content of the claims.

Claims

1. A strain of Bacillus velez Bacillus velezensis ), which is characterized by: The bacteria are deposited in China Center for Type Culture Collection, the deposit date is September 7, 2023, the address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No.M20231632.

2. Use of the Bacillus Velezii as claimed in claim 1 in the preparation of an antimicrobial peptide; the antimicrobial peptide is an anti-Enterobacterial tridecapeptide, the structural formula of which is shown in (I): 。 3. The method for preparing the anti-Enterobacterial tridecapeptide prepared by Bacillus Velezii as claimed in claim 1, wherein the anti-Enterobacterial tridecapeptide has the structural formula (I) as shown in the formula (I), characterized in that The steps include: (1) Cultivation and fermentation The Velez subspecies described in claim 1 is inoculated in an LB slant medium for activation, cultured in an environment of 37°C for 12 h, inoculated into a sterilized LB medium shake flask, cultured at 37°C, 200 rpm, for 12 h to obtain a seed solution, inoculated into a liquid culture medium at an inoculum volume ratio of 1%, cultured in an environment of 37°C for 16 h, centrifuged the fermentation solution to obtain a supernatant, and freeze-dried to obtain a lyophilized powder of the thirteen peptide fermentation solution; (2) Crude extract extraction Add ultrapure water to the lyophilized powder obtained in step (1) to dissolve it, extract it with an equal volume of n-butanol, add ether to the n-butanol extract and incubate it at 4°C overnight to precipitate the tridecapeptide; (3) Separation and preparation of tridecapeptides The 13-peptide precipitate obtained in step (2) is dissolved in the initial mobile phase and then separated and purified by semi-preparative reversed-phase high performance liquid chromatography. The obtained fraction is subjected to rotary evaporation to remove acetonitrile and then freeze-dried to obtain the purified 13-peptide pure product, and the purity is determined by reversed-phase high performance liquid chromatography; The preparation process of the reversed-phase high performance liquid chromatography is as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phases contain 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution mode is shown in Table 1: The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

4. The method for preparing the anti-Enterobacterial tridecapeptide by Bacillus Velezii as claimed in claim 3, characterized in that: The liquid culture medium described in step (1) is: 3.3 g soluble starch, 10 g yeast extract powder, 10 g ammonium sulfate, and 3 g anhydrous magnesium sulfate are dissolved in 1 L ultrapure water, and after dissolution, the pH is adjusted to 6.5 with hydrochloric acid.

5. The preparation method according to claim 4, characterized in that: The ratio of the lyophilized powder to ultrapure water in step (2) is 1:1 to 1:5 (W / V). After dissolution, an equal volume of n-butanol is added and extracted 1 to 3 times. The n-butanol extract is transferred to a new conical flask and an equal volume of ether is added to precipitate the tridecapeptide.

6. Use of the Bacillus Velezii as claimed in claim 1 in the preparation of a drug or feed additive for resisting infection by Gram-negative Enterobacteriaceae; the Gram-negative Enterobacteriaceae is Escherichia coli, Salmonella, Shigella dysenteriae or Pseudomonas aeruginosa.

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