CN115746090A - Bacillus subtilis carrier protein antibacterial peptide BCP4 and application thereof - Google Patents

Abstract

The invention discloses a bacillus subtilis carrier protein antibacterial peptide BCP4, the amino acid sequence of which is KGKTLLQ, and the molecular weight of the antibacterial peptide BCP4 is 787.5036 daltons. Experiments prove that the antibacterial peptide BCP4 can generate a strong inhibiting effect on the bacillus cereus, and lays a foundation for the development of food preservatives, biological medicines and feed additives in the preparation of medicines for treating or preventing diseases caused by the bacillus cereus.

Description

Bacillus subtilis carrier protein antibacterial peptide BCP4 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a bacillus subtilis carrier protein antimicrobial peptide BCP4 and application thereof.

Background

Food-borne pathogenic microorganisms are a main reason for causing food poisoning and influencing the health and life safety of people, and the food-borne pathogenic microorganisms refer to some pathogenic microorganisms which can be eaten by human bodies and carried into the human bodies, wherein 60 percent of the pathogenic microorganisms belong to bacteria. Bacillus cereus is a gram-positive Bacillus capable of producing spores, is widely distributed in nature, is a common food-borne pathogenic bacterium, is easy to pollute starch foods such as residual rice, rice flour and flour, and foods such as desserts, meat products and dairy products which are rich in lipid and protein, thereby causing food poisoning. According to statistics of a Chinese food-borne disease monitoring network and a national food pollutant monitoring network platform, the food poisoning event caused by bacillus cereus accounts for the 4 th of the bacterial food poisoning event and has a growing trend.

At present, aiming at the condition that food-borne pathogenic bacteria are increasingly polluted, antibiotic medicines are the most effective method. However, with the improvement of drug resistance of pathogenic bacteria, the original antibiotic drugs cannot achieve the effect of killing the pathogenic bacteria. Therefore, the search for chemical drug substitutes is also a major focus of current research. Antimicrobial peptides (AMPs) are a natural class of small molecule bioactive peptides with antibacterial or antifungal activity, also known as host defense peptides, produced by organisms such as bacteria, plants, vertebrates, and invertebrates. The compound has the characteristics of good water solubility, low toxicity to higher animals, good thermal stability and the like, is considered as a potential substitute of antibiotics, and has good application prospect in the fields of food preservatives, feed additives and the like.

The Bacillus subtilis is a non-pathogenic bacterium widely existing in nature, has low requirement on pH value and good thermal stability, has little influence on the activity of an organic solvent, and can generate various antibacterial substances in the growth and metabolism process. Most of the active antibacterial products of bacillus subtilis are small-molecule antibacterial peptides, and the active antibacterial products have the advantages of wide antibacterial spectrum, low drug resistance, high thermal stability, small toxic and side effects and the like, so the active antibacterial products are concerned by researchers.

Therefore, the antibacterial peptide which can effectively inhibit the bacillus cereus infection and simultaneously can keep good stability has important research significance.

Disclosure of Invention

The invention aims to provide a bacillus subtilis carrier protein antibacterial peptide BCP4 and application thereof, and provides experimental basis for searching new food preservatives, biological medicines and aquatic feed additives and promoting the healthy and sustainable development of food, medicine and aquaculture industries in China through the research on the antibacterial activity of the bacillus cereus carrier protein antibacterial peptide BCP4.

One of the technical schemes adopted by the invention for solving the technical problems is as follows: provides a bacillus subtilis carrier protein antimicrobial peptide BCP4. The amino acid sequence is KGKTLLQ, such as SEQIDNO:1 is shown.

A feasible theoretical basis exists for searching antibacterial peptide in the sequence of the bacillus subtilis carrier protein by taking the bacillus subtilis carrier protein as a target. Therefore, the bacillus subtilis is used as a leaven to carry out liquid fermentation on the fresh shrimp head homogenate of the penaeus chinensis. Centrifuging 100g for 15min, collecting supernatant, and filtering with 1000Da filter membrane. Stored at-20 ℃ for further analysis. The samples were then subjected to protein sequence analysis using liquid chromatography-mass spectrometry. Then, an APD3 online server is used for screening a bacillus subtilis protein sequence according to the charge property and the hydrophobicity of the antibacterial peptide, and then a Swiss-model server is used for predicting the structure of the bacillus subtilis carrier protein, so that an antibacterial peptide sequence KGKTLLQ with a strong antibacterial effect on bacillus cereus is found and named as BCP4.

The molecular weight of the antibacterial peptide BCP4 is 787.50361Da, the charge is +2, and the total hydrophobicity ratio is 29%.

The antibacterial peptide BCP4 can cause damage to bacteria from the following actions: on one hand, the antibacterial peptide BCP4 destroys bacterial cell membranes, changes the permeability of the bacterial cell membranes, and inhibits the generation of the cell membranes, so that bacteria die; on the other hand, the antimicrobial peptide BCP4 binds to bacterial genomic DNA, inhibiting the synthesis of bacterial DNA, thereby causing bacterial death.

The second technical scheme adopted by the invention for solving the technical problems is as follows: provides an application of bacillus subtilis carrier protein antibacterial peptide BCP4 in preparing antibacterial drugs, and the antibacterial drugs are used for inhibiting and/or killing bacillus cereus.

The third technical scheme adopted by the invention for solving the technical problems is as follows: the effective component of the antibacterial drug comprises bacillus subtilis carrier protein antibacterial peptide BCP4, and the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

in a preferred embodiment of the present invention, the active ingredient of the antibacterial drug is bacillus subtilis carrier protein antibacterial peptide BCP4, and the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

in a preferred embodiment of the invention, the antibacterial agent is used for inhibiting and/or killing bacillus cereus.

The fourth technical scheme adopted by the invention for solving the technical problems is as follows: the effective components of the feed additive comprise bacillus subtilis carrier protein antibacterial peptide BCP4, and the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

in a preferred embodiment of the invention, the effective component of the feed additive is bacillus subtilis carrier protein antimicrobial peptide BCP4, and the amino acid sequence of the antimicrobial peptide BCP4 is SEQ ID NO:1.

in a preferred embodiment of the invention, the feed additive is used for inhibiting and/or killing bacillus cereus.

The fifth technical scheme adopted by the invention for solving the technical problems is as follows: the food preservative comprises the effective component of bacillus subtilis carrier protein antibacterial peptide BCP4, wherein the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

in a preferred embodiment of the invention, the food preservative is used to inhibit and/or kill bacillus cereus.

The antimicrobial peptides of the invention can be synthesized, e.g., by solid phase synthesis, using methods known to those skilled in the art, and purified, e.g., by high performance liquid chromatography, using methods known to those skilled in the art.

The implementation of the invention has the following beneficial effects:

the invention takes bacillus subtilis carrier protein as a research object and discovers polypeptide BCP4 with a brand new amino acid sequence by screening. The bacteriostatic activity of BCP4 on bacillus cereus was studied. The experimental result shows that the peptide has strong inhibiting effect on bacillus cereus, and the bacteriostasis mechanism of the peptide firstly penetrates through the cell membrane of bacteria to change the permeability of the cell membrane, and then is combined with the bacterial genome DNA to inhibit the synthesis of the bacterial DNA, thereby causing the death of the bacteria. The invention provides experimental basis for BCP4 serving as a food preservative, a biological medicine and an aquatic feed additive.

Drawings

FIG. 1 is a diagram of mass spectrometry of the antimicrobial peptide BCP4 of the present invention.

Fig. 2 is a view of the structure of a prediction model of the antimicrobial peptide BCP4 of the present invention.

FIG. 3 is a comparison chart of the antimicrobial peptide BCP4 of the present invention against the Minimum Inhibitory Concentration (MIC) assay of Bacillus cereus. Wherein the content of the first and second substances,

a: the concentration of the antibacterial peptide is 0 mug/mL;

b: the concentration of the antibacterial peptide is 500 mug/mL;

c: the concentration of the antibacterial peptide is 250 mu g/mL;

d: the concentration of the antibacterial peptide is 125 mug/mL;

e: the concentration of the antibacterial peptide is 62.5 mu g/mL;

f: the concentration of the antimicrobial peptide was 31.25. Mu.g/mL.

FIG. 4 is a graph of the Time-kill curve (Time kill) of the antimicrobial peptide BCP4 of the present invention against Bacillus cereus. Diluting to 10 in 0.01M phosphate buffer (pH 7.2) ^6-7 CFU/mL. The concentration of the antimicrobial peptide was 62.5. Mu.g/mL.

FIG. 5 is an electrophoretogram of the combination of the antibacterial peptide BCP4 of the present invention and Bacillus cereus DNA, wherein,

the strip 7 is: blank control;

strips 1-6: the mass ratio of BCP4 to DNA is 100/1, 50/1, 25/1, 25/2, 25/4 and 25/8 respectively.

FIG. 6 is a fluorescence spectrum of DNA competitively bound by the antimicrobial peptide BCP4 of the present invention and EB.

And measuring the fluorescence spectrum of the sample in the range of excitation wavelength 535nm and emission wavelength 550-750 nm by using a multifunctional microplate reader.

FIG. 7 is a fluorescence spectrum of the antibacterial peptide BCP4 of the present invention analyzed by PI staining for the effect of the antibacterial peptide BCP4 on the permeability of the bacterial cell membrane of Bacillus cereus.

And measuring the fluorescence spectrum of the sample in the range of the excitation wavelength of 535nm and the emission wavelength of 550-750 nm by using a multifunctional microplate reader.

FIG. 8 is a graph showing the absorbance of the antimicrobial peptide BCP4 of the present invention for the leakage of reducing sugar in bacterial cell membranes.

The OD value of the sample at 540nm is measured by a multifunctional microplate reader.

FIG. 9 is a graph showing the absorbance of the antimicrobial peptide BCP4 of the present invention measured for nucleic acid leakage in bacterial cell membranes.

The OD value of the sample at 260nm is measured by a multifunctional microplate reader.

FIG. 10 is an absorbance chart of the antimicrobial peptide BCP4 of the present invention measured for protein leakage in bacterial cell membranes.

The OD value of the sample at 280nm is measured by a multifunctional microplate reader.

Detailed Description

For better understanding of the present invention, the present invention is further described in detail with reference to the following examples and the accompanying drawings, but those skilled in the art will appreciate that the following examples are not intended to limit the scope of the present invention, and that any changes and modifications based on the present invention are within the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: screening of bacillus subtilis carrier protein antibacterial peptide

Firstly, fermenting the shrimp head homogenate of the penaeus chinensis by using bacillus subtilis to prepare a sample. Taking bacillus subtilis as a leaven, and carrying out liquid state fermentation on fresh shrimp heads of the Chinese prawn homogenate at 37 ℃ and 200 r/min. After fermentation, 100g of the mixture is centrifuged for 15min, and the supernatant is filtered through a 3000Da filter membrane. Stored at-20 ℃ for further analysis.

Chromatographic conditions are as follows: sample introduction amount: 5.0. Mu.L

A chromatographic column: c18 analytical column, length 25cm, internal diameter 75 μm.

Mobile phase: a:0.1% methanol aqueous solution B: acetonitrile (ACN)

Combining with library searching software: maxquantv1.6.5.0, database: the uniprot Bacillus subtilis protein library distinguishes and identifies the obtained peptide fragment mass spectrogram to obtain 5 peptide fragments, as shown in figure 1. Then, an APD3 online server is used for screening the sequence of the bacillus subtilis, and a peptide fragment structure is predicted through a Swiss-model server, so that an antibacterial peptide with a strong antibacterial effect on the bacillus subtilis is found, wherein the antibacterial peptide has a sequence KGKTLLQ, is named BCP4, and has a molecular weight of 787.50361Da.

Example 2: 3D Structure prediction of antimicrobial peptide BCP4

Predicting the structure of the antimicrobial peptide BCP4 by using an online structure prediction server Swiss-model, and editing and modifying by using Pymol software to obtain the secondary structure of the antimicrobial peptide BCP4, as shown in figure 2.

Example 3: minimum Inhibitory Concentration (MIC) assay for antimicrobial peptide BCP4

Culturing Bacillus cereus at 37 deg.C for 12h to logarithmic growth phase, and diluting to 10 in 0.01M phosphate buffer solution with pH of 7.2 ^6-7 CFU/mL. The peptide was dissolved in phosphate buffer and mixed with the bacteria at 37 ℃ in equal volume for 2h. The Minimum Inhibitory Concentration (MIC) is the lowest concentration of the antimicrobial peptide at which no bacterial growth is visible from the microtiter plate after incubation at 37 ℃. As shown in FIG. 3, the Minimum Inhibitory Concentration (MIC) of BCP4 against Bacillus cereus was 62.5. Mu.g/mL (as shown in FIG. 3).

Example 4: time-kill Curve (Time kill) assay for the antimicrobial peptide BCP4

The time-kill curve for peptide BCP4 was determined using plate colony counting. Culturing Bacillus cereus at 37 deg.C for 12h to logarithmic growth phase in 0.01M phosphate buffer (pH7.2)Diluting to 10 ^6-7 CFU/mL. Dissolving the peptide in phosphate buffer, diluting to 125 mug/mL and 250 mug/mL, mixing with the bacterial liquid in equal volume respectively, and incubating in a constant-temperature biochemical incubator at 37 ℃. At various time points (i.e., 0.5, 1, 1.5, 2, 2.5, and 3), 0.02mL of bacterial suspension was obtained and colonies were counted after 24 hours of incubation on nutrient broth plates at 37 ℃. As shown in fig. 4. The antibacterial peptide BCP4 has an obvious effect on the bacillus cereus at the beginning of 1 h; then continues to trend downward. Under the action of the antibacterial peptide BCP4, the number of bacteria is reduced more quickly. The antibacterial peptide BCP4 is shown to have obvious inhibition effect on the bacillus cereus along with the increase of action time.

Example 5: interaction of antimicrobial peptide BCP4 with bacterial DNA

The interaction of BCP4 with Bacillus cereus genomic DNA was studied using the DNA gel retardation method. Bacillus cereus was cultured in 50mL of Nutrient Broth (NB) at 37 ℃ for 12h, and bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. The purity of the extracted genomic DNA was evaluated at an optical density ratio of 260 to 280nm (OD 260/OD 280. Gtoreq.1.90). Next, 10. Mu.L of DNA (218 ng/. Mu.L) was mixed with a continuous amount of the antimicrobial peptide BCP4 at 25 ℃ for 90min, and the mixture was subjected to electrophoresis on 0.8% agarose gel. Gel retardation was observed under UV irradiation using a GelDocXR gel imaging system (Bio-Rad, USA), as shown in FIG. 5. After the antibacterial peptide BCP4 with different mass concentrations acts on the bacillus cereus, the DNA bands of the thalli do not have obvious migration phenomenon. In the band 6-1, the DNA band brightness was significantly reduced with the increase in the concentration of the antimicrobial peptide BCP4, indicating that the antimicrobial peptide BCP4 has a degrading effect on Bacillus cereus DNA.

Example 6: fluorescence spectrum experiment of antibacterial peptide BCP4 and EB competitive binding DNA

The mode of action of the antibacterial peptide BCP4 and the Bacillus cereus genome DNA is analyzed by a fluorescence spectrum experiment of competitive binding of the antibacterial peptide BCP4 and Ethidium Bromide (EB) to the DNA: the Bacillus cereus genomic DNA was diluted to 50. Mu.g/mL with TE buffer. The reaction was performed in a 96-well plate by first adding 5. Mu.L of DNA solution and 10. Mu.L of EB solution with a concentration of 100. Mu.g/mL to each well, mixing them well, and incubating them in a biochemical incubator at 37 ℃ for 10min in the absence of light. Then 50 μ L of peptide solutions with different concentrations were added, the blank control group was replaced with 50 μ L of distilled water, mixed well and placed in a biochemical incubator to incubate for 30min at 37 ℃ in the dark. After the incubation is finished, the fluorescence spectrum of the sample in the range of excitation wavelength 535nm and emission wavelength 550-750 nm is measured by a multifunctional microplate reader. In aqueous solution, EB has very weak fluorescence, but its fluorescence intensity increases greatly when it binds to double-stranded DNA by intercalation with higher affinity. If there is a substance that acts similarly to DNA in the EB-DNA complex system and EB bound to DNA competes, the fluorescence intensity of the system decreases, indicating that the competitive substance binds to DNA in the same intercalation mode as EB. Therefore, by measuring the change in the fluorescence spectrum of the interaction between the DNA-EB complex system and the competitor, it was determined whether the competitor also binds to DNA by intercalation in the same manner as EB. As can be seen from FIG. 6, the fluorescence intensity of the EB-DNA complex decreased with the increase in the concentration of the antimicrobial peptide BCP4, indicating that the antimicrobial peptide BCP4 was intercalate-bound to the Bacillus cereus DNA, and EB, which was previously bound to a DNA base pair, competed with the DNA and was intercalated with EB instead, thereby decreasing the fluorescence intensity of the entire system.

Example 7: effect of antimicrobial peptide BCP4 on bacterial cell membrane permeability

PI staining analysis of the effect of the antimicrobial peptide BCP4 on the permeability of the bacterial cell membrane of Bacillus cereus. Bacillus cereus was inoculated into LB broth and cultured at 37 ℃ at 200r/min for 12 hours to grow to logarithmic phase. Taking a certain amount of bacteria liquid in logarithmic phase, centrifuging, discarding supernatant, washing collected bacteria three times with 0.1M PBS, and adjusting the concentration of bacteria liquid to 10 ⁷ CFU/mL. Peptides were dissolved in phosphate buffer to concentrations of 1 × MIC, 2 × MIC, blank controls were replaced with sterile normal saline, and incubated with bacteria at 37 deg.C in equal volume for 3h. After the incubation, 100. Mu.L of the supernatant was removed by centrifugation, 100. Mu.L of PI (propidium iodide) dye was added thereto, and the mixture was vortexed at 25 ℃ and incubated at 4 ℃ for 15min in the dark. After staining was complete, washed 2 times with 0.1M PBS and finally resuspended to 100. Mu.L. And measuring the fluorescence spectrum of the sample in the range of excitation wavelength 535nm and emission wavelength 550-750 nm by using a multifunctional microplate reader. PI (Propidium Iodide) is a nucleic acid dye that is present in the excitationThe wavelength is 535nm, and the emission wavelength is 615nm, and the strongest fluorescence absorption is achieved. It cannot cross normal cell membranes, but when the cell membranes are damaged or ruptured, PI can enter the cell membranes to bind to DNA, showing red fluorescence. The degree of cell damage can be determined based on the magnitude of the fluorescence intensity. The binding of PI to the antimicrobial peptide-treated Bacillus cereus DNA at different concentrations is shown in FIG. 7. As can be seen from FIG. 7, the fluorescence intensity of Bacillus cereus treated with different concentrations of the antimicrobial peptide BCP4 is higher than that of the blank control group, which indicates that the antimicrobial peptide BCP4 treatment damages the bacterial cell membrane and increases the permeability of the cell membrane. The fluorescence intensity of the sample treated by 2 × MIC antimicrobial peptide BCP4 is greater than that of the sample treated by 1 × MIC antimicrobial peptide, which indicates that the high dose of antimicrobial peptide BCP4 increases the permeability of the bacterial cell membrane, resulting in more PI entering the cell to bind to DNA and emit fluorescence.

Example 8: effect of antibacterial peptide BCP4 on leakage of intracellular components of bacteria

The integrity of the bacterial cell membrane was judged by measuring leakage of reducing sugars within the bacterial cells. Bacillus cereus was inoculated into 50mL of Nutrient Broth (NB) at 37 ℃ and cultured for 12h to grow to logarithmic phase. Taking a certain amount of bacteria liquid in logarithmic phase, centrifuging, removing supernatant, washing collected bacteria three times with 0.1M PBS, and adjusting the concentration of the bacteria liquid to 10 ⁷ CFU/mL. Dissolving the peptide in phosphate buffer to make the concentration of the peptide be 1 XMIC and 2 XMIC, replacing a blank control group with sterile normal saline, mixing the peptide with bacteria at 37 ℃ in equal volume, incubating and culturing, taking out 120 mu L of bacterial liquid after culturing for 0, 30, 60 and 90min, centrifuging and taking supernatant, reacting by using a DNS colorimetric method, measuring the absorbance at 540nm by using a multifunctional microplate reader, and comparing with a glucose standard curve. As can be seen from fig. 8, the content of reducing sugar in the bacillus cereus liquid of the blank control group is low, while the amount of reducing sugar in the bacillus cereus liquid treated by the antimicrobial peptide BCP4 is significantly more than that of the control group, and the amount of reducing sugar in the liquid increases as the concentration of the antimicrobial peptide BCP4 increases.

The integrity of the bacterial cell membrane is judged by measuring the leakage of nucleic acid and protein in the bacterial cell. Inoculating Bacillus cereus in LB broth, 37 deg.CAnd culturing at 200r/min for 12h to grow to logarithmic phase. Taking a certain amount of bacteria liquid in logarithmic phase, centrifuging, removing supernatant, washing collected bacteria three times with 0.1M PBS, and adjusting the concentration of the bacteria liquid to 10 ⁷ CFU/mL. Dissolving antibacterial peptide BCP4 in phosphate buffer to make its concentration be 1/2 XMIC, 1 XMIC, 2 XMIC, replacing blank control group with sterile physiological saline, mixing with bacteria in equal volume, placing in multifunctional enzyme labeling instrument, incubating at 37 deg.C, and measuring absorbance at 260nm and 280nm at different time points. From fig. 9 and fig. 10, the content of nucleic acid and protein in the bacillus cereus solution of the blank control group is low, while the amount of nucleic acid and protein permeation in the bacillus cereus solution treated by the antimicrobial peptide BCP4 is significantly larger than that of the control group, and the amount of nucleic acid and protein permeation in the solution increases with the increase of the concentration of the antimicrobial peptide BCP4, and the amount of nucleic acid and protein permeation in the solution also increases with the increase of the action time of the antimicrobial peptide BCP4 in the first 20 min. However, after 20min, the nucleic acid and protein permeation amount of the antibacterial peptide BCP4 treated bacteria solution is reduced until the bacteria solution is leveled with the blank control group, and probably caused by the lysis of the antibacterial peptide after the leakage of the nucleic acid and the protein.

In conclusion, the invention provides a brand-new antibacterial peptide BCP4 which has the minimum inhibitory concentration MIC of 62.5 mu g/mL on Bacillus cereus and can inhibit the growth of the Bacillus cereus. The antibacterial peptide BCP4 firstly penetrates through the cell membrane of bacteria, changes the permeability of the cell membrane, and then is combined with bacterial genome DNA to inhibit the synthesis of the bacterial DNA, thereby causing the death of the bacteria.

While specific embodiments of the invention have been described, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, as equivalent modifications and variations as will be made by those skilled in the art in light of the spirit of the invention are intended to be included within the scope of the appended claims.

Claims (10)

1. The amino acid sequence of the bacillus subtilis carrier protein antibacterial peptide BCP4 is shown as SEQ ID NO:1 is shown.

2. The use of the bacillus subtilis carrier protein antimicrobial peptide BCP4 in the preparation of an antimicrobial medicament according to claim 1, wherein: the antibacterial drug is used for inhibiting and/or killing bacillus cereus.

3. An antibacterial drug, which is characterized in that: the effective components of the bacillus subtilis carrier protein antibacterial peptide BCP4 comprise bacillus subtilis carrier protein antibacterial peptide BCP4, wherein the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

4. the antibacterial agent according to claim 3, characterized in that: the effective component of the bacillus subtilis carrier protein antibacterial peptide BCP4 is bacillus subtilis carrier protein antibacterial peptide BCP4, and the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

5. an antibacterial agent as claimed in any one of claims 3 or 4, wherein: the antibacterial drug is used for inhibiting and/or killing bacillus cereus.

6. A feed additive, characterized in that: the effective components of the bacillus subtilis carrier protein antibacterial peptide BCP4 comprise bacillus subtilis carrier protein antibacterial peptide BCP4, wherein the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

7. the feed additive of claim 6, wherein: the active ingredient of the bacillus subtilis carrier protein antibacterial peptide BCP4 is bacillus subtilis carrier protein antibacterial peptide BCP4, and the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

8. a feed additive according to any one of claims 6 or 7 wherein: the feed additive is used for inhibiting and/or killing bacillus cereus.

9. A food preservative characterized by: the active ingredient of the bacillus subtilis carrier protein antibacterial peptide BCP4 is bacillus subtilis carrier protein antibacterial peptide BCP4, and the amino acid sequence of the antibacterial peptide BCP4 is SEQ ID NO:1.

10. the food preservative of claim 9, wherein: the food preservative is used for inhibiting and/or killing bacillus cereus.

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