An enzymatic hydrolysate antibacterial peptide from the dark muscle of chub mackerel, and its preparation method and use

Abstract

The present invention discloses a scomber japonicus dark muscle enzymolysis antibacterial peptide, its preparation method and use. The characteristics are that the amino acid sequence of the antibacterial peptide is Arg-Ser-Cys-His and / or Lys-Ser-Cys-Gly. The steps of its preparation method are as follows: Add deionized water with a mass 2-4 times that of the fish meat to the defatted scomber japonicus dark muscle and adjust the pH to 7-11, then add trypsin and bacillus licheniformis protease for enzymolysis, then incubate to inactivate the enzyme and centrifuge, and take the supernatant to obtain the crude enzymolysis solution; Add C-terminal endo-double enzyme cysteine protease ArgC and serine protease LysC to the crude enzymolysis solution for enzymolysis, incubate to inactivate the enzyme and centrifuge to obtain the upper enzymolysis clear solution, then filter it with an ultrafiltration system to obtain an enzymolysis solution of 1-3 kDa, collect the filtrate and freeze-dry it, and the powder is the scomber japonicus dark muscle enzymolysis antibacterial peptide. The advantages are high extraction efficiency and significant inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria.

Description

Technical Field

[0001] The present invention relates to a preparation technology of a broad-spectrum antimicrobial peptide, in particular to an enzymatically hydrolyzed antimicrobial peptide from dark-colored meat of mackerel, a preparation method thereof and application thereof. Background Art

[0002] As a new type of biological preservative, antimicrobial peptides are environmentally friendly, safe, and highly effective. They are generally derived from microbial metabolites, animal oral secretions, and animal protein hydrolysates. However, currently commercially valuable antimicrobial peptides, such as lysozyme, nisin, and natamycin, are much more effective against Gram-positive bacteria than Gram-negative bacteria. However, the dominant spoilage bacteria in aquatic foods are Gram-negative bacteria such as Shewanella putrefaciens, Pseudomonas aeruginosa, and Flavobacterium. Therefore, the development of commercial antimicrobial peptides for aquatic foods is highly desirable. Oligopeptides with basic amino acids such as lysine or arginine at their carbon or nitrogen termini have demonstrated excellent antibacterial effects against Gram-negative bacteria.

[0003] The muscles of pelagic fish are rich in basic amino acids such as lysine, arginine, and histidine. Su Yang et al.'s study, "Nutritional Analysis and Evaluation of Normal and Dark Meat from Three Tuna Species in the South China Sea," showed that after removing fat, polysaccharides, and other components from the muscle, the proportion of basic amino acids in dark meat was significantly higher than in normal meat. However, dark meat is often a processing waste, used solely as a raw material for fish meal feed, and has low added value. The commercial potential of using these processing waste from pelagic fish to produce green, safe, and highly effective bio-antimicrobial peptides is promising. Currently, no research has been published domestically or internationally on enzymatic antimicrobial peptides from dark meat of mackerel, their preparation methods, and their applications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an enzymatically hydrolyzed antimicrobial peptide from dark-colored meat of mackerel with high extraction efficiency and significant inhibitory effect on both Gram-positive and Gram-negative bacteria, as well as a preparation method and use thereof.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: an enzymatically hydrolyzed antimicrobial peptide from dark-colored meat of mackerel, wherein the amino acid sequence of the antimicrobial peptide is Arg-Ser-Cys-His (RSCH) and / or Lys-Ser-Cys-Gly (KSCG).

[0006] The method for preparing the enzymatically hydrolyzed antimicrobial peptide from dark-colored mackerel meat comprises the following steps:

[0007] (1) Preparation of defatted dark mackerel meat

[0008] Mackerel were steamed at 100°C for 20 minutes, then sprayed with deionized water and cooled to 40-50°C. The dark meat from the back of the fish was collected and homogenized in a homogenizer for 3-4 minutes. After that, isopropyl alcohol was added in an amount equal to the weight of the fish meat. The fish was defatted at 50°C for 90 minutes, and the defatted dark meat was obtained by vacuum filtration.

[0009] (2) Dual enzyme hydrolysis with non-directional endonuclease

[0010] Deionized water in an amount of 2 to 4 times the mass of the fish meat is added to the defatted dark meat of mackerel obtained in step (1), and the pH is adjusted to 7 to 11 with NaOH. Then, trypsin and Bacillus licheniformis protease are added, and enzymatic hydrolysis is carried out at 43-47°C for 4 hours. The mixture is then heated to above 90°C, kept warm for 20 minutes to inactivate the enzyme, and centrifuged at 6500 r / min for 10 minutes. The supernatant is taken to obtain a crude enzymatic solution. First, trypsin and Bacillus licheniformis protease are used for dual-enzyme hydrolysis. Both enzymes can efficiently degrade the dark meat of mackerel, and the synergistic hydrolysis of the dual enzymes can greatly improve the degree of hydrolysis. This is because mackerel protein contains more basic amino acids. There is aspartic acid in the active center of trypsin, which is conducive to combining with positively charged arginine and lysine. Bacillus licheniformis protease is a polar, positively charged amino acid. Therefore, after the fish protein is hydrolyzed by Bacillus licheniformis protease, it provides more action sites for trypsin, greatly improving the enzymatic hydrolysis effect and increasing the extraction rate of mackerel dark meat oligopeptides to more than 95%.

[0011] (3) Dual enzyme hydrolysis with directional endonuclease

[0012] The C-terminal endonuclease double enzyme cysteine protease ArgC and serine protease LysC were added to the crude enzymatic solution obtained in step (2), and the enzymatic hydrolysis was carried out at 43-47°C for 4 h, followed by heating to above 90°C, keeping the temperature for 20 min to inactivate the enzyme, and centrifuging at 5000 r / min for 15 min to obtain the upper enzymatic hydrolysis supernatant; the C-terminal endonuclease double enzyme cysteine protease ArgC and serine protease LysC were then used for enzymatic hydrolysis, which can efficiently and specifically cut the C-terminus of arginine and lysine. Since lysine and arginine are widely distributed in the protein sequence, the protein sample can produce peptides with an average length of 14 amino acid residues and ending with basic amino acids after enzymatic hydrolysis. The lysine or arginine residues at the carboxyl end and the amino end of these peptides carry a positive charge, which greatly improves their antibacterial activity;

[0013] (4) Collection of antimicrobial peptides

[0014] The upper-layer enzymatic hydrolysate obtained in step (3) is filtered with an ultrafiltration system to obtain an enzymatic hydrolysate with a molecular weight of 1-3 kDa. After collecting the filtrate, it is freeze-dried, and the powder is the enzymatic hydrolysate antibacterial peptide from mackerel dark muscle. The amino acid sequences of the antibacterial peptide are Arg-Ser-Cys-His (RSCH) and / or Lys-Ser-Cys-Gly (KSCG).

[0015] Furthermore, the dosages of the trypsin and the bacillus licheniformis protease in step (2) are 1000-3000 U / g respectively.

[0016] Furthermore, the dosages of the cysteine protease ArgC and the serine protease LysC in step (3) are 1000-3000 U / g respectively.

[0017] Use of the above-mentioned enzymatic hydrolysate antibacterial peptide from mackerel dark muscle in the preparation of an antibacterial agent, and the bacterial strains include Enterobacter, Shewanella putrefaciens, Pseudomonas, Lactobacillus, Staphylococcus aureus and Bacillus subtilis.

[0018] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses an enzymatic hydrolysate antibacterial peptide from mackerel dark muscle and its preparation method. The preparation method includes the preparation of defatted mackerel dark muscle, double enzymatic hydrolysis with non-directional endonucleases, double enzymatic hydrolysis with directional endonucleases, and collection of the antibacterial peptide, etc. The obtained enzymatic hydrolysate antibacterial peptide from mackerel dark muscle has broad-spectrum properties and has significant inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria. The MIC values for Enterobacter ( Enterobacteriaceae ), Shewanella putrefaciens ( Shewanella putrefaciens ), and Pseudomonas ( Pseudomonas spp. ) are 10 ug / mL, the MIC value for Lactobacillus ( Lactobacillus ) is 20 ug / mL, and the MIC values for Staphylococcus aureus ( Staphylococcus aureus ) and Bacillus subtilis ( Staphylococcus aureus ) are 80 ug / mL. It is a green, safe and highly efficient broad-spectrum antibacterial peptide. Description of the Drawings

[0019] Figure 1 It is the molecular weight distribution diagram of the crude enzymatic hydrolysate from mackerel dark muscle;

[0020] Figure 2 It is the molecular weight distribution diagram of the upper-layer supernatant from mackerel dark muscle. Detailed Embodiments

[0021] The present invention will be further described in detail below with reference to the embodiments in the drawings.

[0022] Specific Embodiment 1

[0023] A scomber japonicus dark muscle enzymatic hydrolysis antibacterial peptide, the amino acid sequence of the antibacterial peptide is Arg-Ser-Cys-His (abbreviated as RSCH) and / or Lys-Ser-Cys-Gly (abbreviated as KSCG). Specific Example 2

[0025] Example 1

[0026] The preparation method of the scomber japonicus dark muscle enzymatic hydrolysis antibacterial peptide in the above Specific Example 1 is as follows:

[0027] (1) Preparation of defatted scomber japonicus dark muscle

[0028] The scomber japonicus is steamed at 100 °C for 20 min and then sprayed and cooled with deionized water to 40 - 50 °C. Take the darker dark muscle on the back of the fish, process it in a homogenizer for 3 - 4 min, add isopropanol with the same mass as the fish meat, and degrease at 50 °C for 90 min. Vacuum filter to obtain defatted scomber japonicus dark muscle;

[0029] (2) Double enzymatic hydrolysis with non-directional endonucleases

[0030] Add 4 times the mass of deionized water to the defatted scomber japonicus dark muscle obtained in step (1), adjust the pH to 9.5 with NaOH, then add trypsin and bacillus licheniformis protease with an enzyme activity unit of 1250 U / g each, enzymatically hydrolyze at 45 °C for 4 h, then raise the temperature to above 90 °C and keep warm to inactivate the enzyme for 20 min. Centrifuge at 6500 r / min for 10 min, and take the supernatant to obtain the crude enzymatic hydrolysis solution; filter with an ultrafiltration system to obtain the molecular weight distribution of the crude enzymatic hydrolysis solution with 1 kDa < molecular weight < 3 kDa as Figure 1 . Among the peptides obtained by double enzymatic hydrolysis with non-directional endonucleases, more than 90% of the peptides have a molecular weight greater than 1 kDa. The peptides with a molecular weight greater than 3 kDa are the most, with a content of 52.7%; the peptides with a molecular weight of 1 kDa - 3 kDa have a content of 41.1%; the peptides with a molecular weight less than 1 kDa have a content of 6.2%. The yield of soluble peptides in the enzymatic hydrolysis solution / % = (polypeptide content in the enzymatic hydrolysis solution / protein content in scomber japonicus dark muscle) * 100 (the soluble peptide content is measured by trichloroacetic acid precipitation combined with the Folin-phenol method, and the protein content in scomber japonicus dark muscle is measured by the Kjeldahl nitrogen method). The extraction rate of soluble peptides in the enzymatic hydrolysis solution is greater than 95%.

[0031] Freeze-dry the crude enzymatic hydrolysis solution and measure the minimum inhibitory concentration of the freeze-dried powder. The specific measurement method is as follows: Use the micro-dilution method to measure the minimum inhibitory concentration of the freeze-dried powder of the scomber japonicus dark muscle enzymatic hydrolysis crude solution against several Gram-positive bacteria and Gram-negative bacteria. Take the bacterial liquid in the logarithmic growth phase by the McFarland turbidity method, and dilute the bacterial liquid to 10 5CFU / mL. Add 100 μL of MH broth medium to each well of the sterilized enzyme-linked immunosorbent assay (ELISA) plate. Add 100 μL of the freeze-dried powder solution of the crude enzymatic hydrolysate with a mass concentration of 320 μg / mL to the first well, and then perform two-fold dilution on the freeze-dried powder solution of the crude enzymatic hydrolysate to adjust the mass concentration of the freeze-dried powder solution of the crude enzymatic hydrolysate to: 320, 160, 80, 40, 20, 10, 5, 2.5 μg / mL in sequence, and do 3 parallel experiments. Then add another 100 μL of MH broth medium to a row of wells as a negative control. Add 100 μL of the bacterial solution to another row of wells as a positive control. Place the ELISA plates of Shewanella putrefaciens and Pseudomonas in an incubator at 30 °C, and place the rest in an incubator at 37 °C for 12 h. Table 1 shows the determination results of the minimum inhibitory concentration of the freeze-dried powder of the crude enzymatic hydrolysate,

[0032] Table 1 Antibacterial effects of the freeze-dried powder of the crude enzymatic hydrolysates of trypsin and Bacillus licheniformis protease

[0033] .

[0034] As can be seen from Table 1 above, the minimum inhibitory concentration (MIC) value of the freeze-dried powder of the crude enzymatic hydrolysate of chub mackerel dark muscle against Enterobacter ( Enterobacteriaceae ), Shewanella putrefaciens ( Shewanella putrefaciens ), Pseudomonas ( Pseudomonas spp. ), and Lactobacillus ( Lactobacillus ) is 160 μg / mL, and the MIC value against Staphylococcus aureus ( Staphylococcus aureus ) and Bacillus subtilis ( Staphylococcus aureus ) is 320 μg / mL.

[0035] (3)Double enzymatic hydrolysis of site-directed endonucleases

[0036] Add cysteine protease ArgC and serine protease LysC with an enzyme activity unit of 1250 U / g to the crude enzymatic hydrolysate obtained in step (2), and perform enzymatic hydrolysis at 45 °C for 4 h. Then raise the temperature to above 90 °C and keep warm for 20 min to inactivate the enzyme. Centrifuge at 5000 r / min for 15 min to obtain the upper-layer enzymatic hydrolysate supernatant; filter the upper-layer enzymatic hydrolysate supernatant obtained by enzymatic hydrolysis with an ultrafiltration system to obtain an enzymatic hydrolysate with a molecular weight of 1 kDa < molecular weight < 3 kDa. The molecular weight distribution of the obtained upper-layer enzymatic hydrolysate supernatant is as Figure 2 . Among the peptides obtained by the double enzymatic hydrolysis of site-directed endonucleases, more than 90% of the peptides have a molecular weight less than 3 kDa. Among them, the content of peptides with a molecular weight of 1 kDa - 3 kDa is 84.4%, the content of peptides with a molecular weight less than 1 kDa is 9.2%, and the content of peptides with a molecular weight greater than 3 kDa is 6.4%. Therefore, the use of double enzymatic hydrolysis of site-directed endonucleases can obtain more peptides with a smaller molecular weight.

[0037] The enzymatic hydrolysis supernatant was freeze-dried, and the minimum inhibitory concentration of the freeze-dried powder was determined. The specific determination method was as follows: The micro-dilution method was used to determine the minimum inhibitory concentration of the freeze-dried powder of the enzymatic hydrolysis supernatant of mackerel dark muscle against several Gram-positive bacteria and Gram-negative bacteria. The bacterial suspension in the logarithmic growth phase was taken by the McFarland turbidity method, and the bacterial suspension was diluted to 10 5 CFU / mL with MH broth medium. 100 μL of MH broth medium was added to each well of the sterilized enzyme-labeled plate. 100 μL of the freeze-dried powder solution of the enzymatic hydrolysis supernatant with a mass concentration of 320 μg / mL was added to the first well, and then the freeze-dried powder solution of the enzymatic hydrolysis supernatant was serially diluted to adjust the mass concentration of the freeze-dried powder solution of the enzymatic hydrolysis supernatant to 320, 160, 80, 40, 20, 10, 5, 2.5 μg / mL in turn, and 3 parallel tests were conducted. Then, 100 μL of MH broth medium was added to another row of wells as a negative control. 100 μL of the bacterial suspension was added to another row of wells as a positive control. The enzyme-labeled plates of Shewanella putrefaciens and Pseudomonas were placed in an incubator at 30 °C, and the others were placed in an incubator at 37 °C for 12 h. Table 2 is the determination table of the minimum inhibitory concentration of the freeze-dried powder of the enzymatic hydrolysis supernatant obtained in step A3.

[0038] Table 2 Antibacterial effects of freeze-dried powders of the enzymatic hydrolysis supernatants of cysteine protease ArgC and serine protease LysC in step A3

[0039] 。

[0040] As can be seen from Table 2 above, the MIC values of the freeze-dried powder of the enzymatic hydrolysis supernatant obtained in step (3) against Enterobacter ( Enterobacteriaceae ), Shewanella putrefaciens ( Shewanella putrefaciens ), Pseudomonas ( Pseudomonas spp. ), and Lactobacillus ( Lactobacillus ) are 40 μg / mL, and the MIC values against Staphylococcus aureus ( Staphylococcus aureus ) and Bacillus subtilis ( Staphylococcus aureus ) are 160 μg / mL.

[0041] (4) Collection of antibacterial peptides

[0042] The upper-layer enzymatic hydrolysate obtained in step (3) was filtered with an ultrafiltration system to obtain an enzymatic hydrolysate with a molecular weight of 1 kDa < molecular weight < 3 kDa. After collecting the filtrate, it was freeze-dried, and the powder was the enzymatic antibacterial peptide from mackerel dark muscle. The yield percentage of antibacterial peptide in the enzymatic hydrolysate = (content of antibacterial peptide in the enzymatic hydrolysate / protein content in mackerel dark muscle) * 100 (the content of antibacterial peptide in the enzymatic hydrolysate was determined by trichloroacetic acid precipitation combined with the Folin-phenol method, and the protein content in mackerel dark muscle was determined by the Kjeldahl nitrogen method). The extraction rate of antibacterial peptide in the enzymatic hydrolysate was greater than 80%. The obtained antibacterial peptide from mackerel dark muscle was sent to Shanghai OE Biotech Co., Ltd. for amino acid sequence determination, and the amino acid sequence of this antibacterial peptide was RSCH and / or KSCG.

[0043] The minimum inhibitory concentration of the antibacterial peptide from mackerel dark muscle was determined. The specific determination method was as follows: The micro-dilution method was used to determine the minimum inhibitory concentration of the freeze-dried powder of the antibacterial peptide from mackerel dark muscle against several Gram-positive bacteria and Gram-negative bacteria. The bacterial liquid in the logarithmic growth phase was taken by the McFarland turbidity method, and the bacterial liquid was diluted to 〖10〗^ 5 CFU / mL with MH broth medium. 100 μL of MH broth medium was added to each well of the sterilized enzyme-linked immunosorbent assay (ELISA) plate. 100 μL of the freeze-dried powder solution of the antibacterial peptide from mackerel dark muscle with a mass concentration of 320 μg / mL was added to the first well, and then the freeze-dried powder solution of the antibacterial peptide from mackerel dark muscle was serially diluted twice, so that the mass concentrations of the freeze-dried powder solution of the antibacterial peptide from mackerel dark muscle were adjusted to: 320, 160, 80, 40, 20, 10, 5, 2.5 μg / mL in sequence, and a total of 3 parallel experiments were done. Then, 100 μL of MH broth medium was added to another row of wells as a negative control. 100 μL of the bacterial liquid was added to another row of wells as a positive control. The ELISA plates of Shewanella putrefaciens and Pseudomonas were placed in an incubator at 30 °C, and the rest were placed in an incubator at 37 °C for 12 h. Table 3 is the determination table of the minimum inhibitory concentration of the freeze-dried powder of the antibacterial peptide from mackerel dark muscle obtained in step A4.

[0044] Table 3 Antibacterial effect of the freeze-dried powder of the antibacterial peptide from mackerel dark muscle obtained in step A4

[0045] 。

[0046] As can be seen from Table 3 above, the antibacterial peptide from mackerel dark muscle has a strong inhibitory effect on both Gram-positive bacteria and Gram-negative bacteria, showing broad-spectrum antibacterial properties, and the antibacterial effect on Gram-negative bacteria is generally stronger than that on Gram-positive bacteria. And the MIC value of the antibacterial peptide from mackerel dark muscle against Enterobacter ( Enterobacteriaceae ), Shewanella putrefaciens ( Shewanella putrefaciens ), and Pseudomonas ( Pseudomonas spp. ) is 10 μg / mL, and the MIC value against Lactobacillus ( Lactobacillus) The MIC value against it is 20 μg / mL, and the MIC values against Staphylococcus aureus ( Staphylococcus aureus ) and Bacillus subtilis ( Staphylococcus aureus ) are 80 μg / mL.

[0047] Example 2

[0048] Same as Example 1 above, the difference is that

[0049] In step (2), deionized water three times the mass of the fish meat was added to the defatted dark meat of mackerel, the pH was adjusted to 7 with NaOH, then trypsin and Bacillus licheniformis protease with an enzyme activity unit of 1000 U / g were added, and enzymolysis was carried out at 43 °C for 4 h. Then the temperature was raised to above 90 °C, and the enzyme was inactivated by heat preservation for 20 min. Centrifugation was carried out at 6500 r / min for 10 min, and the supernatant was taken to obtain the crude enzyme solution;

[0050] In step (3), cysteine protease ArgC and serine protease LysC with an enzyme activity unit of 1000 U / g were added to the crude enzyme solution, and enzymolysis was carried out at 43 °C for 4 h. Then the temperature was raised to above 90 °C, and the enzyme was inactivated by heat preservation for 20 min. Centrifugation was carried out at 5000 r / min for 15 min to obtain the upper-layer enzymolysis clear solution.

[0051] Example 3

[0052] Same as Example 1 above, the difference is that

[0053] In step (2), deionized water twice the mass of the fish meat was added to the defatted dark meat of mackerel, the pH was adjusted to 11 with NaOH, then trypsin and Bacillus licheniformis protease with an enzyme activity unit of 3000 U / g were added, and enzymolysis was carried out at 47 °C for 4 h. Then the temperature was raised to above 90 °C, and the enzyme was inactivated by heat preservation for 20 min. Centrifugation was carried out at 6500 r / min for 10 min, and the supernatant was taken to obtain the crude enzyme solution;

[0054] In step (3), cysteine protease ArgC and serine protease LysC with an enzyme activity unit of 3000 U / g were added to the crude enzyme solution, and enzymolysis was carried out at 47 °C for 4 h. Then the temperature was raised to above 90 °C, and the enzyme was inactivated by heat preservation for 20 min. Centrifugation was carried out at 5000 r / min for 15 min to obtain the upper-layer enzymolysis clear solution.

[0055] In summary, for the antibacterial peptide from the dark meat of mackerel prepared by the preparation method of the present invention, the extraction rate of the antibacterial peptide was measured to be greater than 80%. The antibacterial activity detection results of the antibacterial peptide from the dark meat of mackerel obtained by the above method showed strong inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria, with broad-spectrum antibacterial properties, and the antibacterial effect on Gram-negative bacteria was generally stronger than that on Gram-positive bacteria.

[0056] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A scomber japonicus dark muscle enzymatically hydrolyzed antibacterial peptide, characterized in that: The amino acid sequences of the antibacterial peptides are Arg-Ser-Cys-His and Lys-Ser-Cys-Gly.

2. The preparation method of the scomber japonicus dark muscle enzymolysis antibacterial peptide according to claim 1, characterized in that The steps are as follows: (1)Preparation of defatted dark muscle of mackerel The mackerel is steamed at 100 °C for 20 min and then sprayed and cooled with deionized water to 40-50 °C. The darker dark muscle on the back of the fish is taken, processed in a homogenizer for 3-4 min, and then isopropanol with the same mass as the fish meat is added. It is defatted at 50 °C for 90 min, and defatted dark muscle of mackerel is obtained by vacuum filtration. (2)Double enzyme hydrolysis with non-directional endonucleases Deionized water with a mass 2-4 times that of the fish meat is added to the defatted dark muscle of mackerel obtained in step (1), and the pH is adjusted to 7-11 with NaOH. Then, trypsin and Bacillus licheniformis protease are added, and enzymatic hydrolysis is carried out at 43-47 °C for 4 h. Then, the temperature is raised to above 90 °C and the enzymes are inactivated by heat preservation for 20 min. Centrifugation is carried out at a rotation speed of 6500 r / min for 10 min, and the supernatant is taken to obtain the crude enzymatic hydrolysate. (3)Double enzyme hydrolysis with directional endonucleases C-terminal endonuclease double enzyme cysteine protease ArgC and serine protease LysC are added to the crude enzymatic hydrolysate obtained in step (2), and enzymatic hydrolysis is carried out at 43-47 °C for 4 h. Then, the temperature is raised to above 90 °C and the enzymes are inactivated by heat preservation for 20 min. Centrifugation is carried out at 5000 r / min for 15 min to obtain the upper clear enzymatic hydrolysate. (4)Collection of antibacterial peptides The upper clear enzymatic hydrolysate obtained in step (3) is filtered with an ultrafiltration system to obtain an enzymatic hydrolysate with a molecular weight of 1-3 kDa. After collecting the filtrate, it is freeze-dried, and the powder is the antibacterial peptide obtained by enzymatic hydrolysis of dark muscle of mackerel. The amino acid sequences of the antibacterial peptides are Arg-Ser-Cys-His and Lys-Ser-Cys-Gly.

3. The preparation method of the scomber japonicus dark muscle enzymolysis antibacterial peptide according to claim 2, wherein: The dosages of the trypsin and the Bacillus licheniformis protease described in step (2) are 1000-3000 U / g respectively.

4. The preparation method of a scomber japonicus dark muscle enzymolysis antibacterial peptide according to claim 2, characterized in that: The dosages of the cysteine protease ArgC and the serine protease LysC described in step (3) are 1000-3000 U / g respectively.

5. Use of the scomber japonicus dark muscle enzymolysis antibacterial peptide according to claim 1 in the preparation of an antibacterial agent, characterized in that The antibacterial agents are antibacterial agents against Enterobacter, antibacterial agents against Shewanella putrefaciens, antibacterial agents against Pseudomonas, antibacterial agents against Lactobacillus, antibacterial agents against Staphylococcus aureus, and antibacterial agents against Bacillus subtilis.

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