CN118165078A - Bacillus subtilis source antibacterial peptide NDYT-8 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a bacillus subtilis source antibacterial peptide NDYT-8 and application thereof. According to the invention, the bacillus subtilis source antibacterial peptide NDYT-8 is finally obtained through mass spectrum identification, genome data mining, database prediction and synthesis verification of bacillus subtilis fermentation metabolite, the amino acid sequence is PRKILLMVKA, the molecular weight is 1168.551 daltons, and an amphipathic alpha helical structure can be formed; the antibacterial peptide NDYT-8 provided by the invention has obvious inhibition effect on a plurality of food-borne pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like, can target to destroy bacterial cell membranes, and has low hemolysis effect; the antibacterial peptide NDYT-8 provided by the invention has L-shaped amino acid, and has lower synthesis cost, so that the antibacterial peptide is expected to replace antibiotics and chemical preservatives, and has great excavation potential and application prospect in the fields of biological medicine, food, agricultural product preservation and the like.

Description

Bacillus subtilis-derived antimicrobial peptide NDYT-8 and its application

Technical Field

The invention belongs to the technical field of bioengineering, and specifically relates to a Bacillus subtilis-derived antimicrobial peptide NDYT-8 and an application thereof.

Background technique

Foodborne pathogens refer to a type of microorganisms that cause human illness by using food as a carrier. Typical foodborne pathogens include Salmonella, diarrheagenic Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Vibrio parahaemolyticus. The prevention and control of foodborne pathogens is a rigid demand for ensuring food safety. At present, the common method for preventing and controlling foodborne pathogens is to add chemical preservatives. However, more and more studies have shown that the use of chemical preservatives has great safety hazards. The development of green, safe and efficient natural preservatives is becoming more and more urgent. The most in-depth natural preservative studied in the food industry is nisin. Nisin has been approved by the US Food and Drug Administration as a food additive and is also one of the natural food preservatives approved for use in my country. However, Nisin can only inhibit Gram-positive bacteria and has no activity against Gram-negative bacteria and fungi; Nisin's antibacterial activity is also unstable and is only suitable for acidic environments; these have greatly limited the application of Nisin in the food industry. Therefore, the development of broad-spectrum natural preservatives and the enhancement of their antibacterial stability and scope of application are issues that need to be addressed today.

Antimicrobial peptides (AMPs) are a class of small molecule polypeptides with strong cationic characteristics, also known as cationic host defense peptides. They are widely found in bacteria, animals, and plants. The sequence consists of only a few to dozens of amino acids. As an emerging class of natural biological preservatives, antimicrobial peptides have a unique membrane destruction mechanism: most antimicrobial peptides have both cationic and hydrophobic amino acid residues, which makes them amphiphilic in polar biological environments, and can then quickly lyse bacteria through electrostatic interaction with negative charges on bacterial cell membranes. Because of its unique mechanism of action, it is unlikely to be affected by bacterial resistance. In addition, unlike the negatively charged cell membrane of bacteria, the surface of normal mammalian cells is mainly composed of neutrally charged phospholipids, so antimicrobial peptides will only selectively kill bacteria without damaging normal cells. Therefore, antimicrobial peptides are considered to be natural preservatives with great potential for development due to their broad-spectrum antibacterial properties and strong biocompatibility.

Bacillus subtilis natto is a strain isolated from natto, and its scientific name is Bacillus subtilis natto. It is certified by the FDA as a generally recognized as safe (GRAS) microorganism. Studies have shown that some strains of Bacillus subtilis natto have good ability to produce antimicrobial peptides, and are expected to become safe and green products that replace preservatives/antibiotics, with great development prospects.

Summary of the invention

In order to overcome the shortcomings of the prior art and provide a new antimicrobial peptide, the primary purpose of the present invention is to provide an antimicrobial peptide NDYT-8 derived from Bacillus subtilis.

The second object of the present invention is to provide an application of the antimicrobial peptide NDYT-8 derived from Bacillus subtilis.

The primary purpose of the present invention can be achieved through the following technical solutions:

A Bacillus subtilis-derived antimicrobial peptide NDYT-8, whose amino acid sequence is as follows: Pro-Arg-Lys-Ile-Leu-Leu-Met-Val-Lys-Ala, and the single-letter abbreviation sequence of the amino acid sequence is PRKILLMVKA;

The antimicrobial peptide NDYT-8 has a molecular weight of 1168.551 Da, a hydrophobicity of 60%, a positive charge of +3, a molecular formula of C ₅₄ H ₁₀₁ N ₁₅ O ₁₁ S ₁ , an average hydrophilicity of 0.61, an isoelectric point of 11.63, and is predicted by APD3 to form an alpha helix.

Preferably, the Bacillus subtilis-derived antimicrobial peptide NDYT-8 comprises a nucleic acid fragment encoding the antimicrobial peptide NDYT-8.

Preferably, the nucleic acid sequence fragment encoding the antimicrobial peptide NDYT-8 is as follows:

ATTGAAGAATTTGTCCAATCCTTGGAAACA

CCGCGCAAAATCTTATTAATGGTTAAAGCG

GGAACTGCAACAGATGCGACAATTCAATCT

CTTCTTCCTCATCTAGAGAAGGATGATATT (SEQ ID NO: 2).

Preferably, the method for preparing the Bacillus subtilis-derived antimicrobial peptide NDYT-8 comprises the following steps:

The peptide resin was obtained by solid phase chemical synthesis through a peptide synthesizer, and the obtained peptide resin was cut with trifluoroacetic acid to obtain the antimicrobial peptide NDYT-8; after purification by reverse phase high performance liquid chromatography, the preparation of the Bacillus subtilis-derived antimicrobial peptide NDYT-8 was completed.

Preferably, the preparation method of the Bacillus subtilis-derived antimicrobial peptide NDYT-8 comprises the following specific steps:

(1) Weigh 2-chlorotrityl chloride resin (2-CTC resin), swell it with dichloromethane in a reactor for half an hour, drain it, and wash it with DMF (N,N-dimethylformamide) three times;

(2) Take 1 eq of Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-alanine) and DMF (N,N-dimethylformamide) as solvent, and 1.5 eq of DIEA (N,N-diisopropylethylamine) to catalyze the reaction on the resin, remove the reaction solution, and wash with DMF 6 times;

(3) Seal the resin with methanol + DIEA for 1 hour and wash with DMF 6 times;

(4) Remove Fmoc (amino acid protecting group) with 20% piperidine in DMF solution and wash with DMF 8 times;

(5) 3eq of Fmoc-Lys(Boc)-OH (tert-butyloxycarbonyl-fluorenylmethoxycarbonyl-lysine), DMF as solvent, 3eq of DIC+HoBT (N,N'-diisopropylcarbodiimide+1-hydroxybenzotriazole) react with the resin, drain and wash;

(6) Remove the amino acid protecting groups with 20% piperidine in DMF solution and wash;

(7) Repeat steps 5-6 according to the amino acid sequence until the N-terminal Pro is attached and the N-terminal Fmoc is removed;

(8) After the reaction is completed, wash the resin and drain it;

(9), 95% TFA + 2% Tis (triisopropylsilyl chloride) + 2% EDT (1,2-ethanedithiol) + 1% H ₂ O to cut and cleave the resin, and wash with ether to obtain a crude product;

(10) purifying and separating the crude product by high performance liquid chromatography to obtain a pure product;

(11) Freeze-drying and testing.

The second object of the present invention can be achieved by the following technical solutions:

The invention discloses an application of an antimicrobial peptide NDYT-8 derived from Bacillus subtilis in antimicrobial drugs or food preservatives.

Specifically, the Bacillus subtilis-derived antimicrobial peptide NDYT-8 is used in inhibiting and/or killing one or more antimicrobial drugs of Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enteritidis.

Specifically, the Bacillus subtilis-derived antimicrobial peptide NDYT-8 is used in inhibiting and/or killing one or more food preservatives of Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enteritidis.

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

The antimicrobial peptide NDYT-8 of the present invention has a significant inhibitory effect on a variety of foodborne pathogens such as Escherichia coli and Staphylococcus aureus, and has the advantages of low hemolysis and high stability. The antimicrobial peptide is screened from FDA-certified "Generally Recognized as Safe (GRAS)" microorganisms, and its safety is guaranteed. In addition, the amino acids of the antimicrobial peptide NDYT-8 provided by the present invention are all L-configuration, and the synthesis cost is low. Therefore, it is expected to replace antibiotics and chemical preservatives, and has great potential for exploration and application value in the fields of biomedicine, food, and agricultural product preservation. The antimicrobial peptide NDYT-8 of the present invention can be made into an antimicrobial drug or food preservative for the prevention and control of Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enteritidis.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

Figure 1 is a diagram of the antimicrobial peptide NDYT-8 helical wheel;

FIG2 is a high performance liquid chromatogram of solid phase synthesis antimicrobial peptide NDYT-8;

FIG3 is an electrospray ionization mass spectrometry (ESI-MS) diagram of solid phase synthesized antimicrobial peptide NDYT-8;

FIG4 is a graph showing the antibacterial effect of the antimicrobial peptide NDYT-8 on Escherichia coli ATCC25922;

FIG5 is a graph showing the sterilization curve of the antimicrobial peptide NDYT-8 against Escherichia coli ATCC25922;

FIG. 6 is a graph showing the results of an experiment on the hemolytic effect of the antimicrobial peptide NDYT-8 on mouse erythrocytes.

Detailed ways

In order to better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

Example 1: Screening of antimicrobial peptide NDYT-8

Combined with the whole genome sequence (CP121266.1) of Bacillus subtilis natto subspecies ND107105 and the mass spectrometry identification of its antibacterial metabolites, the antimicrobial peptide prediction website APD3 (https://aps.unmc.edu/) and DRAMPDatabase (http://dramp.cpu-bioinfor.org/) were used to screen cationic short peptides with an antibacterial activity probability higher than 99%, an α-helical structure and less than 15 amino acids. The DBAASP database (https://dbaasp.org/tools?page=linear-amp-prediction) was further used to screen peptides with positive antibacterial activity against Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 based on the "clustering" method and AI learning method. Several short peptide sequences including the antimicrobial peptide NDYT-8 were obtained by this method, and the short peptide with a chemical synthesis purity higher than 95% was obtained.

The antimicrobial peptide NDYT-8 of the present invention is a natural antimicrobial peptide derived from Bacillus subtilis natto subspecies. Combining metabolite mass spectrometry identification and antimicrobial peptide database analysis and prediction, a novel antimicrobial peptide NDYT-8 with high efficiency and broad spectrum was screened out from the natural genome of Bacillus subtilis natto subspecies. The antimicrobial peptide NDYT-8 provided by the present invention has 10 amino acid residues, and its amino acid sequence is specifically: Pro-Arg-Lys-Ile-Leu-Leu-Met-Val-Lys-Ala, and the single-letter abbreviation sequence of the amino acid sequence is PRKILLMVKA. The molecular weight of the antimicrobial peptide NDYT-8 is 1168.551Da, the hydrophobicity is 60%, the total electrostatic charge is +3, the molecular formula is C ₅₄ H ₁₀₁ N ₁₅ O ₁₁ S ₁ , the average hydrophilicity is 0.61, and the isoelectric point is 11.63. The antimicrobial peptide NDYT-8 of the present invention has significant antibacterial activity against both Gram-positive pathogenic bacteria and Gram-negative pathogenic bacteria, and has the advantages of low hemolysis, high stability and high efficiency. The antimicrobial peptide is screened from FDA-certified "Generally Recognized as Safe (GRAS)" microorganisms, and its safety is guaranteed. It is expected to replace antibiotics and chemical preservatives, and has great potential for development and application value in the fields of biomedicine, food, and agricultural product preservation.

Example 2: Preparation method of the antimicrobial peptide NDYT-8 described in Example 1

The antimicrobial peptide NDYT-8 was synthesized by solid phase chemical synthesis: the preparation of the antimicrobial peptide NDYT-8 was carried out one by one from the C-terminus to the N-terminus by a peptide synthesizer, and the specific steps are as follows:

(1) Weigh 2-CTC resin, swell it with DCM in a reactor for 0.5 h, drain it, and wash it with DMF three times;

(2) Take 1 eq of Fmoc-Ala-OH, use DMF as solvent, and catalyze the reaction on the resin with 1.5 eq of DIEA. Draw off the reaction solution and wash with DMF 6 times.

(3) Seal the resin with methanol + DIEA for 1 hour and wash with DMF 6 times;

(4) Remove Fmoc with 20% piperidine in DMF solution and wash with DMF 8 times;

(5) 3eq of Fmoc-Lys(Boc)-OH, DMF as solvent, 3eq (DIC+HoBT) reacted with the resin, drained, and washed;

(6) Remove Fmoc with 20% piperidine in DMF solution and wash;

(7) Repeat steps (5) to (6) from the C-terminus to the N-terminus in the order of amino acids P-R-K-I-L-L-M-V-K-A until the Pro at the N-terminus is connected and the Fmoc at the N-terminus is removed; and a resin with the side chain protection of the Fmoc group removed is obtained;

(8) After the reaction is completed, wash the resin and drain it;

(9) Add a cleavage reagent to the peptide resin obtained above, 95% TFA (trifluoroacetic acid) + 2% Tis (triisopropylsilyl chloride) + 2% EDT + 1% H ₂ O to cleave the resin, and wash with ether precipitation to obtain a crude polypeptide;

(10) Dissolve the peptide in 15% ACN solution, eluent A is 0.1% TFA/water solution; eluent B is 0.1% TFA/acetonitrile solution, the elution concentration is 10% to 70%, the elution time is 30 min, the detection wavelength is 220 nm, the flow rate is 1 mL/min, collect the main peak, and freeze-dry; obtain the purified antimicrobial peptide NDYT-8 (as shown in Figure 2);

(11) Identification of antimicrobial peptide NDYT-8: The antimicrobial peptide NDYT-8 obtained above was analyzed by electrospray ionization mass spectrometry. The theoretical molecular weight was basically consistent with the measured molecular weight. The purity of the antimicrobial peptide NDYT-8 was greater than 95% (as shown in FIG3 ).

Example 3: Determination of antibacterial activity of antimicrobial peptide NDYT-8

The antibacterial activity of the antimicrobial peptide NDYT-8 was detected by the micro-two-fold dilution method. The minimum inhibitory concentration, that is, the lowest drug concentration that can inhibit bacterial growth and reproduction, was determined. The strains involved in this embodiment include Gram-positive pathogenic bacteria Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19115; Gram-negative pathogenic bacteria Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 14028, Vibrio parahaemolyticus ATCC 17802;

Use LB medium to culture the indicator bacteria to the exponential growth phase, dilute the bacterial solution with MH medium to adjust the concentration to 107 CFU/mL, and dispense 90 μL of the bacterial solution/well into a 96-well plate;

Use DMSO to dissolve the antimicrobial peptide NDYT-8 (4 mg/mL), use MH medium to gradient dilute the antimicrobial peptide NDYT-8 to (2-1000 μg/mL), add 90 μL to the 96-well culture plate containing the bacterial solution to be tested according to the concentration gradient, and set three wells for each concentration; 90 μL sterile PBS + 90 μL MH medium is used as a negative blank control, and 90 μL MH medium + 90 μL bacterial suspension is used as a positive blank control.

The 96-well plate was placed in a constant temperature incubator at 37°C and 160 rpm for 24 h;

The light absorption value of the bacterial solution at 600 nm was detected using an ELISA instrument, and the average value of the sample concentrations of the well where no bacterial growth was detected and the adjacent wells was taken as the minimum inhibitory concentration (MIC).

The antibacterial activity of the antimicrobial peptide NDYT-8 against a variety of foodborne pathogens was verified by three repeated experiments as shown in the following table, and its MIC value was 16-128 μg/mL. The antibacterial activity of the antimicrobial peptide NDYT-8 against Gram-negative bacteria was relatively strong, and its MIC value was less than 40 μg/mL.

Example 4: Determination of the antibacterial peptide NDYT-8 inhibition of bacterial growth curve

The strains involved in this example are Gram-positive bacteria Staphylococcus aureus subsp. Aureus ATCC 29213 and Gram-negative bacteria Escherichia coli ATCC 25922.

The bacteria to be tested were placed in LB medium and cultured at 37°C and 220 rpm for 12 h; then transferred to MH medium at a ratio of 1:100 and cultured for another 2.5 h at 37°C and 220 rpm; the bacterial solution was diluted with MH medium to OD600 = 0.1;

Take 150 μL MH culture medium and put it into a 96-well plate. Add 10 μL of the test bacterial solution to each well. Add the antimicrobial peptide NDYT-8 with final concentrations of 0 MIC, 1 MIC, 3 MIC, and 5 MIC in sequence, mix well, put it into a microplate reader, culture at 37°C, measure the OD600 reading every 2 hours, and measure until 10 hours; draw a growth curve.

The results are shown in FIG5 . Three repeated experiments showed that as the concentration of the antimicrobial peptide NDYT-8 increased, bacterial growth was inhibited. When the concentration of the antimicrobial peptide increased to 3MIC (50-200 μg/mL), the growth of Escherichia coli and Staphylococcus aureus was significantly inhibited.

Example 5: Hemolytic activity of antimicrobial peptide NDYT-8 on fresh mouse erythrocytes

Fresh mouse blood was collected and centrifuged at 1000 rpm for 10 min in a centrifuge tube. The supernatant was removed and the red blood cell sediment was taken out. PBS was added to wash twice, and the supernatant was removed again by centrifugation at 1000 rpm for 10 min. Finally, physiological saline was added to the centrifuge tube to prepare a cell suspension containing 6% (v/v) red blood cells, and 100 μL/well was dispensed into a 96-well plate.

PBS buffer (pH 7.4) was prepared with different concentration gradients of antimicrobial peptides (0 μg/mL, 40 μg/mL, 100 μg/mL, 200 μg/mL); PBS solution was used as the negative control group, and polymyxin (200 μg/mL) was used as the positive control.

Add 100 μL of antimicrobial peptides and positive controls to a 96-well plate containing mouse blood; incubate at 37°C for 60 min;

The 96-well plate was removed and centrifuged (3000×g, 5 min, 4°C), 100 μL of the supernatant was transferred to a new 96-well plate, and the hemoglobin release in the supernatant was evaluated by measuring the absorbance at 540 nm using a microplate reader.

As shown in Figure 6, three repeated experiments showed that the antimicrobial peptide solution was incubated with the mouse red blood cell suspension. The OD540 test results showed that compared with the control group, the antimicrobial peptide NDYT-8 did not cause obvious hemolysis of red blood cells when the concentration reached 200 μg/mL, and was relatively safe.

In summary, the present invention provides an antimicrobial peptide NDYT-8, the amino acid sequence of which is shown in SEQ ID NO.1, specifically PRKILLMVKA.

The antimicrobial peptide NDYT-8 provided by the present invention is a short sequence that can form an amphipathic α-helical structure, contains 10 amino acids, has a molecular weight of 1168.551 Daltons, is a straight-chain polypeptide, and all amino acids are L-type. In vitro antibacterial experiments show that the antimicrobial peptide NDYT-8 has a broad-spectrum antibacterial activity, and exhibits good antibacterial effects on standard strains of Staphylococcus aureus, Listeria monocytogenes, Salmonella enteritidis, and Vibrio parahaemolyticus, with a minimum inhibitory concentration (MIC) value of 16 μg/mL; at the same time, the hemolytic effect is low.

In the present invention, the preparation method of the antimicrobial peptide NDYT-8 is a polypeptide solid phase synthesis method. The present invention has no special requirements for the polypeptide solid phase synthesis method, and a method well known to those skilled in the art can be used.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (8)

1. A Bacillus subtilis-derived antimicrobial peptide NDYT-8, characterized in that its amino acid sequence is as follows: Pro-Arg-Lys-Ile-Leu-Leu-Met-Val-Lys-Ala, the single-letter abbreviation sequence of the amino acid sequence is PRKILLMVKA; The antimicrobial peptide NDYT-8 has a molecular weight of 1168.551 Da, a hydrophobicity of 60%, a positive charge of +3, a molecular formula of C54H101N15O11S1, an average hydrophilicity of 0.61, an isoelectric point of 11.63, and is predicted by APD3 to form an alpha helix. 2. The Bacillus subtilis-derived antimicrobial peptide NDYT-8 according to claim 1, characterized in that the Bacillus subtilis-derived antimicrobial peptide NDYT-8 comprises a nucleic acid fragment encoding the antimicrobial peptide NDYT-8. 3. The Bacillus subtilis-derived antimicrobial peptide NDYT-8 according to claim 1, characterized in that the nucleic acid sequence fragment encoding the antimicrobial peptide NDYT-8 is as follows: ATTGAAGAATTTGTCCAATCCTTGGAAACA CCGCGCAAAATCTTATTAATGGTTAAAGCG GGAACTGCAACAGATGCGACAATTCAATCT CTTCTTCCTCATCTAGAGAAGGATGATATT (SEQ ID NO: 2). 4. The Bacillus subtilis-derived antimicrobial peptide NDYT-8 according to any one of claims 1 to 3, characterized in that the preparation method of the Bacillus subtilis-derived antimicrobial peptide NDYT-8 comprises the following steps: The peptide resin was obtained by solid phase chemical synthesis through a peptide synthesizer, and the obtained peptide resin was cut by TFA to obtain the antimicrobial peptide NDYT-8; after purification by reverse phase high performance liquid chromatography, the preparation of the Bacillus subtilis-derived antimicrobial peptide NDYT-8 was completed. 5. The Bacillus subtilis-derived antimicrobial peptide NDYT-8 according to claim 4, characterized in that the preparation method of the Bacillus subtilis-derived antimicrobial peptide NDYT-8 comprises the following specific steps: (1) Weigh 2-CTC resin, swell it with DCM in a reactor for half an hour, drain it, and wash it with DMF three times; (2) Take 1 eq of Fmoc-Ala-OH, use DMF as solvent, and catalyze the reaction on the resin with 1.5 eq of DIEA. Draw off the reaction solution and wash with DMF 6 times. (3) Seal the resin with methanol + DIEA for 1 hour and wash with DMF 6 times; (4) Remove Fmoc with 20% piperidine in DMF solution and wash with DMF 8 times; (5) 3eq of Fmoc-Lys(Boc)-OH, DMF as solvent, 3eq (DIC+HoBT) reacted with the resin, drained, and washed; (6) Remove Fmoc with 20% piperidine in DMF solution and wash; (7) Repeat steps 5-6 according to the amino acid sequence until the N-terminal Pro is attached and the N-terminal Fmoc is removed; (8) After the reaction is completed, the resin is washed and dried; (9) The resin was cut and cleaved with 95% TFA + 2% Tis + 2% EDT + 1% H ₂ O, and washed with ether to obtain a crude product; (10) purifying and separating the crude product by high performance liquid chromatography to obtain a pure product; (11) Freeze-drying and testing. 6. Use of the Bacillus subtilis-derived antimicrobial peptide NDYT-8 according to any one of claims 1 to 5. 7. The use of the Bacillus subtilis antimicrobial peptide NDYT-8 according to claim 6, characterized in that the Bacillus subtilis antimicrobial peptide NDYT-8 is used in inhibiting and/or killing one or more antimicrobial drugs of Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enteritidis. 8. The use of the Bacillus subtilis antimicrobial peptide NDYT-8 according to claim 6, characterized in that the Bacillus subtilis antimicrobial peptide NDYT-8 is used as a food preservative to inhibit and/or kill one or more of Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enteritidis.