CN118221766A - Fish source antibacterial peptide and screening method and application thereof - Google Patents

Abstract

The invention relates to a fish-source antibacterial peptide, a screening method and application thereof, wherein the antibacterial peptide comprises the following components: at least one of antibacterial peptide P1, antibacterial peptide P2, antibacterial peptide P3 and antibacterial peptide P4, wherein the amino acid sequence of the antibacterial peptide P1 is SEQ ID NO.1: KLCQLCAGKGT, the amino acid sequence of the antibacterial peptide P2 is SEQ ID NO.2: KKQCANLQNA, the amino acid sequence of the antibacterial peptide P3 is SEQ ID NO.3: KSHQSVMGF, and the amino acid sequence of the antibacterial peptide P4 is SEQ ID NO.4: KFCKQLFGGF. According to the invention, by taking the fish byproducts as raw materials, 4 fish-source antibacterial peptide sequences with excellent antibacterial potential are identified, and can change the permeability and fluidity of pathogenic bacteria cell membranes and reduce the activity of pathogenic bacteria somatic cells, so that the antibacterial and bacteriostatic effects are exerted. The invention combines bioinformatics technology to find the antibacterial peptide from fish protein, and compared with the traditional method, the screening method has the advantages of high screening efficiency, simplicity, practicability, high safety, lower cost and the like.

Description

Fish source antibacterial peptide and screening method and application thereof

Technical Field

The invention relates to the technical field of foods and medicines, in particular to a fish-source antibacterial peptide, a screening method and application thereof.

Background

Antibacterial peptides (AMPs), also known as host defense peptides, are a diverse class of natural molecules that are the first line of defense for all multicellular organisms. These active peptides have a broad range of biological activities that kill pathogenic bacteria, fungi, viruses and even cancer cells. The first antimicrobial peptides in the world were discovered by swedish scientists g.boman et al in 1980 when studying the immune mechanism of north american silkworms, and then the sources of antimicrobial peptides were continuously expanded, and to date 2700 antimicrobial peptides derived from bacteria, archaebacteria, protozoa, fungi, plants, animals, and the like have been discovered. The host-defense peptides are structurally amphiphilic and generally have a molecular weight of 2000-7000 Da and consist essentially of 20-60 amino acid residues, most of which have a net positive charge of +2 to +9 when exposed to physiological conditions at pH 7.4, and are therefore also referred to as cationic host-defense peptides.

The Chinese water area is wide, the aquatic resources are various, and the total yield is high. The aquatic resources in China can be roughly divided into: ① Fish, ② crustaceans, ③ molluscs, ④ algae, ⑤ mammals. Wherein fish are the largest group of aquatic resources. The fish source proteins are derived from abundant aquatic products, a large amount of byproducts such as fish scales, fish bones and fish skins can be produced in the aquatic product industrialization, and the processing byproducts of the products contain abundant proteins, so that if the fish source proteins can be utilized with high values, the problem of environmental pollution can be solved, the comprehensive utilization of the waste in the aquatic product processing can be promoted, and the great economic benefit is brought.

In the prior art, how to find the antibacterial peptide by using the fish by-product as the protein raw material is reported, so that the development of the systematic research of the related fish-source antibacterial peptide is of great significance.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: the invention provides a fish-source antibacterial peptide, a screening method and application thereof, wherein the fish-source antibacterial peptide has the characteristics of short sequence length, excellent antibacterial activity, good biocompatibility and the like, and the screening method is simple and efficient, thereby providing theoretical basis and experimental basis for the application of the fish-source antibacterial peptide in the food or medicine industry in the later period.

In order to solve the technical problems, the invention adopts the following technical scheme:

In one aspect, the invention provides a fish-derived antibacterial peptide, which comprises at least one of antibacterial peptide P1, antibacterial peptide P2, antibacterial peptide P3 and antibacterial peptide P4, wherein the amino acid sequence of the antibacterial peptide P1 is SEQ ID NO.1: KLCQLCAGKGT, the amino acid sequence of the antibacterial peptide P2 is SEQ ID NO.2: KKQCANLQNA, the amino acid sequence of the antibacterial peptide P3 is SEQ ID NO.3: KSHQSVMGF, and the amino acid sequence of the antibacterial peptide P4 is SEQ ID NO.4: KFCKQLFGGF.

Preferably, the antimicrobial peptide is of an alpha-helical structure.

According to the fish-derived antibacterial peptide provided by the invention, 4 fish-derived antibacterial peptide sequences with excellent antibacterial potential are identified by taking fish byproducts as raw materials, and can change the permeability and fluidity of pathogenic bacteria cell membranes and reduce the activity of pathogenic bacteria cells, so that the antibacterial and bacteriostatic effects are exerted. Among them, the antibacterial effect of the antibacterial peptide P3 is most remarkable, which is probably due to the fact that the antibacterial peptide P3 has a more typical alpha-helix structure than the other three antibacterial peptides, is easier to insert into pathogenic bacteria cell membranes, and causes the pathogenic bacteria membrane structure to be damaged to a greater extent. In addition, the 4 fish-derived antibacterial peptides have good biocompatibility on mammalian cells.

In another aspect, the present invention provides a method for screening the above-mentioned fish-derived antimicrobial peptide, comprising the steps of:

s1, preparing a fish protein peptide primary extract, and carrying out mass spectrometry analysis on the fish protein peptide primary extract;

s2, screening a peptide sequence with excellent antibacterial activity potential and hydrophobicity, positively charged property and amphipathy by means of an antibacterial peptide database and a prediction tool;

S3, modeling a three-dimensional structure by using modeling software, selecting the three-dimensional structure with the lowest energy by combining coarse-grain molecular dynamics simulation software, and screening the alpha-helical antibacterial peptide with hydrophobicity, positive charge and amphipathy.

Preferably, in step S1, the preparation method of the primary extract of fish protein peptide may be at least one of an acid method, an alkali method, an enzyme method and a hot water method.

The invention combines bioinformatics technology to find the antibacterial peptide from fish protein, and compared with the traditional method, the screening method has the advantages of high screening efficiency, simplicity, practicability, high safety, lower cost and the like.

In still another aspect, the invention provides an application of the fish-derived antibacterial peptide in preparation of vibrio harveyi antibacterial or bacteriostatic products.

Preferably, the minimum inhibitory concentration of the antibacterial peptide P1, the antibacterial peptide P2 and the antibacterial peptide P4 on the Vibrio harveyi is 0.25mg/mL, and the minimum inhibitory concentration of the antibacterial peptide P3 on the Vibrio harveyi is 0.125mg/mL.

The fish source antibacterial peptide provided by the invention can be used as an antibacterial or bacteriostatic product, and particularly has good antibacterial and bactericidal effects on Vibrio harveyi.

In view of the above properties and functions of the antibacterial peptide, a person of ordinary skill in the art can use the antibacterial peptide as a template, and directionally modify or synthesize a derivative of the antibacterial peptide of fish origin by changing the sequence of the antibacterial peptide so as to improve the antibacterial activity of the antibacterial peptide, and the antibacterial peptide can be applied to the fields of foods and medicines.

Drawings

FIG. 1 is a spatial structural diagram of an antibacterial peptide of example 1;

FIG. 2 is a spiral wheel view of the antibacterial peptide of example 1;

FIG. 3 is a high performance liquid chromatogram of the antimicrobial peptide of example 2;

FIG. 4 is a mass spectrum of the antibacterial peptide of example 2;

FIG. 5 is a MIC diagram of a synthetic antimicrobial peptide;

FIG. 6 shows the effect of synthetic antimicrobial peptides on cell membrane permeability, wherein (A) is the outer membrane and (B) is the inner membrane;

FIG. 7 is a graph showing the effect of synthetic antimicrobial peptides on cell membrane fluidity;

FIG. 8 is the effect of synthetic antimicrobial peptides on cell membrane integrity;

FIG. 9 is a graph showing cytotoxicity of synthetic antimicrobial peptides on human skin fibroblasts;

FIG. 10 is a view showing hemolysis of synthetic antimicrobial peptides;

FIG. 11 is a circular dichroism spectrum of synthetic antimicrobial peptides in TFE solution;

FIG. 12 is a graph showing the secondary structure profile of synthetic antimicrobial peptides in TFE solution.

Detailed Description

In order that the above-described aspects may be better understood, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The embodiment provides a fish-derived antibacterial peptide, which comprises at least one of antibacterial peptide P1, antibacterial peptide P2, antibacterial peptide P3 and antibacterial peptide P4, wherein the amino acid sequence of the antibacterial peptide P1 is SEQ ID NO.1: KLCQLCAGKGT, the amino acid sequence of the antibacterial peptide P2 is SEQ ID NO.2: KKQCANLQNA, the amino acid sequence of the antibacterial peptide P3 is SEQ ID NO.3: KSHQSVMGF, and the amino acid sequence of the antibacterial peptide P4 is SEQ ID NO.4: KFCKQLFGGF.

The antibacterial peptide has an alpha-helical structure.

The method for screening the rice comprises the following steps of:

s1, preparing a fish protein peptide primary extract, and carrying out mass spectrometry analysis on the fish protein peptide primary extract;

The preparation method of the fish source protein peptide primary extract can be at least one of an acid method, an alkali method, an enzyme method and a hot water method.

S2, screening out a peptide sequence with excellent antibacterial activity potential and hydrophobicity, positively charged property and amphipathy by means of an antibacterial peptide database and a prediction tool.

S3, modeling a three-dimensional structure by using modeling software, selecting the three-dimensional structure with the lowest energy by combining coarse-grain molecular dynamics simulation software, and screening the alpha-helical antibacterial peptide with hydrophobicity, positive charge and amphipathy.

In a preferred embodiment of the invention, the antimicrobial peptide database is CAMP3 (http:// www.camp.bicnirrh.res.in /), the prediction tool is Heliquest (https:// helix. Ipmc. Cnrs. Fr /), the modeling software is Rosseta, and the coarse-grained molecular dynamics simulation software is Gromacs.

The final antimicrobial peptide selected by the above method may be one or more polypeptides comprising the amino acid sequences shown in SEQ ID NO.1-SEQ ID NO. 4.

The fish source antibacterial peptide can be applied to preparation of Ha Weishi vibrio antibacterial or bacteriostatic products.

The minimum inhibitory concentration of the antibacterial peptide P1, the antibacterial peptide P2 and the antibacterial peptide P4 on the Vibrio harveyi is 0.25mg/mL, and the minimum inhibitory concentration of the antibacterial peptide P3 on the Vibrio harveyi is 0.125mg/mL.

Example 1: screening method of fish-source antibacterial peptide

S1, preparing a fish protein peptide primary extract, and determining peptide sequences of the fish protein peptide primary extract by means of nano liter liquid chromatography-quadrupole orbitrap mass spectrometry;

The preparation method of the fish source protein peptide primary extract specifically comprises the following steps:

Cleaning fresh fish source protein scales, weighing 5g of fish scales, dissolving in 0.5MNaOH solution at a feed liquid ratio of 1:20, stirring for 2 hours, and replacing the solution every 1 hour to remove impurities in the fish scales and soften the fish source protein scales. The fish protein flakes are washed to be neutral by distilled water, dissolved in 0.1MEDTA-2Na for 2 hours with the aid of ultrasonic decalcification at a ratio of 1:15g/mL, and washed to be neutral by distilled water. The pretreated fish protein flakes were dissolved in 10% citric acid (w/w) at a ratio of 10:1mL/g in a constant temperature water bath at 50℃for 12h. Cooling the sample solution to 37 ℃, adding 1% pepsin for enzymolysis for 2 hours at the pH of=1.5, heating to 100 ℃ for inactivating the enzyme for 10 minutes, finally filtering with gauze, taking supernatant, and freeze-drying to obtain the fish-derived protein flake protein hydrolysate;

The peptide sequence determination method is specifically as follows:

The freeze-dried component of the protein hydrolysate of the fish protein flake purified by the gel filtration chromatography system is dissolved in 10 mu L of loading buffer (99.9% H ₂ O,0.1% formic acid) for the next test; placing the sample on a C18 reverse phase analysis column (50 mm multiplied by 50cm monolithic column), and washing with 2-100% acetonitrile solution gradient at a running speed of 500nL/min; full scan acquisition (350-1800 m/z) works in orbital wells with a resolution of 70,000 (automatic gain control at 3 x 10 ⁶) using regular reflectors.

S2, comparing and analyzing the obtained peptide sequence with an antibacterial peptide database CAMP3 (http:// www.camp.bicnirrh.res.in /), and primarily screening out a peptide sequence with antibacterial activity potential; and then carrying out spiral round and physicochemical property analysis on the identified peptide sequence by utilizing Heliquest tool (https:// helix. Ipmc. Cnrs. Fr /), and further screening out the preferred peptide sequence with hydrophobicity, positive charge and amphipathy.

S3, carrying out three-dimensional structure modeling on a peptide sequence by using Rosseta software, simulating 100ns by using Gromacs coarse-particle molecular dynamics, selecting the three-dimensional structure with the lowest energy, and finally screening out 4 alpha-helical antibacterial peptides with hydrophobicity, positive charge and amphipathy, namely antibacterial peptides P1, P2, P3 and P4.

The spatial structure and helix pattern of the peptides P1-P4 obtained in this example are shown in FIGS. 1 and 2, respectively, and the relevant physicochemical properties are recorded in Table 1.

TABLE 1 four antibacterial peptide sequences and related physicochemical Properties

Example 2: synthesis of the corresponding polypeptide from the antibacterial peptide sequence obtained by the screening in example 1

The antibacterial peptide sequences obtained by screening in example 1 are synthesized into corresponding polypeptides by using a solid-phase chemical synthesis method by Jier biochemical company (Shanghai), the synthesized polypeptides are purified to have the purity of more than 95% by reverse-phase high performance liquid chromatography, the amino acid sequences of the polypeptides are further identified by electrospray mass spectrometry, the molecular weights of the obtained synthetic antibacterial peptides P1, P2, P3 and P4 are 1121.37, 1117.27, 1174.41 and 1020.16 respectively, and the predicted amino acid sequences are consistent with the sequences of the antibacterial peptides P1, P2, P3 and P4 respectively.

The chromatographic conditions are as follows: the chromatographic column was 4.6X 250mm,Kromasil C18 5. Mu.m; liquid chromatography conditions: mobile phase: a pump: 0.1% by volume of acetonitrile trifluoroacetate solution, B pump: 0.1% by volume of aqueous trifluoroacetic acid. The flow rate is 1.0mL/min; the sample injection amount is 20 mu L; detection wavelength: 220nm; elution time: 30min; the elution mode is gradient elution: the antibacterial peptides P1, P2, P3 and P4 are fed with 15% of A+85% of B, 8% of A+92% of B, 25% of A+75% of B and 20% of A+80% of B at the beginning, the antibacterial peptides are fed with 40% of A+60% of B at 25.0min, the antibacterial peptides are fed with 100% of A+0% of B at 25.1min, and the reaction is stopped when the reaction is maintained for 30 min. The high performance liquid chromatograms of the antibacterial peptides P1, P2, P3 and P4 are shown in FIG. 3.

The mass spectrum conditions are as follows: mobile phase: water: acetonitrile = 1:1, nebulizer gas flow: 1.5L/min, sample injection amount: the CDL and Block temperatures were 250℃and 400℃respectively at 0.2 mL/min. The liquid chromatography mass spectrum of the antibacterial peptides P1, P2, P3 and P4 is shown in FIG. 4.

The antibacterial peptide synthesized in example 2 was subjected to antibacterial activity, antibacterial mechanism, safety and secondary structure study analysis.

1. Determination of antibacterial Activity

The inhibitory activity of the antibacterial peptide synthesized in example 2 of the present invention against bacteria was examined by a microtiter method: the antibacterial activity of the antibacterial peptides P1, P2, P3 and P4 on the aquatic pathogenic bacteria Vibrio harveyi 2510 is evaluated by using the aquatic pathogenic bacteria as indicator bacteria. The specific operation is as follows:

Inoculating the strains stored in the freezing tube one by one to an LB broth culture medium for activation culture, culturing in a shaking table at 30 ℃ and rotating at 180r/min until the strains reach a logarithmic phase, transferring the activated strains to a fresh LB broth culture medium for secondary culture, shaking and culturing in the shaking table at 30 ℃ until the strains reach the logarithmic phase, and diluting the strain culture medium cultured until the strains reach the logarithmic phase to 1X 10 ⁵ cfu/mL with the fresh LB broth culture medium for later use.

Adding 90 mu L of LB broth culture medium and 10 mu L of 2500 mu g/mL of antibacterial peptide into the first column of a 96-well plate, adding 50 mu L of culture medium into 2-9 columns, uniformly mixing the 1 st column, sucking 50 mu L of mixed liquid of LB broth culture medium and P1-P4 to the 2 nd column, continuously uniformly mixing the 2 nd column, removing 50 mu L of sample to the 3 rd column, and the like until the 9 th column; then, the diluted bacterial liquid was added to 2 to 9 columns in an amount of 50. Mu.L. Column 10 was added with 100 μl of LB broth as a blank. Column 11 was added with 100. Mu.L of bacterial liquid as positive control. Finally, the 96-well plate was placed in an incubator at 30℃overnight with slow shaking for 12 hours, and light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible. The results are shown in Table 2 and FIG. 5.

TABLE 2 Minimum Inhibitory Concentration (MIC) of antibacterial peptides P1-P4 against Vibrio harveyi

2. Cell membrane permeability study

First outer membrane permeability assay

Outer membrane permeability was determined by measuring beta-lactamase activity using cefdithien (Nitrocefin) as substrate. The log phase bacterial cells were collected by centrifugation, washed and resuspended in PBS, the bacterial concentration was adjusted to an absorbance at 600nm of about 0.4, and then the final concentration of MIC of the antimicrobial peptide was added to the bacterial suspension, and the control group was added with an equal volume of PBS. mu.L of the bacterial suspension was mixed with 50. Mu.L of cefditoren (0.5 mg/mL) in 96-well plates and incubated at 37 ℃. After incubation, absorbance at 500nm was measured using a SpectraMax iD3 multifunctional microplate reader.

(II) inner Membrane permeability measurement

Endomembrane permeability was determined by measuring the β -galactosidase activity released by the bacteria into the culture medium using o-nitrobenzene- β -D-galactopyranoside (ONPG) as substrate. Bacteria grown to log phase were centrifuged, washed and resuspended in ONPG solution (1.5 mM) and added to 96-well plates (50 μl/well). The final concentration of MIC of the antimicrobial peptide was added to a 96-well plate (50. Mu.L/well), and the control was added to an equal volume of PBS solution. The absorbance at 420nm was measured by a SpectraMax iD3 multifunctional microplate reader.

As can be seen from FIG. 6, the antibacterial peptide disclosed by the invention acts on the cell membrane of Vibrio harveyi, can change the cell membrane structure, and leads to the remarkable increase of the permeability of the inner membrane and the outer membrane, so that the cell content of the thalli is leaked, thereby inhibiting the growth and propagation of the thalli cells and reducing the infection rate of pathogenic bacteria.

3. Cell membrane fluidity study

The logarithmic phase of the bacterial cells were collected by centrifugation, washed twice with PBS solution, then the bacterial concentration was adjusted to about 0.4 absorbance at 600nm, and 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) was added to a final concentration of 4mM, and incubated at 37℃for 30 minutes in the absence of light. The final MIC concentration of the antimicrobial peptide was then added to the bacterial suspension and incubated for a period of time, centrifuged to remove excess dye, and the cells resuspended in PBS. Fluorescence intensities were recorded using a SpectraMax iD3 multifunctional microplate reader with excitation and emission wavelengths of 358nm and 428nm, respectively.

As can be seen from fig. 7, in this measurement, the decrease in membrane fluidity increases the polarization of DPH fluorescence, resulting in an increase in fluorescence intensity, and the cell membrane fluidity of vibrio harveyi treated with the antimicrobial peptides P1-P4 of the present invention is significantly decreased compared to the control group, which suggests that the antimicrobial peptides P1-P4 act on the cell membrane of the pathogenic bacteria, resulting in rearrangement of the membrane phospholipid bilayer, resulting in a decrease in cell membrane fluidity.

4. Cell membrane integrity study

The log phase somatic cells were collected by centrifugation, washed with PBS and resuspended. Then 50. Mu.L of the cell suspension was mixed with 50. Mu.L of propidium iodide (PI, initial concentration 12. Mu.g/mL) in 96-well plates, incubated at room temperature for 15min, centrifuged to remove excess dye, and the cells resuspended in PBS and added with the final concentration of the antimicrobial peptide MIC. Fluorescence intensity was measured with SpectraMaxiD multifunctional microplate reader, excitation and emission wavelengths 535 and 617nm, respectively.

As shown in FIG. 8, the Vibrio harveyi cell membrane integrity treated with the antimicrobial peptides P1-P4 of the present invention is disrupted compared with the control group, and the results of the above-described studies on cell membrane permeability and fluidity are interpretable. The antibacterial peptide acts on the cell membrane of vibrio harveyi to damage the integrity of the cell membrane, thereby affecting the functions of the cell membrane, namely leading to the increase of the permeability of the cell membrane and the reduction of the fluidity of the cell membrane, and reducing the infection capability of pathogenic bacteria to a host.

5. Safety study

The cytotoxicity of the antibacterial peptides P1-P4 is measured by adopting a CCK-8 method, and the specific steps are as follows:

And (3) paving: the logarithmic phase Human Skin Fibroblasts (HSF) are digested by 0.25% pancreatin, washed by PBS buffer (0.01M, pH=7.2), centrifuged (2000 r/min, 3 min) and collected to prepare cell suspension, the cell culture medium ratio is 45mL DMEM, 5mL serum, 0.5mL diabody, and the concentration is adjusted to 1X 10 ⁵/mL by adopting a cell counting method; the cell suspension was aspirated into 96-well plates (100. Mu.L/well), and cultured in a cell incubator at 37℃under a CO ₂ concentration and a humidity of 95% for 24 hours.

Sample adding: the prepared 96-well plate was removed from the incubator, column 1 was added with 100. Mu.L of cell culture medium as a blank. Antibacterial peptide samples were diluted with sterile water to concentrations of 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μg/mL, 20 μl of sample per well, and 80 μl of medium were added and mixed well, and then added to 96-well plates, 5 sets of parallel groups were set for each concentration, and the outermost periphery of each 96-well plate was sealed with PBS buffer (0.01 m, ph=7.2). The 96-well plate was then placed in an incubator for further incubation for 24 hours.

Adding CCK-8: adding CCK-8 solution according to 10% of the total volume of each hole of culture medium, namely 20 mu L, placing the culture medium into an incubator for continuous culture for 2 hours, and measuring the absorbance at 450nm and 650nm by using an enzyme-labeled instrument.

Cell viability calculation formula:

note that: m is an antibacterial peptide treatment group; c is a blank control group; s is cell viability.

As can be seen from fig. 9, the antibacterial peptide of the present invention is less cytotoxic to mammalian cells over a range of concentrations.

(II) hemolysis test the specific steps are as follows:

P1-P4 was diluted in 96-well plates with PBS as solvent to a concentration of 32.25. Mu.g/mL and 96.75. Mu.g/mL, respectively, with ultrapure water as a blank and Triton-X as a positive control; then preparing 8% erythrocyte suspension, namely taking 10mL of fresh mouse blood (preserved by heparin sodium anticoagulation tube) and centrifuging for 10min, lightly flushing with PBS for 2 times, re-suspending with 10mL of PBS, and adding 1.6mL of erythrocyte into 18.4mL of PBS; finally, 8% of the red blood cell suspension was added to the prepared 96-well plate containing the drug and incubated at 37℃for 1 hour, and 120mL of the supernatant was centrifuged and removed to a new 96-well plate, and the absorbance of the newly prepared 96-well plate at 540nm was measured to calculate the hemolysis ratio by the following formula.

As shown in figure 10, the antibacterial peptide provided by the invention has low hemolysis and high safety, and is suitable for being used in various fields.

6. Secondary structure study

The secondary structure of the antibacterial peptide is determined by adopting a round two-chromatography method, and the specific steps are as follows:

The antimicrobial peptide was dissolved in sterile water and the sample was mixed with trifluoroethanol (Aladin) in a 1:1 volume ratio to give a final antimicrobial peptide concentration of 125. Mu.g/mL. The circular dichroism spectrum (lambda=190-260 nm) of the antimicrobial peptides was measured at a temperature of 25℃and a frequency of 50nm/min for an optical path of 0.1cm using J1500 spectropolarimeter (Jasco, tokyo, japan). 3 replicates were measured.

As can be seen from the circular dichroism measurement (figure 11), the antibacterial peptides P1-P3 form a negative peak at 220-230 nm and a strong positive peak at 190-200 nm, and the antibacterial peptides P1-P3 are of alpha-helical structures, which have great influence on the antibacterial activity. As can be seen from FIG. 12, the content of the alpha-helix structure of each of the antibacterial peptides P1, P3 and P4 is more than 20%, and the content of the alpha-helix structure of the antibacterial peptide P2 is 1%, which indicates that each of the antibacterial peptides P1 to P4 has an alpha-helix structure.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (6)

1. The fish-source antibacterial peptide is characterized by comprising at least one of antibacterial peptide P1, antibacterial peptide P2, antibacterial peptide P3 and antibacterial peptide P4, wherein the amino acid sequence of the antibacterial peptide P1 is SEQ ID NO.1: KLCQLCAGKGT, the amino acid sequence of the antibacterial peptide P2 is SEQ ID NO.2: KKQCANLQNA, the amino acid sequence of the antibacterial peptide P3 is SEQ ID NO.3: KSHQSVMGF, and the amino acid sequence of the antibacterial peptide P4 is SEQ ID NO.4: KFCKQLFGGF.

2. The fish-derived antimicrobial peptide of claim 1, wherein the antimicrobial peptide is an α -helical structure.

3. A method of screening a fish-derived antibacterial peptide according to claim 1, comprising the steps of:

s1, preparing a fish protein peptide primary extract, and carrying out mass spectrometry analysis on the fish protein peptide primary extract;

s2, screening a peptide sequence with excellent antibacterial activity potential and hydrophobicity, positively charged property and amphipathy by means of an antibacterial peptide database and a prediction tool;

S3, modeling a three-dimensional structure by using modeling software, selecting the three-dimensional structure with the lowest energy by combining coarse-grain molecular dynamics simulation software, and screening the alpha-helical antibacterial peptide with hydrophobicity, positive charge and amphipathy.

4. The method according to claim 3, wherein in step S1, the primary fish-derived protein peptide extract is prepared by at least one of acid method, alkali method, enzyme method and hot water method.

5. Use of the fish-derived antibacterial peptide of claim 1 for preparing a vibrio harveyi antibacterial or bacteriostatic product.

6. The use according to claim 5, wherein the minimum inhibitory concentration of the antibacterial peptide P1, the antibacterial peptide P2 and the antibacterial peptide P4 on vibrio harveyi is 0.25mg/mL and the minimum inhibitory concentration of the antibacterial peptide P3 on vibrio harveyi is 0.125mg/mL.