Hippophae rhamnoides seed meal antibacterial peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of antibacterial peptide preparation, and particularly discloses a hippophae rhamnoides seed meal antibacterial peptide and a preparation method and application thereof.The hippophae rhamnoides seed meal antibacterial peptide is prepared through the steps that hippophae rhamnoides seed meal is smashed, sieved, degreased, dissolved and centrifuged to obtain hippophae rhamnoides seed meal crude protein, then protease is used for enzymolysis, and the hippophae rhamnoides seed meal crude protein is obtained; centrifuging, taking supernatant, and freeze-drying to obtain the sea-buckthorn seed meal antibacterial peptide; the protease is selected from any one of trypsin, alpha-chymotrypsin and alkaline protease. The hippophae rhamnoides seed meal antibacterial peptide prepared by the invention can inhibit staphylococcus aureus, escherichia coli and salmonella.

Description

Technical Field

[0001] The invention relates to the technical field of antibacterial peptide preparation, and in particular to a seabuckthorn seed meal antibacterial peptide and a preparation method and application thereof. Background Art

[0002] In recent years, the extensive use and even abuse of antibiotics has led to the emergence of many drug-resistant bacteria, posing a threat to environmental pollution and human health and safety. To address this problem, people have begun to actively explore efficient, safe, and environmentally friendly alternatives to antibiotics to seek solutions.

[0003] In this context, antimicrobial peptides have become a hot topic of research. As a class of small molecule peptide chains with the activity of killing or inhibiting microorganisms, antimicrobial peptides are an important component of the innate immunity of organisms and are widely present in animals, plants and microorganisms. Bacteria have developed various bacterial resistance mechanisms against antimicrobial peptides, including modification of cell surface components, degradation of antimicrobial peptides, and antimicrobial peptide efflux. Therefore, it is very necessary to develop new antimicrobial peptides that can inhibit bacteria. Summary of the invention

[0004] In order to develop an antibacterial peptide that inhibits bacteria, the present invention provides a seabuckthorn seed meal antibacterial peptide and a preparation method and application thereof. The seabuckthorn seed meal antibacterial peptide prepared by the present invention can inhibit Staphylococcus aureus, Escherichia coli and Salmonella.

[0005] The present invention provides a seabuckthorn seed meal antibacterial peptide, wherein the seabuckthorn seed meal antibacterial peptide is obtained by defatting, dissolving and centrifuging the seabuckthorn seed meal to obtain the seabuckthorn seed meal crude protein, then enzymolyzing the seabuckthorn seed meal with a protease, centrifuging and taking the supernatant for freeze-drying to obtain the seabuckthorn seed meal antibacterial peptide;

[0006] The protease is selected from any one of trypsin, α-chymotrypsin and alkaline protease.

[0007] The present invention obtains seabuckthorn seed meal antibacterial peptides by enzymatic extraction from seabuckthorn seed meal, and the seabuckthorn seed meal antibacterial peptides have significant inhibitory effects on Staphylococcus aureus, Escherichia coli and Salmonella. The seabuckthorn seed meal antibacterial peptides act on Staphylococcus aureus and can cause the cell membrane of Staphylococcus aureus to be deformed and damaged and ruptured.

[0008] Furthermore, the seabuckthorn seed meal antibacterial peptides contain 8 antibacterial peptides whose sequences are shown as SEQ ID NO.1 to SEQ ID NO.8.

[0009] The present invention also provides a method for preparing the antibacterial peptide from seabuckthorn seed meal, comprising the following steps:

[0010] The seabuckthorn seed meal is crushed and sieved, defatted with an organic solvent and dried to obtain seabuckthorn seed meal powder, dissolved in distilled water and then bathed in 45°C to 55°C for 60min to 70min, and the precipitate is collected by centrifugation and freeze-dried to obtain the seabuckthorn seed meal crude protein;

[0011] The crude protein of seabuckthorn seed meal is prepared into an antibacterial peptide solution with distilled water, the pH is adjusted to 8-10, 4000U-8000U of protease is added, and enzymolysis is carried out at 35℃-45℃ for 1h-3h. After the enzyme is inactivated, the supernatant is centrifuged and freeze-dried to obtain the seabuckthorn seed meal antibacterial peptide.

[0012] The enzymatic hydrolysis conditions of the present invention are set as follows: pH 8-10, adding 4000U-8000U of protease, and enzymatic hydrolysis at 35°C-45°C for 1h-3h. The antibacterial activity of the seabuckthorn seed meal antibacterial peptide prepared by the present invention is significantly higher than the antibacterial activity of the seabuckthorn seed meal antibacterial peptide prepared under other enzymatic hydrolysis conditions.

[0013] Furthermore, the crude protein of seabuckthorn seed meal was prepared into an antibacterial peptide solution with distilled water, the pH was adjusted to 9, 6000U of protease was added, and enzymatic hydrolysis was performed at 40°C for 2h. After the enzyme was inactivated, the supernatant was centrifuged and freeze-dried to obtain the seabuckthorn seed meal antibacterial peptide.

[0014] Furthermore, the protease is trypsin, and the seabuckthorn seed meal antibacterial peptide obtained by trypsin hydrolysis has the best antibacterial effect.

[0015] Furthermore, the organic solvent is any one of petroleum ether, acetone and ether.

[0016] The present invention also provides an application of the antibacterial peptide of seabuckthorn seed meal in the preparation of antibacterial drugs, wherein the antibacterial peptide is used to inhibit Staphylococcus aureus, Escherichia coli and Salmonella.

[0017] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0018] Furthermore, the auxiliary material is one or a combination of starch, lactose, sucrose, mannitol, cellulose and gelatin.

[0019] The present invention also provides an antimicrobial peptide, wherein the antimicrobial peptide is selected from any one of the following 8 antibacterial peptides;

[0020] Antimicrobial peptide 1: AIGVDILK;

[0021] Antibacterial peptide 2: ALEACVKARNEGR;

[0022] Antimicrobial peptide 3: ARPAADER;

[0023] Antimicrobial peptide 4: IGGIGTVPVGR;

[0024] Antimicrobial peptide 5: ILSLSPSR;

[0025] Antimicrobial peptide 6: VGLAETIR;

[0026] Antimicrobial peptide 7: VLQLETAAGAAIR;

[0027] Antibacterial peptide 8: VPTVDVSVVDLTVR.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses seabuckthorn seed meal as raw material, and the seabuckthorn seed meal antibacterial peptide obtained by enzymatic extraction can inhibit Staphylococcus aureus, Escherichia coli and Salmonella. The seabuckthorn seed meal antibacterial peptide obtained by trypsin enzymatic hydrolysis has the best antibacterial effect. The seabuckthorn seed meal antibacterial peptide acts on Staphylococcus aureus and can cause the cell membrane of Staphylococcus aureus to be deformed and damaged and ruptured. The seabuckthorn seed meal antibacterial peptide contains 8 antibacterial peptides, and the sequences are shown in SEQ ID NO.1 to SEQ ID NO.8. In the method for preparing seabuckthorn seed meal antibacterial peptide provided by the present invention, the enzymatic hydrolysis conditions are limited to: pH 8 to 10, adding 4000U to 8000U of protease, and enzymatic hydrolysis at 35°C to 45°C for 1h to 3h, so that the antibacterial activity of the obtained seabuckthorn seed meal antibacterial peptide is significantly improved compared with other conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The antibacterial effect of each component of seabuckthorn seed meal antibacterial peptide ultrafiltration on Staphylococcus aureus;

[0032] In the figure, A shows the inhibition of Staphylococcus aureus by four polypeptide components with different molecular weights;

[0033] B is a statistical graph of the inhibitory diameters of four different molecular weights F-Ⅰ, F-Ⅱ, F-Ⅲ and F-Ⅳ against Staphylococcus aureus.

[0034] Figure 2 The results of gel chromatography separation and antibacterial activity of F-Ⅲ component are shown;

[0035] In the figure, A is the gel chromatography separation chromatogram of the F-III component;

[0036] The effect diagram of BF-Ⅲ-A and F-Ⅲ-B in inhibiting Staphylococcus aureus;

[0037] C is the statistical graph of the inhibition diameter of F-Ⅲ-A and F-Ⅲ-B against Staphylococcus aureus.

[0038] Figure 3 This is the total ion current diagram of component F-Ⅲ-A.

[0039] Figure 4 This is the mass spectrum of antibacterial peptide 1, antibacterial peptide 2, antibacterial peptide 3 and antibacterial peptide 4. In the figure, a, b, c and d represent antibacterial peptide 1, antibacterial peptide 2, antibacterial peptide 3 and antibacterial peptide 4, respectively.

[0040] Figure 5 It is the mass spectrum of antibacterial peptide 5, antibacterial peptide 6, antibacterial peptide 7 and antibacterial peptide 8. In the figure, a, b, c and d represent antibacterial peptide 5, antibacterial peptide 6, antibacterial peptide 7 and antibacterial peptide 8, respectively.

[0041] Figure 6 The simulation results of docking of antibacterial peptide with Staphylococcus aureus receptor molecule;

[0042] In the figure, A is the molecular docking simulation result of antibacterial peptide 1 and Staphylococcus aureus receptor;

[0043] B is the molecular docking simulation result of antibacterial peptide 2 against Staphylococcus aureus receptor;

[0044] C is the molecular docking simulation result of the antibacterial peptide 3 Staphylococcus aureus receptor;

[0045] D is the molecular docking simulation result of antibacterial peptide 4 Staphylococcus aureus receptor;

[0046] E is the molecular docking simulation result of the antibacterial peptide 5 Staphylococcus aureus receptor;

[0047] F is the molecular docking simulation result of the antibacterial peptide 6 Staphylococcus aureus receptor;

[0048] G is the molecular docking simulation result of the antibacterial peptide 7 Staphylococcus aureus receptor;

[0049] H is the molecular docking simulation result of antibacterial peptide 8 on Staphylococcus aureus receptor.

[0050] Figure 7 The antibacterial activity of component F-Ⅲ-A against Staphylococcus aureus was determined by the microplate dilution method. In the figure, A represents the antibiotic positive group, B represents the F-Ⅲ-A group; and C represents the sterile saline negative group.

[0051] Figure 8 The results are as follows: the effect of antibacterial peptides from seabuckthorn seed meal on Staphylococcus aureus;

[0052] In the figure, A is a laser confocal micrograph of Staphylococcus aureus treated with the control group;

[0053] B is the laser confocal micrograph of Staphylococcus aureus treated with 1×MIC, where 1×MIC means 1 times the MIC value, i.e., 2.5 mg / mL F-Ⅲ-A;

[0054] C is the laser confocal micrograph of Staphylococcus aureus treated with 4×MIC. 4×MIC means 4 times the MIC value, i.e., 10 mg / mL F-Ⅲ-A.

[0055] Fig. 9 The scanning electron micrographs of Staphylococcus aureus after being treated with different concentrations of antibacterial peptides;

[0056] In the figure, A is the SEM image of the control group after S. aureus, (a), (b), and (c) represent three repeated treatments;

[0057] B is a scanning electron micrograph of Staphylococcus aureus after treatment with an antibacterial peptide at a concentration of 1×MIC, (a), (b) and (c) represent three repeated treatments, respectively;

[0058] C is a scanning electron micrograph of Staphylococcus aureus after treatment with an antibacterial peptide at a concentration of 4×MIC, where (a), (b) and (c) represent three repeated treatments, respectively.

[0059] Fig.10 The inhibitory effects of peptides prepared by enzymatic hydrolysis with different proteases on different pathogens;

[0060] In the figure, a is the inhibitory effect of peptides prepared by different protease hydrolysis on Staphylococcus aureus, A, B, C, D, E, F, G and H represent trypsin, neutral protease, acid protease, alkaline protease, α-chymotrypsin, papain, pepsin and composite protease, respectively;

[0061] b is the inhibitory effect of peptides prepared by different protease hydrolysis on Bacillus subtilis, A, B, C, D, E, F, G and H represent trypsin, neutral protease, acid protease, alkaline protease, α-chymotrypsin, papain, pepsin and composite protease, respectively;

[0062] c is the inhibitory effect of peptides prepared by different protease hydrolysis on Escherichia coli, A, B, C, D, E, F, G and H represent trypsin, neutral protease, acid protease, alkaline protease, α-chymotrypsin, papain, pepsin and composite protease, respectively;

[0063] d is the inhibitory effect of peptides prepared by different protease hydrolysis on Salmonella. A, B, C, D, E, F, G and H represent trypsin, neutral protease, acid protease, alkaline protease, α-chymotrypsin, papain, pepsin and composite protease, respectively.

[0064] Fig.11 The effect of trypsin hydrolysis conditions on the antibacterial activity and hydrolysis degree of Staphylococcus aureus;

[0065] In the figure, A shows the effect of the antibacterial activity and hydrolysis degree of the peptides obtained by trypsin hydrolysis at different pH values ​​on Staphylococcus aureus;

[0066] B is the effect of trypsin on the antibacterial activity and hydrolysis degree of peptides obtained at different enzymatic hydrolysis temperatures against Staphylococcus aureus;

[0067] C is the effect of the antibacterial activity and hydrolysis degree of the peptides obtained by trypsin at different enzymatic hydrolysis times on Staphylococcus aureus;

[0068] D is the effect of trypsin on the antibacterial activity and hydrolysis degree of peptides obtained by adding different enzyme amounts against Staphylococcus aureus.

[0069] Fig.12 The stability study results of the antibacterial activity of the antibacterial peptides from seabuckthorn seed meal prepared by the present invention;

[0070] In the figure, A is the effect of temperature on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal;

[0071] B is the effect of ultraviolet irradiation time on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal;

[0072] C is the effect of pH value on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal;

[0073] D is the effect of repeated freezing and thawing times on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal. DETAILED DESCRIPTION

[0074] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0075] Experimental materials used in the present invention: seabuckthorn seed meal (byproduct of supercritical CO2 low-temperature extraction of oil) was provided by Xinjiang Zhongke Seabuckthorn Technology Co., Ltd.; Staphylococcus aureus CMCC (B) 26003, Escherichia coli CMCC (B) 44102, Bacillus subtilis CMCC (B) 63501, and Salmonella CMCC (B) 50335 were provided by the Drug and Instrument Supervision and Inspection Station of the Support Department of the Xinjiang Military Region; sodium hydroxide, hydrochloric acid, and petroleum ether were all domestically produced analytical grade; alkaline protease (200U / mg), pepsin (Porcine gastric mucosa) (30 U / mg), neutral protease (100 U / mg), papain (800 U / mg), composite protease (120 U / mg): Shanghai Yuanye Biotechnology Co., Ltd.; α-chymotrypsin (800 U / mg): Shanghai McLean Biochemical Technology Co., Ltd.; trypsin (250 U / mg), agar: Beijing Solebow Technology Co., Ltd.; acid protease (50 U / mg): Shanghai Blue Season Biological Reagent Co., Ltd.; LB broth medium: Nantong Kaiheng Biotechnology Development Co., Ltd.

[0076] Example 1: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0077] Preparation of crude protein from seabuckthorn seed meal: grind the seabuckthorn seed meal through a 60-mesh sieve, defatted with petroleum ether and then dried to obtain seabuckthorn seed meal powder; weigh 100g of seabuckthorn seed meal powder, add distilled water at a solid-liquid ratio of 1:9, adjust the solution pH to 10, water bath at 50℃ for 60min, centrifuge at 6000r / min for 10min, collect the supernatant and adjust the pH to 5, centrifuge at 6000r / min for 10min, and freeze-dry the precipitate to obtain crude protein from seabuckthorn seed meal.

[0078] Preparation of antibacterial peptides from seabuckthorn seed meal: crude protein from seabuckthorn seed meal was mixed with distilled water to prepare an antibacterial peptide solution with a mass concentration of 3%, the pH of the antibacterial peptide solution was adjusted to 9.0, 6000U / g of trypsin was added, enzymatic hydrolysis was carried out in a 40°C water bath for 2.0h, the enzyme was inactivated in a boiling water bath for 10min, centrifuged at 6000r / min for 10min, and the supernatant was freeze-dried to obtain the antibacterial peptides from seabuckthorn seed meal.

[0079] Example 2: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0080] Preparation of crude protein from seabuckthorn seed meal: Grind the seabuckthorn seed meal through a 60-mesh sieve, defatted with petroleum ether and then dried to obtain seabuckthorn seed meal powder; weigh 100g of seabuckthorn seed meal powder, add distilled water at a solid-liquid ratio of 1:9, adjust the solution pH to 10, place in a 50℃ water bath for 60min, centrifuge at 6000r / min for 10min, adjust the supernatant pH to 5, centrifuge at 6000r / min for 10min, and freeze-dry the precipitate to obtain crude protein from seabuckthorn seed meal.

[0081] Preparation of antibacterial peptides from seabuckthorn seed meal: crude protein from seabuckthorn seed meal was mixed with distilled water to prepare an antibacterial peptide solution with a mass concentration of 3%, the pH of the antibacterial peptide solution was adjusted to 8.9, 6240 U / g of trypsin was added, and enzymatic hydrolysis was carried out in a 39.5°C water bath for 2.2 h, the enzyme was inactivated in a boiling water bath for 10 min, centrifuged at 6000 r / min for 10 min, and the supernatant was freeze-dried to obtain the antibacterial peptides from seabuckthorn seed meal.

[0082] The seabuckthorn seed meal antibacterial peptide prepared in Example 1 was used as the object to study its inhibitory effect on Staphylococcus aureus. The seabuckthorn seed meal antibacterial peptide was separated, purified and peptide segments were identified by ultrafiltration, gel chromatography filtration (Sephadex G-25), liquid chromatography and tandem mass spectrometry (LC-MS / MS), and potential antibacterial peptide segments were screened out through the antibacterial peptide database (APD3) and combined with molecular docking methods to analyze their interaction force and mode of action. The minimum inhibitory concentration (MIC) of the antibacterial peptide was determined; combined with laser confocal scanning microscopy, scanning electron microscopy and other methods, its antibacterial mechanism was preliminarily explored from the aspects of the permeability and integrity of the cell membrane of Staphylococcus aureus. The specific experimental methods and results are as follows:

[0083] 1. Test methods

[0084] 1. Study on the inhibitory effect of antibacterial peptides from seabuckthorn seed meal on Staphylococcus aureus

[0085] A single colony of Staphylococcus aureus was selected and activated in LB liquid medium. The colony was placed in a 37°C constant temperature oscillator and cultured until the logarithmic growth phase. The concentration was determined by comparing with a McFarland turbidimeter tube and the activated test strain was diluted to a concentration of 110 with sterile saline. 6 CFU / mL bacterial suspension, stored at 4℃ for later use. Pour in LB solid culture medium melted at about 50℃, with a thickness of 5mm, shake gently and evenly, wait for it to solidify, take 100uL of bacterial suspension and spread it evenly on the surface of LB solid culture medium with a bacteria stick, punch holes in the plate with a 6mm sterilized metal puncher, pick out the agar in the hole, seal the bottom with an alcohol lamp, and add 80uL of sample solution with a concentration of 80mg / mL to each well. The sample solution is obtained by dissolving seabuckthorn seed meal antibacterial peptide in sterile water. Place the culture dish in a 37℃ constant temperature incubator and culture it for 24h, and measure the diameter of the inhibition zone by the cross method. The average value of the diameter of the inhibition zone of 3 parallel holes is taken as the measurement result.

[0086] 2. Sephadex G-25 chromatography separation and purification

[0087] The antibacterial peptides from seabuckthorn seed meal were prepared into a 20 mg / mL polypeptide solution and filtered through a 0.45 μm microporous filter membrane. The polypeptide solution was then divided into four components, >30 KDa (F-Ⅳ), 30-10 KDa (F-Ⅲ), 10-3 KDa (F-Ⅱ) and <3 KDa (F-Ⅰ), by ultrafiltration membranes with different molecular retention rates. The antibacterial activity was determined after freeze-drying. The results were repeated three times and the average value was taken.

[0088] The swollen Sephadex G-25 was loaded into a 1.6cm×60cm chromatography column, and the components with better antibacterial activity after ultrafiltration were mixed with ultrapure water to prepare a (10mg / mL) peptide solution. After passing through a 0.45um water-based microporous filter membrane, the solution was added to the chromatography column by rotating along the tube wall. The sample volume was 2mL each time, and the ultrapure water was eluted at a flow rate of 1mL / min. UV detection was performed at a wavelength of 280nm. At the same time, different elution peaks were collected according to the UV absorption signal, freeze-dried, and the antibacterial activity was determined. Repeat three times and take the average value.

[0089] 3. LC-MS / MS Identification Analysis

[0090] (1) Chromatographic separation

[0091] Liquid A used in the liquid phase was 0.1% formic acid in water, and liquid B was 0.1% formic acid in acetonitrile (84% acetonitrile). The liquid chromatography column (0.15 mm × 150 mm, RP-C18, Column Technology Inc.) was equilibrated with 95% liquid A, and the sample was loaded into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an automatic sampler and then separated by the liquid chromatography column. The relevant liquid phase gradient was set as follows:

[0092] 0 min - 50 min, linear gradient of solution B from 4% to 50%;

[0093] 50-54 minutes, linear gradient of solution B from 50% to 100%;

[0094] 54 minutes---60 minutes, liquid B was maintained at 100%.

[0095] (2) Mass spectrometry identification

[0096] The enzymatic hydrolysis product (the antibacterial peptide component of seabuckthorn seed meal after ultrafiltration) was separated by capillary high performance liquid chromatography and analyzed by mass spectrometry using a QExactive HF-X mass spectrometer (Thermo Fisher). The analysis time was 60 min. Detection method: positive ion. The mass-to-charge ratio of the peptide and its fragments was collected according to the following method: 10 fragment spectra (MS2 scan) were collected after each full scan (full scan).

[0097] (3) Database search

[0098] The raw files of mass spectrometry test were searched into the corresponding database using the software MaxQuant 1.5.5.1, and finally the protein identification and quantitative analysis results were obtained. Potential antibacterial peptides were screened out through the antibacterial peptide database (APD3).

[0099] (4) Molecular docking

[0100] The 3D structure of the peptide was constructed using Chemdraw and Chem3D. Then, the RCSB PDB database (http: / / www.rcsb.org / ) was used to screen the crystal structure of the original strain of Staphylococcus aureus with high resolution (PDB: 7ASN) as the molecular docking receptor. The protein was dehydrated and dephosphorylated using PyMOL software and saved as a PDB file. The Molecular Operating Environment 2019 software was used to minimize the energy of the peptide, pre-process the Staphylococcus aureus receptor and search for active pockets. Finally, MOE 2019 was run for molecular docking. The binding activity of the two was evaluated based on the binding energy, and the results were visualized using PyMOL software.

[0101] (5) Determination of MIC

[0102] The antibacterial peptides from seabuckthorn seed meal were prepared into an 80 mg / mL antibacterial peptide solution with sterile water, and 100uL was added to a 96-well plate. The positive group was added with the same volume of antibiotic solution, and the blank group was a sterile aqueous solution. Diluted with sterile water in turn, and 100uL of the solution was aspirated from the last well. The final concentration in the wells was 80, 40, 20, 10, 5, 2.5, 1.25, and 0.625 mg / mL. Take Staphylococcus aureus in the logarithmic growth period and measure its concentration with a McFarland turbidimeter tube, and dilute it to 1×10 with fresh LB medium. 6 CFU / mL, put the diluted bacterial solution into a 96-well plate, 100uL per well, put the 96-well plate into a constant temperature incubator at 37℃ and culture for 15h, add resazurin dye, observe the color change of the dye, the lowest concentration when the dye is still blue is the minimum inhibitory concentration (MIC) of the component, and repeat the test 3 times.

[0103] (6) Laser confocal microscopy and scanning electron microscopy

[0104] Prepare 0.5 mL of peptide solution with a mass concentration of 2.5 mg / mL (MIC) and 10 mg / mL (4 MIC) with sterile PBS in corresponding 1.5 mL centrifuge tubes for later use. Dilute the bacterial solution to 10 8 CFU / mL, add the same volume of diluted bacterial suspension to the centrifuge tube, vortex mix, and the control group is sterile PBS, placed in a 37℃ constant temperature incubator for shaking culture for 6h. After the culture is completed, the bacteria are collected by centrifugation (8000r / min3min), the supernatant is discarded, and 300μL of sterile PBS is added to resuspend the bacteria. Prepare the staining working solution (SYTO-9 working concentration is 5μM and PI working concentration is 2μg / mL), and stain at 37℃ in the dark for 30min. After staining, the sample is placed on a laser confocal microscope (400 times) for photography.

[0105] Staphylococcus aureus was cultured to the logarithmic growth phase, the cells were collected by centrifugation (5000r / min, 10min), and resuspended in sterile saline to prepare a bacterial suspension (10 8 CFU / mL), add 4mL of bacterial suspension to a 10mL sterile centrifuge tube, and then add the same volume of peptide solution with a mass concentration of 2.5mg / mL (MIC) and 10mg / mL (4MIC) respectively, and shake and culture in a 37℃ constant temperature incubator for 6h. Then centrifuge to collect the bacteria, wash the bacteria three times with sterile saline, add 4% glutaraldehyde solution and fix it in a 4℃ refrigerator overnight. After washing three times with sterile saline again, ethanol gradient dehydration was performed, freeze-dried, and the bacteria morphology was observed under a scanning electron microscope after gold spraying on the adhesive platform. The saline group was used as the control group.

[0106] 4. Data processing and analysis

[0107] Origin 2021 software was used for drawing, and IBM SPSS statistics 26 software was used for ANOVA significance analysis. Three parallels were made for each sample, and the results were expressed as mean ± standard error.

[0108] 2. Experimental Results

[0109] 1. Study on the inhibitory effect of antibacterial peptides from seabuckthorn seed meal on Staphylococcus aureus

[0110] The results are as follows Figure 1 As shown, the antibacterial peptides from seabuckthorn seed meal prepared by the present invention are separated into four polypeptide components with different molecular weights after ultrafiltration: >30KDa (F-Ⅳ), 30KDa~10KDa (F-Ⅲ), 10KDa~3KDa (F-Ⅱ) and <3KDa (F-Ⅰ). These four components all have inhibitory effects on Staphylococcus aureus, among which F-Ⅲ has better antibacterial activity than other components (P<0.05) and the antibacterial diameter is 18.73mm.

[0111] 2. Separation and purification results of Sephadex G-25 chromatography

[0112] The results are as follows Figure 2 As shown, by further gel chromatography separation of the F-Ⅲ component, two components F-Ⅲ-A and F-Ⅲ-B were obtained. After the antibacterial activity of the two components against Staphylococcus aureus was determined, it was found that the antibacterial activity of component F-Ⅲ-A was significantly stronger than that of F-Ⅲ-B (P < 0.05), so component F-Ⅲ-A was selected for further experiments.

[0113] 3. LC-MS / MS identification and analysis results

[0114] The peptide sequence of component F-Ⅲ-A was identified and analyzed using LC-MS / MS technology. The total ion current of F-Ⅲ-A is shown in Figure 3 As shown in Table 1, the peptides identified were subjected to homology analysis by the antibacterial peptide database (APD3) and potential antibacterial peptides were screened. The results are shown in Table 1. The present invention obtained 8 antibacterial peptides with sequences as shown in SEQ ID NO.1-SEQ ID NO.8, which were respectively recorded as antibacterial peptide 1, antibacterial peptide 2, antibacterial peptide 3, antibacterial peptide 4, antibacterial peptide 5, antibacterial peptide 6, antibacterial peptide 7 and antibacterial peptide 8. The mass spectra of the 8 antibacterial peptides are shown in Table 1. Figure 4 and Figure 5 All 8 antibacterial peptides contain arginine, and their specific information is shown in Table 1.

[0115] Table 1 Screening of antibacterial peptides from seabuckthorn seed meal

[0116]

[0117] 4. Molecular docking simulation results

[0118] In order to explore the interaction between the eight antibacterial peptides and the Staphylococcus aureus receptor, molecular docking was performed using MOE 2019 software. According to the binding results, the eight peptides can enter the binding domain of the Staphylococcus aureus receptor respectively. Figure 6 The molecular docking energy range of the 8 peptides and receptor binding is -7.4156-10.0265 kcal / mol, and the average binding energy is -8.3608 kcal / mol. It may be that these 8 peptides have different side chain groups that bind to the receptor active site. The docking energy values ​​of antibacterial peptide 7+Staphylococcus aureus, antibacterial peptide 8+Staphylococcus aureus, and antibacterial peptide 2+Staphylococcus aureus are lower, which means that the peptide compound has a stronger binding force with the Staphylococcus aureus receptor. The binding of peptides to receptors is mainly through hydrogen bond interactions and electrostatic interactions.

[0119] Among them, the antimicrobial peptide 7 (VLQLETAAGAAIR) with the highest binding strength interacts with the S. aureus receptor and can form hydrogen bond interactions with multiple binding sites (Glu23, Pro91, Arg89, Lys70, Lys24, Arg53 and Lys10). Lys24 and Glu23 in the S. aureus receptor form electrostatic interactions with the compound antimicrobial peptide 7. Antimicrobial peptide 2 (ALEACVKARNEGR) also has a strong binding ability with the S. aureus receptor, with a binding energy of -9.6447 kcal / mol. Residues Pro81, Trp83, Thr10, Arg18, Arg21, and Arg16 form hydrogen bonds with the ligand antimicrobial peptide 2 (ALEACVKARNEGR). The Arg16 and Arg14 residues in the S. aureus receptor form electrostatic interactions with the ligand. The ligand antibacterial peptide 8 (VPTVDVSVVDLTVR) also showed strong binding strength with Staphylococcus aureus, with a binding energy of -9.3121 kcal / mol, and was able to interact with the residues of the receptor. As shown in the figure, the Lys69 residue in the Staphylococcus aureus receptor formed a hydrogen bond interaction with the antibacterial peptide 8 (VPTVDVSVVDLTVR), and the residue Lys73 formed an electrostatic interaction with the ligand.

[0120] 5. Determination of MIC

[0121] Resazurin was used as an indicator (a blue non-fluorescent, non-toxic dye that can be reduced by oxidoreductase in bacteria to appear pink) to determine the MIC of F-Ⅲ-A against Staphylococcus aureus.

[0122] The results are as follows Figure 7 As shown, the antibiotic (A) positive group all showed blue, indicating that all the bacteria in the well plate were dead; the sterile saline (C) group all showed pink, indicating that the bacteria in the well plate were not inhibited; the F-Ⅲ-A (B) group showed pink fluorescence at a concentration of 1.25 mg / mL, so the MIC of component F-Ⅲ-A was 2.5 mg / mL.

[0123] 6. Laser confocal microscopy observation

[0124] The results of the effect of seabuckthorn seed meal antibacterial peptides on Staphylococcus aureus are as follows Figure 8As shown in the figure, the cell membrane of normal bacteria is not permeable to PI. Only when the bacteria die or apoptosis and the integrity of the biofilm is destroyed can PI enter, thus showing up through laser confocal microscopy. The green fluorescence after SYTO9 staining indicates that Staphylococcus aureus has a complete cell membrane, and the red fluorescence after PI staining indicates that the integrity of the cell membrane is destroyed. Compared with the negative control group, the 1×MIC treatment group PI single channel shows red fluorescence, indicating that the integrity of the Staphylococcus aureus cell membrane is changing. Under 4×MIC treatment, it can be seen that the red fluorescence of the PI single channel is significantly enhanced, and the range is increased, indicating that with the increase in concentration, the integrity of the Staphylococcus aureus cell membrane has changed significantly.

[0125] 7. Scanning electron microscope observation

[0126] like Fig. 9 As shown, the Staphylococcus aureus in the control group was spherical, with a smooth surface and a relatively regular shape, without obvious depressions and breakages. After the Staphylococcus aureus was treated with an antibacterial peptide at a concentration of 1×MIC, it can be observed that the surface of the Staphylococcus aureus was rough and some cells were shrunk and depressed. After the Staphylococcus aureus was treated with an antibacterial peptide at a concentration of 4×MIC, the degree of depression on the cell surface was further deepened, some cells were more severely deformed, the surface was rugged and irregular, and some cells were ruptured, accompanied by the leakage of internal solutions. Therefore, the seabuckthorn seed meal antibacterial peptide prepared by the present invention acts on Staphylococcus aureus, which can cause the cell membrane of Staphylococcus aureus to be distorted, damaged and ruptured. Therefore, the antibacterial action mode of the seabuckthorn seed meal antibacterial peptide may be directly related to the change in the cell membrane structure.

[0127] Example 3: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0128] The operation steps are the same as those in Example 1, except that the pH of the antibacterial peptide solution is adjusted to 10.

[0129] Example 4: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0130] The operation steps are the same as those in Example 1, except that the pH of the antibacterial peptide solution is adjusted to 8.

[0131] Example 5: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0132] The operation steps are the same as those in Example 1, except that 4000 U / g of trypsin is added.

[0133] Example 6: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0134] The operation steps are the same as those in Example 1, except that 8000 U / g of trypsin is added.

[0135] Example 7: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0136] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 35°C water bath for 2.0 h.

[0137] Example 8: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0138] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 45° C. water bath for 2.0 h.

[0139] Example 9: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0140] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 40° C. water bath for 1.0 h.

[0141] Example 10: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0142] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 40° C. water bath for 3.0 h.

[0143] Example 11: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0144] The operation steps are the same as those in Example 1, except that the added protease is alkaline protease.

[0145] Example 12: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0146] The operation steps are the same as those in Example 1, except that the added protease is α-chymotrypsin.

[0147] Comparative Example 1: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0148] The operation steps are the same as those in Example 1, except that the pH of the antibacterial peptide solution is adjusted to 7.

[0149] Comparative Example 2: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0150] The operation steps are the same as those in Example 1, except that the pH of the antibacterial peptide solution is adjusted to 11.

[0151] Comparative Example 3: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0152] The operation steps are the same as those in Example 1, except that 2000 U / g of trypsin is added.

[0153] Comparative Example 4: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0154] The operation steps are the same as those in Example 1, except that 10000 U / g of trypsin is added.

[0155] Comparative Example 5: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0156] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 50° C. water bath for 2.0 h.

[0157] Comparative Example 6: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0158] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 55° C. water bath for 2.0 h.

[0159] Comparative Example 7: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0160] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 40° C. water bath for 4.0 h.

[0161] Comparative Example 8: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0162] The operation steps were the same as those in Example 1, except that the enzymatic hydrolysis was carried out in a 40° C. water bath for 5.0 h.

[0163] Comparative Example 9: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0164] The operation steps are the same as those in Example 1, except that the added protease is papain.

[0165] Comparative Example 10: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0166] The operation steps are the same as those in Example 1, except that the added protease is a composite protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd.).

[0167] Comparative Example 11: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0168] The operation steps are the same as those in Example 1, except that the added protease is pepsin.

[0169] Comparative Example 12: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0170] The operation steps are the same as those in Example 1, except that the added protease is acidic protease.

[0171] Comparative Example 13: A seabuckthorn seed meal antibacterial peptide and a preparation method thereof.

[0172] The operation steps are the same as those in Example 1, except that the added protease is a neutral protease.

[0173] The inhibitory effects of the polypeptides prepared by different proteases in Example 1, Example 11-Example 12, and Comparative Examples 9-13 on Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Salmonella were studied, with the diameter of the inhibition zone as the evaluation index; Taking Example 1-Example 10 and Comparative Examples 1-8 as examples, the effects of pH, temperature, time, and enzyme addition in the enzymatic hydrolysis conditions on the antibacterial activity of the prepared polypeptides on Staphylococcus aureus were studied. The specific experimental methods and results are as follows:

[0174] 1. Experimental Methods

[0175] 1. Antibacterial activity detection (determination of the diameter of the inhibition zone)

[0176] Single colonies of the four test strains were selected and activated in LB liquid medium. The culture was placed in a constant temperature oscillator at 37°C until the logarithmic growth phase. The concentration was determined by comparing with McFarland turbidimeter tubes and the activated test strains were diluted to a concentration of 10 with sterile saline. 6 CFU / mL bacterial suspension was stored at 4℃ for future use.

[0177] Pour in LB solid culture medium melted at about 50℃, with a thickness of 5mm, shake it gently and evenly, wait for it to solidify, take 100uL of bacterial suspension on the surface of solid culture medium and spread it evenly with a bacterial stick, use a 6mm sterilized metal puncher to punch holes in the plate, pick out the agar in the hole, seal the bottom with an alcohol lamp, add 80uL of seabuckthorn seed meal antibacterial peptide with a mass concentration of 80mg / mL to each well, place the culture dish in a 37℃ constant temperature incubator and culture for 24h, use physiological saline as blank control, and use the cross method to measure the diameter of the inhibition zone. The average value of the diameter of the inhibition zone of 3 parallel holes is the measurement result.

[0178] 3. Study on the stability of antibacterial peptides from seabuckthorn seed meal

[0179] The antibacterial peptides from seabuckthorn seed meal prepared in Example 2 were used as an example to study the effects of temperature, pH, ultraviolet light and freeze-thaw times on the antibacterial activity of the antibacterial peptides from seabuckthorn seed meal.

[0180] (1) Effect of temperature on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal

[0181] The antibacterial peptides from seabuckthorn seed meal were treated at 20℃, 40℃, 60℃, 80℃, 100℃ and 121℃ for 20min. The untreated antibacterial peptides from seabuckthorn seed meal were used as control. The diameter of the inhibition zone was determined by agar diffusion method to observe the effect of heat treatment on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal.

[0182] (2) Effect of pH on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal

[0183] The pH values ​​of antibacterial peptides from seabuckthorn seed meal were adjusted to 3, 5, 7, 9, and 11, respectively. After 5 hours of treatment, the pH value was adjusted back to the original value. The untreated antibacterial peptides from seabuckthorn seed meal were used as a control. The diameter of the inhibition zone was determined by agar diffusion method to observe the effects of acid and alkali treatments on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal.

[0184] (3) Effect of UV light on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal

[0185] The antibacterial peptides from seabuckthorn seed meal were irradiated under ultraviolet light for 20min, 40min, 60min, 80min and 100min. The untreated antibacterial peptides from seabuckthorn seed meal were used as control. The diameter of the inhibition zone was determined by agar diffusion method to observe the effect of ultraviolet treatment on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal.

[0186] (4) Effects of freeze-thaw cycles on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal

[0187] The antibacterial peptides from seabuckthorn seed meal were prepared into a solution and repeatedly frozen and thawed for 2, 4, 6, 8, and 10 times. The untreated antibacterial peptides from seabuckthorn seed meal were used as a control. The diameter of the inhibition zone was determined by agar diffusion method to observe the effect of freeze-thaw treatment on the antibacterial activity of antibacterial peptides from seabuckthorn seed meal.

[0188] 4. Data processing

[0189] SPSS26.0 was used to perform one-way analysis of variance (ANOVA) on the experimental data, and Duncan's multiple comparison method was used to analyze the significance of differences between samples. The charts were drawn using Origin 2021.

[0190] 2. Experimental Results

[0191] 1. Antibacterial activity of antibacterial peptides obtained by enzymatic hydrolysis with different proteases against different pathogens

[0192] Table 2 Antibacterial activity of antibacterial peptides obtained by enzymatic hydrolysis with different proteases

[0193]

[0194] Note: “-” indicates no inhibition zone, “+” indicates an inhibition zone with a diameter ≤14mm, and “++” indicates an inhibition zone with a diameter >14mm.

[0195] The results are shown in Table 2 and Fig.10As shown in the figure, the antibacterial peptides obtained by enzymatic hydrolysis with trypsin, alkaline protease and α-chymotrypsin have inhibitory effects on Staphylococcus aureus, Escherichia coli and Salmonella, while the peptides obtained by enzymatic hydrolysis with neutral protease, compound protease, papain and acid protease can only inhibit Staphylococcus aureus, but have no inhibitory effects on Escherichia coli and Salmonella; the peptides obtained by enzymatic hydrolysis with pepsin have no inhibitory effects on several pathogens. Therefore, only trypsin, alkaline protease and α-chymotrypsin can inhibit multiple pathogens at the same time, and trypsin has the best effect.

[0196] 2. Effects of pH, temperature, time and enzyme dosage in enzymatic hydrolysis conditions on the antibacterial activity and hydrolysis degree of the prepared peptides against Staphylococcus aureus

[0197] The results are as follows Fig.11 As shown in the figure, the antibacterial diameter gradually increases when the pH value is 7-9, reaches the maximum at 9, and then gradually decreases. The hydrolysis degree gradually increases when the pH value is 7-10, reaches the maximum at 10, and then decreases. The main reason for the analysis is that the conformation of trypsin changes under alkaline conditions, and the enzyme is irreversibly inactivated. The optimal range of enzymatic hydrolysis pH is 8-10 ( Fig.11 A). As the temperature increases, the antibacterial diameter reaches the maximum at 40°C, and then gradually decreases. The degree of hydrolysis reaches the maximum at 50°C and then decreases. The reason may be that when the temperature exceeds 50°C, the secondary bond of the enzyme will break, thereby reducing the enzyme activity and causing the degree of hydrolysis to decrease. Therefore, the optimal range of enzymatic hydrolysis temperature is 35°C to 45°C ( Fig.11 B). With the increase of time and enzyme addition, the antibacterial diameter gradually increased. It is speculated that the increase of enzyme amount and time increased the number of polypeptide fragments with antibacterial activity in the enzymatic hydrolysis product. When the enzyme addition reached 6000U / g and the enzymatic hydrolysis time was 2h, the antibacterial activity was the strongest; when the enzyme addition was further increased, the antibacterial activity of the enzymatic hydrolyzate decreased slightly, but the degree of hydrolysis showed an upward trend. It may be that the enzyme hydrolyzed the seabuckthorn seed meal protein into single amino acids or smaller peptide fragments with low or no antibacterial activity. Therefore, the optimal range of enzyme addition is 4000U~8000U, and the optimal range of enzymatic hydrolysis time is 1h~3h( Fig.11 C and D).

[0198] 3. Study on the stability of antibacterial activity of antibacterial peptides from seabuckthorn seed meal

[0199] The results are as follows Fig.12As shown in the figure, when the temperature ranged from 40℃ to 121℃, the antibacterial activity of the antibacterial peptides from seabuckthorn seed meal against the three bacteria decreased significantly only at 121℃ compared with the CK group (no treatment) (P<0.05), and there was no significant difference in other treatment groups (P>0.05). When the pH value varied from 3.0 to 11.0, the antibacterial activity of the antibacterial peptides treated with different pH values ​​against the three bacteria decreased compared with the CK group. The antibacterial diameter of the antibacterial peptides against Staphylococcus aureus decreased after treatment with pH<5 and pH=11 (P<0.05), but there was no significant difference in the antibacterial diameter against Salmonella and Escherichia coli (P>0.05), which may be because Staphylococcus aureus is more sensitive to the antibacterial peptides from seabuckthorn seed meal than Salmonella and Escherichia coli, resulting in a larger change in its antibacterial diameter. After different UV irradiation time and repeated freeze-thaw treatment, the antibacterial diameter of the antibacterial peptide against the three bacteria was not significantly different from that of the control group (P>0.05), indicating that the antibacterial peptide from seabuckthorn seed meal has good antibacterial stability against temperature, UV, acid and alkali, and repeated freeze-thaw.

[0200] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0201] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A seabuckthorn seed meal antibacterial peptide, characterized in that: The seabuckthorn seed meal antibacterial peptide is obtained by defatting, dissolving and centrifuging the seabuckthorn seed meal to obtain the seabuckthorn seed meal crude protein, then enzymolyzing it with protease, centrifuging it and taking the supernatant for freeze-drying to obtain the seabuckthorn seed meal antibacterial peptide; The protease is selected from any one of trypsin, α-chymotrypsin and alkaline protease.

2. The antibacterial peptide from seabuckthorn seed meal according to claim 1, characterized in that The seabuckthorn seed meal antibacterial peptides contain 8 antibacterial peptides with sequences as shown in SEQ ID NO.1 to SEQ ID NO.

8.

3. A method for preparing the antibacterial peptide from seabuckthorn seed meal according to claim 1 or 2, characterized in that: The steps include: The seabuckthorn seed meal is crushed and sieved, defatted with an organic solvent and dried to obtain seabuckthorn seed meal powder, dissolved in distilled water and then bathed in 45°C to 55°C for 60min to 70min, centrifuged and precipitated, and freeze-dried to obtain the seabuckthorn seed meal crude protein; The crude protein of seabuckthorn seed meal is prepared into an antibacterial peptide solution with distilled water, the pH is adjusted to 8-10, 4000U-8000U of protease is added, and enzymolysis is carried out at 35℃-45℃ for 1h-3h. After the enzyme is inactivated, the supernatant is centrifuged and freeze-dried to obtain the seabuckthorn seed meal antibacterial peptide.

4. The method for preparing antibacterial peptides from seabuckthorn seed meal according to claim 3, characterized in that: The crude protein of seabuckthorn seed meal was prepared into an antibacterial peptide solution with distilled water, the pH was adjusted to 9, 6000U of protease was added, and enzymolysis was carried out at 40℃ for 2h. After the enzyme was inactivated, the supernatant was centrifuged and freeze-dried to obtain the antibacterial peptide of seabuckthorn seed meal.

5. The method for preparing antibacterial peptides from seabuckthorn seed meal according to claim 3, characterized in that: The organic solvent is any one of petroleum ether, acetone and ether.

6. Use of the antibacterial peptide from seabuckthorn seed meal according to any one of claims 1 to 2 in the preparation of antibacterial drugs, characterized in that: The antibacterial agent is used to inhibit Staphylococcus aureus, Escherichia coli and Salmonella.

7. The use of the antibacterial peptide from seabuckthorn seed meal in the preparation of antibacterial drugs according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The use of the antibacterial peptide from seabuckthorn seed meal in the preparation of antibacterial drugs according to claim 7, characterized in that: The auxiliary material is one or a combination of starch, lactose, sucrose, mannitol, cellulose and gelatin.

9. An antimicrobial peptide, characterized in that The antimicrobial peptide is selected from any one of the 8 antibacterial peptides described in claim 2: Antimicrobial peptide 1: AIGVDILK; Antibacterial peptide 2: ALEACVKARNEGR; Antimicrobial peptide 3: ARPAADER; Antimicrobial peptide 4: IGGIGTVPVGR; Antimicrobial peptide 5: ILSLSPSR; Antibacterial peptide 6: VGLAETIR; Antimicrobial peptide 7: VLQLETAAGAAIR; Antibacterial peptide 8: VPTVDVSVVDLTVR.

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