Broad-spectrum antimicrobial peptides from milk and uses thereof

Abstract

The application discloses a broad-spectrum antibacterial peptide from milk and application thereof, and relates to the technical field of biology. The amino acid sequence of the antibacterial peptide is shown in any one of SEQ ID NO. 2-6. On the basis of a template peptide, symmetric structure design and amino acid mutation are performed to obtain five new antibacterial peptides. It is found through experiments that the five new antibacterial peptides have broad-spectrum antibacterial activity and are significantly higher than the template peptide, and are substantially free of cytotoxicity. 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 application still has good antibacterial activity in a physiological salt concentration and a protease environment, has certain serum stability, and has high application value. In addition, the antibacterial peptide has a good inhibitory effect on the formation of bacterial biofilm and has a synergistic effect with traditional antibiotics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to broad-spectrum antimicrobial peptides derived from milk and their applications. Background Technology

[0002] Currently, the overuse of antibiotics in clinical treatment increases pathogen resistance to drugs. Therefore, the need to find novel active molecules with different mechanisms of action represents one of the most pressing challenges in medicine. Continued research into the roles 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 offers a golden opportunity to develop potential antimicrobial peptide candidates that can effectively replace traditional antibiotics.

[0003] This invention aims to develop short-chain antimicrobial peptides with high antimicrobial activity and low hemolytic activity by using milk-derived antimicrobial peptides as templates. Summary of the Invention

[0004] The purpose of this invention is to provide a broad-spectrum antimicrobial peptide derived from milk and its applications, thereby addressing the problems existing in the prior art. This antimicrobial peptide exhibits high antimicrobial activity and low hemolytic activity, providing technical support for the development of candidate drugs that effectively replace traditional antibiotics.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This 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 antimicrobial peptides in the preparation of antimicrobial agents, wherein the amino acid sequence of the antimicrobial peptides is shown in any one of SEQ ID NO. 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 Cronobacter sakazakii; the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes or Bacillus cereus.

[0013] The present invention also provides an antibacterial agent, the active ingredient of which includes an antimicrobial peptide with an amino acid sequence as shown in any one of SEQ ID NO.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 Cronobacter 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] This invention, based on the template peptide L-0 (LKKISQYYQKFA), involves symmetrical structural design and amino acid mutation to obtain five novel antimicrobial peptides. Experiments revealed that these five novel antimicrobial peptides possess broad-spectrum antibacterial activity, all significantly higher than the template peptide, and exhibit virtually no cytotoxicity. The geometric mean (GM) of the minimum inhibitory concentration reaches 3.60, and the therapeutic index (TI) reaches 71.11.

[0018] The antimicrobial peptides prepared by this invention maintain good antimicrobial activity in physiological salt concentrations and protease environments, and have certain serum stability, thus possessing high application value. Furthermore, the antimicrobial peptides have a good inhibitory effect on bacterial biofilm formation and exhibit synergistic effects with traditional antibiotics.

[0019] This invention provides new ideas and effective template sequences for the design of milk-derived antimicrobial peptides. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0022] Figure 2The hemolytic activities of the template peptide, the designed peptide, and the meliofemoral peptide are represented; where A represents the hemolytic activity of the template peptide and the designed peptide; and B represents the hemolytic activity of the meliofemoral peptide.

[0023] Figure 3 A statistical graph showing the survival rate of biological membranes under the action of different template peptides and designed peptides;

[0024] Figure 4 Laser confocal microscopy image of E. coli ATCC 25922 after treatment with FITC-labeled peptide W-4;

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

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

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

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

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

[0034] Using the milk-derived antimicrobial peptide L-0 as a template peptide, its amino acid sequence was mirror-folded and replaced with tyrosine (Tyr), tryptophan (Trp), phenylalanine (Phe), and arginine (Arg) to increase the peptide's positive charge and hydrophobicity, achieving the insertion of aromatic amino acids and the formation of perfect amphiphilicity. A total of five design peptides were obtained. The amino acid sequences and physicochemical parameters of the template peptide and the design peptides are shown in Table 1. The amphiphilicity of the peptide sequences is represented by an α-helix wheel diagram, see [details omitted]. Figure 1 .

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

[0036]

[0037] Note: a: Measured by mass spectrometry; b: Hydrophobicity; c: Average hydrophobic torque.

[0038] As shown in Table 1, L-1 exhibits a mirror-symmetric structure with the positive charge changing from +3 to +4. W-1 and W-2 introduce uncharged tryptophan and phenylalanine respectively, with no change in charge compared to L-1, but the hydrophobicity increases sequentially to 0.635 and 0.802. W-3 and W-4 further introduce arginine, but at different insertion positions. W-3 has a net charge of +6 and a hydrophobicity of 0.335, but a hydrophobic moment of 0.881. The reason for this parameter change may be that W-3 has a perfect amphiphilic structure. Figure 1 The amphiphilicity of peptides is illustrated using an α-helix wheel chart.

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

[0040] Antimicrobial peptides L-0, L-1, W-1, W-2, W-3, and W-4 were prepared using a solid-phase synthesis method, as follows:

[0041] 1. Synthesis and purification of antimicrobial peptides

[0042] The second amino acid located at the C-terminus of the designed sequence was added to the reaction column with a condensing agent and N,N-dimethylformamide. After the reaction was completed, the ninhydrin method was used for detection. Then, the sample was washed once with a protective solution and N,N-dimethylformamide, and the detection was repeated. The above steps were repeated until the sequence was synthesized. The product was precipitated by washing with dichloromethane, methanol, and diethyl ether.

[0043] The synthesized sample was dissolved in pure water in a test tube, filtered through a filter membrane, and analyzed by rapid gradient high-performance liquid chromatography (10-100%). 200 mg of the synthesized sample was dissolved in 15 mL of water and 5 mL of methanol in a beaker and sonicated until completely dissolved, then filtered through a filter membrane. The filtered solution was purified using gradient chromatography. Samples were collected within a 0-30 min time period and analyzed by high-performance liquid chromatography (HPLC). After verifying the purity of the sample, fractions were obtained using a rotary evaporator. The samples were then lyophilized and stored in the dark.

[0044] 2. Identification of antimicrobial peptides

[0045] The purified peptides were collected and the target peaks were identified using an LC 6000 reversed-phase preparative chromatograph and a Waters 2000 mass spectrometer. Finally, the actual peptide molecular weight and purity were analyzed. The results show that the synthesis of each peptide in this invention is correct.

[0046] Example 3: Determination of the antibacterial 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 using the micro-dilution method. 95 μL of BSA solution was added to well A of a 96-well plate, and 50 μL of BSA solution was added to wells B and H. 5 μL of a 2.56 mM antimicrobial peptide solution was added to well A, and after mixing, 50 μL was pipetted into well B and mixed again. This process was repeated until well G was reached. Any excess mixture (more than 50 μL) was discarded. The test bacteria were inoculated onto MHB medium and incubated at 37°C for 12 h, followed by second-generation culture until the bacterial concentration reached OD500. 600 =0.4. In a 96-well plate, 50 μL of cultured bacterial solution was added to the AG row wells as the experimental group. The antimicrobial peptide concentration in the AG row decreased gradually from 64 μM to 1 μM. In the H row, 50 μL of bacterial solution was added to the first 6 wells, and culture medium was added to the last 6 wells as the positive and negative control groups, respectively. After incubation for 24 hours, the absorbance was measured using a microplate reader. The concentration of antimicrobial peptide at which turbidity was not directly observable in the wells was taken as the minimum inhibitory concentration (MIC). Each antimicrobial peptide sample was tested three times.

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

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

[0051]

[0052]

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

[0054]

[0055] All six peptides exhibited antibacterial activity against both selected Gram-negative and Gram-positive bacteria. The antibacterial activity was significantly enhanced after symmetrical structural design and amino acid substitution. The antibacterial effect against Gram-positive bacteria was weaker than that against Gram-negative bacteria, especially since the MIC values ​​of the template peptides were all greater than 64 μM. However, the designed peptide W-4 showed the best antibacterial effect. This indicates that the introduction of mirror-symmetric structure and hydrophobic amino acids is beneficial for improving the antibacterial properties and broad-spectrum antibacterial activity of short peptides.

[0056] Example 4: Determination of the 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, and the specific methods and results are as follows:

[0058] Blood from healthy individuals was centrifuged at 1000×g for 10 min at 4°C. The supernatant was removed, and the sample was washed three times with PBS and resuspended. The antimicrobial peptide solution and the resuspended blood cells were added to a 96-well plate, following the procedure in Example 3, except that the bacterial culture was replaced with the blood cell suspension. After culturing for 4 h, the plate was centrifuged for 10 min, and the supernatant was transferred to a new 96-well plate. The absorbance at 570 nm was measured. The first six wells of the 96-well plate (H row) without antimicrobial peptide served as the negative control, and the last six wells contained blood cells treated with 0.1% Triton X-100 as the positive control. The peptide concentration corresponding to 10% hemolytic activity was defined as the minimum hemolytic concentration (MHC). The experiment was independently repeated three times. 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 assay for the hemolytic activity of antimicrobial peptides are as follows: Figure 2 As shown.

[0061] As peptide concentration increases, its hemolytic activity also increases, indicating an enhanced ability to damage mammalian erythrocytes. However, at the highest peptide concentrations tested, the hemolytic activity of all peptides was below 10%, with L-1, W-1, W-2, and W-3 ​​exhibiting hemolytic activities of less than 5%, while L-0 showed hemolytic activity greater than 5% at concentrations of 32 μM and W-4 at 64 μM. Other antimicrobial peptides, such as melitrix venom peptide (GIGAVLKVLTGLPALISWIKRKRQQ, SEQ ID NO. 7), showed a hemolytic activity of 17.6% at a concentration of 0.5 μM. These results demonstrate that the antimicrobial peptides designed in this invention possess excellent biocompatibility.

[0062] To further evaluate the cell selectivity of antimicrobial peptides, the therapeutic index (TI) for each peptide was calculated by combining the MIC of the antimicrobial peptide and the minimum hemolytic concentration for human erythrocytes. A higher therapeutic index indicates a better antimicrobial effect. The results are shown in Table 4. TI = MHC / GM; GM: the geometric mean of the MIC values ​​of all peptides, with values ​​exceeding 64 μM calculated as 128 μM.

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

[0064]

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

[0066] Among them, the TI of the designed peptide W-4 was 71.11, while the TI of the template peptide L-0 was 5.51, with the designed peptide being nearly 13 times higher than the template peptide. Based on the final comprehensive therapeutic index, the antibacterial effects of the six peptides were ranked as follows: W-4 > W-3 > W-2 > W-1 > L-1 > L-0.

[0067] Example 5: Stability Study of Antimicrobial Peptides

[0068] Stability studies were conducted on antimicrobial peptides (template peptide L-0 and designed peptides L-1, W-1, W-2, W-3, and W-4). The specific methods and results are as follows:

[0069] 1. Salt ion stability

[0070] Salt ion solutions were prepared using BSA diluent at different concentrations: 150 mM, 4.5 mM, 1 mM, 6 mM, 8 mM, 4 mM, and 2.5 mM. The blank control consisted of the antimicrobial peptide without salt ion treatment. The stability of the antimicrobial peptide at different salt ion concentrations was tested, and the results are shown in Tables 5 and 6.

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

[0072]

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

[0074]

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

[0076] 2. Serum stability

[0077] The stability of antimicrobial peptides in serum was determined using the same method as described above for salt ion stability. Fetal bovine serum at different concentrations was prepared using BSA dilution buffer, with final serum concentrations of 50%, 25%, and 12.5%. A blank control was provided, consisting of peptides without serum treatment. The stability of the antimicrobial peptides at different serum concentrations was then assessed, and 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 of the MIC values ​​of different peptides under the same serum concentration was calculated using significance analysis.

[0084] 3. Enzyme stability

[0085] To determine the stability of antimicrobial peptides under enzyme treatment, enzyme-antimicrobial peptide reaction solutions with trypsin, pepsin, papain, and proteinase K at a final concentration of 1 mg / mL were prepared. After 1 hour of reaction, the stability was determined according to the methods for salt ion and serum stability. The blank control was the antimicrobial peptide without enzyme treatment. The stability of antimicrobial peptides under different enzyme treatments was tested, 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: Significance analysis was performed using the MIC values ​​of different peptides in the same enzyme environment.

[0091] The above results indicate that, compared with the template peptide, the five designed peptides, after structural symmetry and amino acid mutation design, still exhibit good antibacterial activity in physiological concentrations of salt ions and high concentrations of serum, demonstrating their high stability in salt ions and serum. Although the antibacterial activity of each peptide decreased in the enzyme stability experiment, the designed peptides generally maintained effective activity, with designed peptides W-3 and W-4 showing stronger stability compared to the other designed peptides and the template peptide.

[0092] Example 6: Inhibitory effect of antimicrobial peptides on biofilms

[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 biofilms were tested. The specific methods and results are as follows:

[0094] To determine the inhibitory effect of antimicrobial peptides on biofilms, this experiment adjusted the bacterial concentration to OD0.05. 600 =0.4 and then diluted 1000 times. Following the procedure in Example 3, after culturing for 24 hours, the supernatant of the 96-well plate was discarded, and it was stained with 0.1% crystal violet dye for 15 minutes. After rinsing with deionized water, the dye was dissolved in 70% alcohol, and the absorbance at 595 nm was measured using an ELISA reader. The experiment was performed in triplicate and repeated 3 times.

[0095] The results are as follows Figure 3 As shown, both the template peptide and the designed peptide ultimately achieved a killing effect in inhibiting the formation of Escherichia coli (E. coli) ATCC 25922 biofilm, producing significant results. However, the template peptide L-0 showed a weaker inhibitory effect on the biofilm compared to other designed peptides. In particular, peptides W-3 and W-4 exhibited extremely high elimination rates of bacterial biofilm in the 1-2 μM range, completely killing the biofilm at 4 μM.

[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 investigated. The specific methods and results are as follows:

[0098] 1. FITC-labeled peptide localization assay

[0099] Adjust the bacterial culture concentration to OD 600 =0.15. The minimum inhibitory concentration (MIC) of the FITC-labeled peptide was incubated with the bacterial culture for 15 min in an incubator, followed by centrifugation and discarding of the supernatant. The resuspended sample was then mixed with 10 μg / mL PI solution and incubated for another 15 min, followed by centrifugation to remove excess dye. The sample was then transferred to a glass slide and incubated overnight at 4°C. Observations and photographs were taken using a laser confocal microscope and a super-resolution fluorescence microscope with excitation wavelengths set to 488 nm and 535 nm, respectively.

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

[0101] 2. Measurement of cell membrane integrity

[0102] The effect of antimicrobial peptides on the cell membrane integrity of the tested bacteria was assessed using flow cytometry (FACScan). The bacterial suspension and antimicrobial peptide were mixed at concentrations of 1 MIC and 1 / 2 MIC, respectively. After incubation, 10 mg / mL of PI dye was added and reacted for 30 min. Excess dye was washed off with PBS, and the suspension was resuspended. Detection was performed in FACScan at an excitation wavelength of 488 nm. Bacterial suspensions without added antimicrobial peptides served as a blank control group.

[0103] The results are as follows Figure 5 As shown, the fluorescence intensity in the blank control group was extremely low, indicating almost no bacterial death. After treatment 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 all six antimicrobial peptides had antibacterial effects. Among them, W-3 and W-4 had fluorescent cell ratios exceeding 70%, with W-4 approaching 80%, exhibiting the strongest bactericidal efficacy.

[0104] 3. Observation of cell membrane morphology

[0105] The extent of damage to the bacterial outer membrane by antimicrobial peptides was investigated by observing the outer membrane morphology of the test strain E. coli ATCC25922 using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The experimental procedures are as follows:

[0106] (1) The morphology of the bacterial cell membrane was observed using a scanning electron microscope (SEM). The bacterial culture was cultured to the second generation, centrifuged, and resuspended to obtain OD values. 600 =0.15 for later use. Dilute the antimicrobial peptide to 1MIC with bacterial solution and incubate for 1 hour. Discard the supernatant and wash and centrifuge for later use. Add 1 mL of 2.5% glutaraldehyde and incubate for 12 hours. Then wash the bacterial sludge with 50%, 70%, 90% and 100% ethanol solutions for 10 minutes each time. Then incubate the bacterial sludge with a mixture of 100% ethanol and tert-butanol solution for 30 minutes. Finally, treat with pure tert-butanol for 1 hour. Dry the obtained bacterial sludge solid, coat it with a film and observe and photograph it with a scanning electron microscope.

[0107] (2) The extent to which antimicrobial peptides damage the structure of the bacterial cells to be tested was observed using transmission electron microscopy (TEM). The bacterial sludge was fixed with 1% osmium tetroxide for 2 hours, centrifuged and washed, and then treated with 50%, 70%, 90% and 100% ethanol solutions for 8 minutes each time. After treatment with 100% ethanol for 10 minutes, it was treated with a mixture of equal volumes of ethanol and acetone for another 10 minutes. Finally, it was treated with 100% acetone for 10 minutes. The bacterial sludge was then embedded with embedding medium for 30 minutes and then embedded in pure resin overnight. Finally, it was stained with lead citrate and uranium acetate and prepared into sections for observation and photography using transmission electron microscopy.

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

[0109] The results showed that the bacteria in the control group had smooth surfaces, no wrinkles or pores, and were fully formed with intact structures. After treatment with different concentrations of W-4, the morphology deteriorated from wrinkled to surface-depressed, and even pore-forming, with cell fragmentation, structural loss, and significant loss of contents. This indicates that W-4 caused greater damage to bacteria and further demonstrates that the antimicrobial peptides disrupted the bacterial cell membrane, leading to bacterial death.

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined 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. The application of antimicrobial peptides in the preparation of antibacterial agents, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.2; The antibacterial agent inhibits pathogens such as Escherichia coli, Salmonella, Cronobacter sakazakii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, or Bacillus cereus.

3. An antibacterial agent, characterized in that, The active ingredients include an antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO.

2.

4. The antibacterial agent according to claim 3, characterized in that, The antibacterial agent inhibits pathogens such as Escherichia coli, Salmonella, Cronobacter sakazakii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, or Bacillus cereus.

5. The antibacterial agent according to claim 3, characterized in that, The antibacterial agent also includes pharmaceutically acceptable excipients.

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