Milk-derived broad-spectrum antibacterial peptide and application thereof

Abstract

The invention discloses a milk-derived broad-spectrum antibacterial peptide and application thereof, and relates to the technical field of biology. The amino acid sequence of the antibacterial peptide is as shown in any one of SEQ ID NO.2-6. Five novel antibacterial peptides are obtained by carrying out symmetrical structure design and amino acid mutation on the basis of a template peptide, and tests find that the five novel antibacterial peptides have broad-spectrum antibacterial activity which is obviously higher than that of the template peptide, and basically have no cytotoxicity. Wherein the geometric mean (GM) of the minimum inhibitory concentration can reach 3.60, and the therapeutic index (TI) can reach 71.11. The antibacterial peptide prepared by the invention still keeps good antibacterial activity in physiological salt concentration and protease environment, has certain serum stability, and has higher application value; in addition, the antibacterial peptide has a good inhibition effect on the formation of a bacterial biofilm, and has a synergistic effect with traditional antibiotics.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a milk-derived broad-spectrum antimicrobial peptide and application thereof. Background Art

[0002] Currently, the overuse of antibiotics in clinical treatment increases the resistance of pathogens to drugs. Therefore, the need to find new active molecules with different mechanisms of action represents one of the most pressing challenges in medicine. Continuing research on the role of these natural immunomodulatory peptides and a deeper understanding of the complexity of immune responses may lead to the development of treatment options for immune-related diseases. The application of antimicrobial peptides provides a golden opportunity to develop potential antimicrobial peptide drug candidates that are effective alternatives to traditional antibiotics.

[0003] The present invention intends to use milk-derived antimicrobial peptides as templates to develop short-chain antimicrobial peptides with high antimicrobial activity and low hemolytic activity. Summary of the invention

[0004] The purpose of the present invention is to provide a milk-derived broad-spectrum antimicrobial peptide and its application to solve the problems existing in the above-mentioned prior art. The antimicrobial peptide has high antimicrobial activity and low hemolytic activity, and provides technical support for the development of candidate drugs that effectively replace traditional antibiotics.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an antimicrobial peptide L-1, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides an antimicrobial peptide W-1, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] The present invention also provides an antimicrobial peptide W-2, the amino acid sequence of which is shown in SEQ ID NO.4.

[0009] The present invention also provides an antimicrobial peptide W-3, the amino acid sequence of which is shown in SEQ ID NO.5.

[0010] The present invention also provides an antimicrobial peptide W-4, the amino acid sequence of which is shown in SEQ ID NO.6.

[0011] The present invention also provides the use of an antimicrobial peptide in the preparation of an antibacterial agent, wherein the amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NOs. 2-6.

[0012] Furthermore, the pathogens inhibited by the antibacterial agent include Gram-negative bacteria and Gram-positive bacteria; the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Salmonella or Enterobacter sakazakii; the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Bacillus cereus.

[0013] The present invention also provides an antibacterial agent, wherein the active ingredient comprises an antibacterial peptide having an amino acid sequence as shown in any one of SEQ ID NOs. 2-6.

[0014] Furthermore, the pathogens inhibited by the antibacterial agent include Gram-negative bacteria and Gram-positive bacteria; the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Salmonella or Enterobacter sakazakii; the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Bacillus cereus.

[0015] Furthermore, the antibacterial agent also includes pharmaceutically acceptable excipients.

[0016] The present invention discloses the following technical effects:

[0017] The present invention obtains five new antimicrobial peptides by symmetric structural design and amino acid mutation based on the template peptide L-0 (LKKISQYYQKFA). The experiments show that the five new antimicrobial peptides have broad-spectrum antibacterial activity and are significantly higher than the template peptide, and are basically non-cytotoxic. Among them, the geometric mean (GM) of the minimum inhibitory concentration can reach 3.60, and the therapeutic index (TI) can reach 71.11.

[0018] The antimicrobial peptide prepared by the present invention still maintains good antimicrobial activity in a physiological salt concentration and protease environment, and has a certain serum stability, and has a high application value; in addition, the antimicrobial peptide has a good inhibitory effect on the formation of bacterial biofilms and has a synergistic effect with traditional antibiotics.

[0019] The invention provides a new idea and an effective template sequence for the design of milk-derived antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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 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.

[0021] Figure 1 α-helical wheel structure diagram of the template peptide and the designed peptide;

[0022] Figure 2is the hemolytic activity of the template peptide, the designed peptide and melittin; wherein A is the hemolytic activity of the template peptide and the designed peptide; B is the hemolytic activity of melittin;

[0023] Figure 3 It is a statistical diagram of the survival rate of biofilm under the action of different template peptides and designed peptides;

[0024] Figure 4 This is a laser confocal micrograph of E. coli ATCC 25922 treated with FITC-labeled peptide W-4;

[0025] Figure 5 PI staining flow cytometric analysis of E. coli ATCC 25922 after treatment with template peptide and designed peptide; A is the control, BG are antimicrobial peptides L-0, L-1, W-1, W-2, W-3 and W-4 respectively;

[0026] Figure 6 The SEM images of E. coli ATCC 25922 after being treated with template peptide and designed peptide;

[0027] Figure 7 TEM images of E. coli ATCC 25922 after treatment with template peptide and designed peptide. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1 Sequence design of broad-spectrum antimicrobial peptides

[0034] The milk-derived antimicrobial peptide L-0 was used as a template peptide, mirror-folded and amino acid substitutions were performed. The amino acids were replaced with tyrosine (Tyr), tryptophan (Trp), phenylalanine (Phe) and arginine (Arg) to increase the positive charge and hydrophobicity of the peptide, thereby achieving the purpose of inserting aromatic amino acids and forming perfect amphipathicity. A total of 5 designed peptides were designed. The amino acid sequences and physicochemical parameters of the template peptide and the designed peptide are shown in Table 1. The amphipathicity of the peptide sequence is represented by an α-helical wheel diagram, see Figure 1 .

[0035] Table 1 Amino acid sequences and physicochemical parameters of template peptides and designed peptides

[0036]

[0037] Note: a: measured by mass spectrometer; b: hydrophobicity; c: average hydrophobic moment.

[0038] As shown in Table 1, L-1 presents a mirror-symmetrical structure and the positive charge number changes from +3 to +4. W-1 and W-2 introduce uncharged tryptophan and phenylalanine respectively. The charge number does not change compared with L-1, but the hydrophobicity increases successively to 0.635 and 0.802. W-3 and W-4 further introduce arginine, but the insertion positions are different. The net charge of W-3 is +6, the hydrophobicity is 0.335, but the hydrophobic moment is 0.881. The reason for the change in this parameter may be that W-3 is a perfect amphipathic structure. Figure 1 The changes in the amphipathicity of the peptides are shown diagrammatically using an α-helical wheel.

[0039] Example 2 Preparation of antimicrobial peptides by solid phase synthesis

[0040] The antimicrobial peptides L-0, L-1, W-1, W-2, W-3 and W-4 were prepared by solid phase synthesis as follows:

[0041] 1. Synthesis and purification of antimicrobial peptides

[0042] The second amino acid at the C-terminus of the designed sequence and a condensing agent, N,N-dimethylformamide, are added to the reaction column. After the reaction is completed, the reaction is detected using the ninhydrin method. After that, the reaction is washed once with a protective solution and N,N-dimethylformamide, and the reaction is detected again. The above steps are repeated until the sequence is synthesized, and the finished product is precipitated by washing with dichloromethane, methanol, and ether.

[0043] The synthesized sample is dissolved in a test tube with pure water, filtered through a filter membrane, and analyzed by rapid gradient high performance liquid chromatography (10-100%); 200 mg of the synthesized sample is dissolved in a beaker with 15 mL of water and 5 mL of methanol, and ultrasonicated until completely dissolved, and then filtered through a filter membrane; the filtered solution is purified by gradient chromatography, and the sample components are analyzed by high performance liquid chromatography (HPLC) after the sample is collected within a time period of 0-30 minutes. After the sample purity is checked, a rotary evaporator is used for fractionation, and the obtained sample is freeze-dried and refrigerated in the dark.

[0044] 2. Identification of antimicrobial peptides

[0045] The purified polypeptides were collected and the target peaks were identified by LC 6000 reverse phase preparative chromatograph and Waters 2000 mass spectrometer, and finally the actual peptide molecular weight and purity were analyzed. The results showed that the polypeptides of the present invention were synthesized correctly.

[0046] Example 3 Determination of antimicrobial activity of antimicrobial peptides

[0047] The antimicrobial activity of antimicrobial peptides (template peptide L-0 and designed peptides L-1, W-1, W-2, W-3 and W-4) was determined. The specific methods and results are as follows:

[0048] The minimum inhibitory concentration (MIC) was determined by the micro-dilution method. 95 μL BSA solution was added to the wells in row A of the 96-well plate, and 50 μL BSA solution was added to the wells in row BH. 5 μL of the antimicrobial peptide solution with a concentration of 2.56 mM was added to the wells in row A, and 50 μL was pipetted with a pipette and added to the wells in row B. The mixture was stirred and mixed. The operation was repeated until the well G was reached. Any mixture exceeding 50 μL was discarded. The bacteria to be tested were inoculated into MHB medium and cultured at 37°C for 12 hours. After that, the second generation culture was performed until the bacterial concentration was OD 600 =0.4, 50 μL of cultured bacterial solution was added to the AG wells of the 96-well plate as the experimental group. At this time, the antimicrobial peptide concentration of the AG wells of the 96-well plate decreased gradually from 64 μM to 1 μM. 50 μL of bacterial solution was added to the first 6 wells of the H wells, and the culture medium was added to the last 6 wells as the positive and negative control groups, respectively. After culturing in the incubator for 24 hours, the absorbance was measured with an ELISA instrument, and the antimicrobial peptide concentration corresponding to the turbidity in the solution in the well was taken as the minimum inhibitory concentration. The experiment was repeated 3 times for each antimicrobial peptide sample.

[0049] The results are shown in Tables 2 and 3.

[0050] Table 2 MIC values ​​of antimicrobial peptides against Gram-negative bacteria (μM)

[0051]

[0052]

[0053] Table 3 MIC values ​​of antimicrobial peptides against Gram-positive bacteria (μM)

[0054]

[0055] The six peptides had antibacterial activity against both selected Gram-negative and Gram-positive bacteria, and the antibacterial ability was significantly improved after symmetrical structure design and amino acid replacement. The antibacterial effect of the six peptides on Gram-positive bacteria was weaker than that on Gram-negative bacteria, especially the MIC values ​​of the template peptides were all greater than 64μM, but the designed peptide W-4 showed the best antibacterial effect. This shows that the introduction of mirror symmetrical structure and hydrophobic amino acids is conducive to improving the antibacterial performance and broad-spectrum antibacterial activity of short peptides.

[0056] Example 4 Determination of hemolytic activity of antimicrobial peptides

[0057] The hemolytic activity of antimicrobial peptides (template peptide L-0 and designed peptides L-1, W-1, W-2, W-3 and W-4) was determined. The specific method and results are as follows:

[0058] Take healthy human blood and centrifuge it at 1000×g and 4℃ for 10 minutes, then remove the supernatant. Rinse the obtained sample with PBS 3 times and resuspend it. Add the antimicrobial peptide solution and the resuspended blood cells to a 96-well plate. Refer to Example 3 for the specific operation, and replace the bacterial solution with the blood cell suspension. After culturing for 4 hours, centrifuge for 10 minutes, take the supernatant to a new 96-well plate, and measure the absorbance at 570nm. The first 6 wells of the 96-well plate H row do not add antimicrobial peptides as the negative control group, and the last 6 wells are added with blood cells treated with 0.1% Triton X-100 as the positive control group. The peptide concentration corresponding to 10% hemolytic activity is defined as the minimum hemolytic concentration (Minimal Hemolytic Concentration, MHC). The experiment was repeated 3 times independently. The formula for calculating hemolytic activity is:

[0059] Hemolytic activity (%) = (OD 测定值 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )×100%.

[0060] The results of the determination of the hemolytic activity of antimicrobial peptides are as follows Figure 2 shown.

[0061] As the peptide concentration increases, its hemolytic activity also increases, which means that the ability to damage mammalian red blood cells is enhanced. However, when the highest peptide concentration of this test is reached, the hemolytic activity of all peptides is below 10%, among which the hemolytic activities of L-1, W-1, W-2, and W-3 ​​are all less than 5%, and the hemolytic activity of L-0 is greater than 5% when the concentration reaches 32μM and the concentration of W-4 ​​reaches 64μM. Other antimicrobial peptides, such as bee venom peptide (GIGAVLKVLTGLPALISWIKRKRQQ, SEQ ID NO.7), have a hemolytic activity of 17.6% at a concentration of 0.5μM. The above results show that the antimicrobial peptides designed by the present invention have excellent biocompatibility.

[0062] To further evaluate the cell selectivity of antimicrobial peptides, the therapeutic index (TI) of each peptide was obtained by comprehensively calculating the MIC of the antimicrobial peptides and the minimum hemolytic concentration for human erythrocytes. The larger the therapeutic index, the better the antibacterial effect of the antimicrobial peptide. The results are shown in Table 4. TI = MHC / GM; GM: the geometric mean of the MIC values ​​of all peptides, and 128 μM is used for calculation when it exceeds 64 μM.

[0063] Table 4 MHC values ​​of antimicrobial peptides (μM)

[0064]

[0065] Note: MHC: (minimum hemolytic concentration) peptide concentration that causes 10% hemolysis.

[0066] Among them, the TI of the designed peptide W-4 is 71.11, and the TI of the template peptide L-0 is 5.51, which is nearly 13 times higher than that of the template peptide. According to the final comprehensive therapeutic index, the antibacterial effects of the six peptides are ranked as follows: W-4>W-3>W-2>W-1>L-1>L-0.

[0067] Example 5 Antimicrobial Peptide Stability Study

[0068] The stability of antimicrobial peptides (template peptide L-0 and designed peptides L-1, W-1, W-2, W-3 and W-4) was studied. The specific methods and results are as follows:

[0069] 1. Salt ion stability

[0070] Salt ion solutions were prepared with BSA diluent at different concentrations. The salt ion concentrations were 150mM, 4.5mM, 1mM, 6mM, 8mM, 4mM and 2.5mM, respectively. The blank control was the antimicrobial peptide that was not treated with salt ions. The stability of the antimicrobial peptide at different salt ion concentrations was tested. The results are shown in Tables 5 and 6.

[0071] Table 5 MIC values ​​(μM) of antimicrobial peptides against E.coil ATCC 25922 in different salt ion environments

[0072]

[0073] Table 6 MIC values ​​(μM) of antimicrobial peptides against S. aureus ATCC 29213 in different salt ion environments

[0074]

[0075] Note: The concentrations of NaCl, KCl, NH4Cl, MgCl2, CaCl2, ZnCl2 and FeCl3 were 150mM, 4.5mM, 6mM, 1mM, 2.5mM, 8mM and 4mM respectively; the significance analysis and calculation were performed based on the MIC of different types of peptides in the same ionic environment.

[0076] 2. Serum stability

[0077] The stability of the antimicrobial peptide in serum was determined by the same method as the salt ion stability described above. Different concentrations of fetal bovine serum were prepared with BSA diluent, and the final serum concentrations were 50%, 25% and 12.5%. The blank control was a peptide not treated with serum, and the stability of the antimicrobial peptide at different serum concentrations was tested. The results are shown in Tables 7 and 8.

[0078] Table 7 MIC values ​​(μM) of antimicrobial peptides against E.coil ATCC25922 in different serum environments

[0079]

[0080]

[0081] Table 8 MIC values ​​(μM) of antimicrobial peptides against S. aureus ATCC 29213 in different serum environments

[0082]

[0083] Note: The significance analysis and calculation were performed based on the MIC values ​​of different peptides under the same serum concentration environment.

[0084] 3. Enzyme stability

[0085] In order to determine the stability of antimicrobial peptides under enzyme treatment, trypsin, pepsin, papain and proteinase K were prepared with a final concentration of 1 mg / mL of enzyme-antimicrobial peptide reaction solution. After the two were allowed to react for 1 hour, the test was carried out according to the salt ion and serum stability determination method. The blank control was the antimicrobial peptide without protease treatment. The stability of the antimicrobial peptides under different enzyme treatments was tested in this way, and the results are shown in Tables 9 and 10.

[0086] Table 9 MIC values ​​(μM) of peptides against E.coil ATCC25922 under different enzyme treatments

[0087]

[0088] Table 10 MIC values ​​(μM) of peptides against S. aureus ATCC 29213 under different enzyme treatments

[0089]

[0090] Note: The significance analysis and calculation were performed based on the MIC values ​​of different peptides in the same enzyme environment.

[0091] The above results show that compared with the template peptide, the five designed peptides after structural symmetry and amino acid mutation design still have good antibacterial activity in physiological concentration salt ion and high concentration serum environment, indicating that they have high salt ion and serum stability. Although the antibacterial activity of each peptide was reduced in the enzyme stability experiment, the designed peptides still maintained effective activity as a whole, and the stability of the designed peptides W-3 and W-4 was stronger than that of other designed peptides and template peptides.

[0092] Example 6 Inhibitory effect of antimicrobial peptides on biofilm

[0093] The inhibitory effects of antimicrobial peptides (template peptide L-0 and designed peptides L-1, W-1, W-2, W-3 and W-4) on biofilm were tested. The specific methods and results are as follows:

[0094] In order to determine the inhibitory effect of antimicrobial peptides on biofilm, the bacterial concentration was adjusted to OD 600 = 0.4 and then diluted 1000 times. Referring to the operation of Example 3, the 96-well plate after culturing for 24 hours was discarded of the supernatant and stained with 0.1% crystal violet dye for 15 minutes, washed with deionized water, and then 70% alcohol was added to dissolve the dye. The absorbance at 595 nm was measured using an ELISA reader. The test was repeated 3 times in 3 parallels.

[0095] The results are as follows Figure 3 As shown, it can be seen that the template peptide and the designed peptide both played a killing role in inhibiting the formation of E. coli ATCC 25922 biofilm, and produced obvious effects. However, the inhibitory effect of template peptide L-0 on biofilm was weaker than that of other designed peptides, especially peptides W-3 and W-4 showed extremely obvious elimination rate of bacterial biofilm between 1-2μM, and they could kill all biofilms when reaching 4μM.

[0096] Example 7 Antibacterial Mechanism of Antimicrobial Peptides

[0097] The antibacterial mechanism of antimicrobial peptides (template peptide L-0 and designed peptides L-1, W-1, W-2, W-3 and W-4) was studied. The specific methods and results are as follows:

[0098] 1. FITC-labeled peptide localization test

[0099] Adjust the bacterial concentration to OD 600 =0.15, the minimum inhibitory concentration of FITC-labeled peptide was incubated with the bacterial solution in an incubator for 15 minutes, then centrifuged and the supernatant was discarded, resuspended and mixed with 10μg / mL PI solution and incubated for 15 minutes, then centrifuged to wash away excess dye, and then the sample was moved to a slide and placed at 4°C overnight. The laser confocal microscope and super-high resolution fluorescence microscope were set to excitation wavelengths of 488nm and 535nm for observation and photography.

[0100] like Figure 4 As shown, the green fluorescence produced by the FITC-labeled peptide W-4 under a fluorescence microscope overlaps with the red fluorescence of propidium iodide and is located on the surface of the bacterial cell membrane, indicating that the antimicrobial peptide is bound to the membrane surface of the antimicrobial peptide and first has a destructive effect on the bacterial cell membrane.

[0101] 2. Determination of cell membrane integrity

[0102] Flow cytometry (FACScan) was used to evaluate the effect of antimicrobial peptides on the integrity of bacterial cell membranes. The concentrations of bacterial solution and antimicrobial peptides were 1MIC and 1 / 2MIC, respectively. After mixed incubation, 10 mg / mL PI dye was added for 30 minutes, and the excess dye was washed away with PBS and resuspended, and detected in FACScan with an excitation wavelength of 488 nm. The bacterial solution without antimicrobial peptides was used as the blank control group.

[0103] The results are as follows Figure 5 As shown, the fluorescence intensity of the blank control group was extremely small, indicating that almost no bacteria died. After being treated with the template peptide and the designed peptide, the bacterial fluorescence ratios were 46.7%, 48.3%, 49.6%, 68.2%, 72.1%, and 79.9%, respectively, indicating that the six antimicrobial peptides all had antibacterial effects. Among them, the fluorescence cell ratios of W-3 and W-4 exceeded 70%, and W-4 was close to 80%, with the strongest bactericidal effect.

[0104] 3. Observation of cell membrane morphology

[0105] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the cell outer membrane morphology of the test strain E. coli ATCC25922 to study the degree of damage of antimicrobial peptides to the bacterial cell outer membrane. The experimental operation is as follows:

[0106] (1) Use a scanning electron microscope (SEM) to observe the cell membrane morphology of the bacteria to be tested. Cultivate the bacterial solution until the second generation and resuspend it by centrifugation to OD 600 =0.15 for later use, dilute the antimicrobial peptide to a concentration of 1MIC with bacterial solution and incubate for 1 hour, discard the supernatant and wash and centrifuge for later use, add 1mL 2.5% glutaraldehyde and incubate for 12 hours, then wash the bacterial sludge with 50%, 70%, 90% and 100% ethanol solutions, respectively, for 10 minutes each time, then mix 100% ethanol and tert-butanol solution and incubate the bacterial sludge for 30 minutes, finally treat with pure tert-butanol for 1 hour, dry the obtained bacterial sludge solid, and then observe and photograph it with a scanning electron microscope after coating.

[0107] (2) Observe the degree of damage of the antimicrobial peptides to the bacterial cell structure under test using a transmission electron microscope (TEM). The bacterial sludge was fixed with 1% osmium tetroxide for 2 hours and then centrifuged and washed. The bacterial sludge was then treated with 50%, 70%, 90% and 100% ethanol solutions for 8 minutes each time, then treated with 100% ethanol for 10 minutes and then treated with a mixture of ethanol and acetone of the same volume for another 10 minutes, and finally treated with 100% acetone for 10 minutes. The bacterial sludge was embedded with embedding solution for 30 minutes and then embedded with pure resin overnight. Finally, it was stained with lead citrate and uranyl acetate and sliced ​​for observation and photography using a transmission electron microscope.

[0108] The results of cell membrane morphology observation are as follows Figure 6 and Figure 7 shown.

[0109] The results showed that the bacterial surface of the control group was smooth, without wrinkles or holes, the cells were full, and the structure was complete. After being treated with different concentrations of W-4, the morphology changed from wrinkles to surface depressions, and even holes appeared. The cell morphology was broken, the structure was missing, and a large amount of content was lost. It can be seen that W-4 has a higher degree of damage to bacteria, and it further proves that the antimicrobial peptide destroys the bacterial cell membrane, thereby killing the bacteria.

[0110] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An antimicrobial peptide L-1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. An antimicrobial peptide W-1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

3. An antimicrobial peptide W-2, characterized in that: The amino acid sequence is shown in SEQ ID NO.

4.

4. An antimicrobial peptide W-3, characterized in that: The amino acid sequence is shown in SEQ ID NO.

5.

5. An antimicrobial peptide W-4, characterized in that: The amino acid sequence is shown in SEQ ID NO.

6.

6. The use of antimicrobial peptides in the preparation of antibacterial agents, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NOs. 2-6.

7. The use according to claim 6, characterized in that: The pathogens inhibited by the antibacterial agent include Gram-negative bacteria and Gram-positive bacteria; the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Salmonella or Enterobacter sakazakii; the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Bacillus cereus.

8. An antibacterial agent, characterized in that The active ingredient comprises an antimicrobial peptide having an amino acid sequence as shown in any one of SEQ ID NOs. 2-6.

9. The antibacterial agent according to claim 8, characterized in that The pathogens inhibited by the antibacterial agent include Gram-negative bacteria and Gram-positive bacteria; the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Salmonella or Enterobacter sakazakii; the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Bacillus cereus.

10. The antibacterial agent according to claim 8, characterized in that The antibacterial agent also includes pharmaceutically acceptable excipients.

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