Antibacterial peptide ATMP23 with improved antibacterial activity, mutant of antibacterial peptide ATMP23 and application of antibacterial peptide ATMP23 in preparation of antibacterial agent

Abstract

The invention provides an antibacterial peptide ATMP23 with improved antibacterial activity, a mutant of the antibacterial peptide ATMP23 and application of the antibacterial peptide ATMP23 in preparation of an antibacterial agent. The amino acid sequence of the antibacterial peptide ATMP23 provided by the invention is shown as SEQ ID NO: 1, amino acid mutation and screening are carried out on the basis of the amino acid sequence, and antibacterial peptide mutants A23-M1, A23-M2 and A23-M3 with significantly improved antibacterial activity are obtained. The antibacterial peptide ATMP23 provided by the invention is small in molecular weight, convenient to artificially synthesize and low in cost, has a good bactericidal effect on vibrio splendidus, vibrio alginolyticus, vibrio parahaemolyticus, streptococcus agalactiae, streptococcus iniae, staphylococcus aureus, escherichia coli and the like, and can remarkably reduce the feed conversion ratio of livestock, poultry and aquatic products when being applied to livestock, poultry and aquatic feed; and the feed additive can improve the indexes of growth performance and immune function of bred animals, can be widely applied to the industries of feed, food, medicine and the like, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an antimicrobial peptide ATMP23 with improved antibacterial activity and a mutant thereof, and an application of the antimicrobial peptide in preparing an antimicrobial agent. Background Art

[0002] In recent years, the long-term use of antibiotic growth-promoting additives in feed has caused more and more attention to problems such as drug resistance, drug residues and environmental pollution, and the call for a ban on the addition of antibiotics to feed has become increasingly louder. Natural antimicrobial peptides have the same broad-spectrum antibacterial effect as antibiotics. They can be used as feed additives to achieve the purpose of inhibiting bacteria and promoting growth. Antimicrobial peptides have the characteristics of no residue and no drug resistance of pathogenic bacteria, and are environmentally friendly feed additives. Therefore, the research and development of antimicrobial peptides as feed additives is of great significance to improving the quality of livestock and poultry products and promoting the development of green animal husbandry. However, the natural content of antimicrobial peptides in animals is extremely small, and natural resources are limited; in addition, antimicrobial peptides have a small molecular weight, are difficult to separate and purify, and have cumbersome extraction steps and low yields; while the artificial design and modification of antimicrobial peptides is a fast and effective way to obtain antimicrobial peptides, and has become an important part of antimicrobial peptide development. Therefore, obtaining antimicrobial peptides with simple structure, high antimicrobial activity and easy preparation is an urgent need in the development and research of antimicrobial peptides. Summary of the invention

[0003] The object of the present invention is to provide an antimicrobial peptide ATMP23 with improved antibacterial activity and a mutant thereof and use thereof in the preparation of an antimicrobial agent. The antimicrobial peptide ATMP23 mutant provided by the present invention is a mutant obtained by random mutation and screening, has stronger antibacterial activity and better stress resistance, and improves its application effect as a feed additive in livestock and poultry and aquaculture and in food preservation.

[0004] In order to achieve the above purpose, the following technical solutions are adopted: The present invention provides an antimicrobial peptide ATMP23 with improved antibacterial activity. The amino acid sequence of the antimicrobial peptide ATMP23 is shown in SEQ ID NO:1.

[0005] The present invention provides an antimicrobial peptide ATMP23 mutant A23-M1 with improved antibacterial activity. The amino acid sequence of the mutant A23-M1 is shown in SEQ ID NO: 2, which is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine.

[0006] The present invention provides an antimicrobial peptide ATMP23 mutant A23-M2 with improved antibacterial activity. The amino acid sequence of the mutant A23-M2 is shown in SEQ ID NO: 3, and the mutant A23-M2 is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine, and changing the 20th amino acid from lysine to arginine.

[0007] The present invention provides an antimicrobial peptide ATMP23 mutant A23-M3 with improved antibacterial activity. The amino acid sequence of the mutant A23-M3 is shown in SEQ ID NO: 4, which is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine, the 20th amino acid from lysine to arginine, and the 12th amino acid from arginine to isoleucine.

[0008] The present invention also provides the use of the antimicrobial peptide ATMP23 and / or the antimicrobial peptide ATMP23 mutant in the preparation of an antimicrobial agent.

[0009] Further: the antibacterial agent is a preparation that inhibits Gram-positive bacteria and Gram-negative bacteria.

[0010] Further: the Gram-positive bacteria include Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dolphinus; the Gram-negative bacteria include Vibrio splendens, Escherichia coli, Vibrio parahaemolyticus and Vibrio alginolyticus.

[0011] The present invention also provides the use of the antimicrobial peptide ATMP23 and / or the antimicrobial peptide ATMP23 mutant in the preparation of feed additives for livestock, poultry and aquatic animals.

[0012] Further: the livestock and poultry include chickens, ducks, pigs, geese, cattle and sheep.

[0013] Further: the aquatic animals include crucian carp, carp, tilapia, turbot, grass carp, silver carp, sea bass, bighead carp, black carp, dace, Atlantic salmon, grouper, rainbow trout, bighead bream, bream, eel, large yellow croaker, yellow catfish, mandarin fish, prawns, crayfish, shrimp, sea cucumber and crab.

[0014] Compared with the prior art, the advantages and technical effects of the present invention are as follows: the antimicrobial peptide ATMP23 mutants A23-M1, A23-M2 and A23-M3 of the present invention have small molecular weight, are easy to synthesize artificially and have low cost, have significantly better bactericidal effects on Gram-positive bacteria than ATMP23, and have better stress resistance. The application of the antimicrobial peptide ATMP23 mutants in the present invention in livestock and poultry and aquatic feed can significantly reduce the feed-to-meat ratio of farmed animals and improve the growth performance indicators of farmed animals.

[0015] The antimicrobial peptide mutant produced by the present invention is a chicken immune peptide, which is safe and environmentally friendly and can be widely used in feed, food, medicine and other industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The plate inhibition results of the antimicrobial peptide ATMP23 and its mutants against Staphylococcus aureus, wherein No. 5-8 are chlortetracycline titers of 3000, 5000, 10000, and 20000 ppm, respectively; No. 1-4 are A23-M3, A23-M2, A23-M1, and ATMP23, respectively; Figure 2 It is the result of the thermoresistance of the antimicrobial peptide ATMP23 and its mutants; Figure 3 The antibacterial effect of antimicrobial peptide ATMP23 and its mutants on Vibrio splendens; Figure 4 It is the MIC inhibitory effect of A23-M3 against Vibrio splendens. DETAILED DESCRIPTION

[0017] The following embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims. The protection scope of the present invention and the scope of claims are not limited to the cases provided.

[0018] Unless otherwise specified, the reagents and biological materials used in the following specific examples can be obtained from commercial sources.

[0019] 1. Strains and vectors Pichia pastoris GS115, plasmid pPIC9K, Escherichia coli DH5α, Escherichia coli BL21, and plasmid pET 21a(+) were purchased from Invitrogen; the optimized gene was synthesized by Shanghai Jierui Biotechnology Co., Ltd.; sequencing analysis and primer ordering were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0020] 2. Reagents and culture media Plasmid extraction kit, fragment purification and recovery kit, restriction endonuclease, etc. were purchased from Bao Biotechnology (Dalian) Co., Ltd.; GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; protein marker: Blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0021] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

[0022] MD medium: 1.34% YNB, 0.4 mg / L biotin, 2% glucose; YPD medium: 1% yeast extract, 2% peptone, 2% glucose; BMGY medium: 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% glycerol; BMMY medium: 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% methanol; When the above culture medium is solid, add 2% agar powder.

[0023] Example 1: Preparation of antimicrobial peptide ATMP23 Chemical synthesis method of antimicrobial peptide ATMP23: According to the sequence of SEQ ID NO: 1, the full sequence of ATMP23 was synthesized using a fully automatic peptide synthesizer (ABI433), and purified by HPLC reverse phase column chromatography desalting. The molecular weight of the purified antimicrobial peptide ATMP23 was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence was analyzed by an automatic amino acid sequence determination instrument.

[0024] The antimicrobial peptide ATMP23 is an artificially designed and synthesized active polypeptide, comprising 31 amino acid residues, with a theoretical molecular weight and isoelectric point of 3.76 KDa and 13.35, respectively. Its amino acid sequence is shown in SEQ ID NO: 1: LVQRGRFGRFLRKIRRFRPKVTITIQGSARF.

[0025] The nucleotide sequence shown in SEQ ID NO: 5 was optimized and synthesized according to the amino acid sequence, and the synthesized nucleotide base sequence was cloned into the pPIC9K plasmid and expressed using GS115 as a host.

[0026] Example 2: Analysis of the antibacterial activity profile of antimicrobial peptide ATMP23 The antibacterial activity of the antimicrobial peptide ATMP23 was determined by the agar plate diffusion method, and the tested microbial strains were: Escherichia coli, Staphylococcus aureus, Vibrio splendens, Vibrio parahaemolyticus 3# (No. 3#), Vibrio parahaemolyticus 4# (No. 4#), Vibrio alginolyticus, Streptococcus agalactiae (Wuxi, commercially available), and Streptococcus iniae (Wuxi, commercially available).

[0027] The above-mentioned test microorganisms (OD600=0.2~0.3) were mixed with 25mL of LB solid culture medium at 55℃ and spread on a plate (bacterial suspension:LB culture medium=1:100). After solidification, holes were punched with a sterilized hole puncher (7mm in diameter). 70μL of antimicrobial peptide ATMP23 (0.2g / mL fermentation spray-dried powder, or fermentation liquid after centrifugation) was added into the holes. The cells were cultured at 30℃ for 8~12h, and the diameter of the transparent circle around the hole was recorded. The measurement was repeated three times for each strain, and the average value was calculated.

[0028] Table 1 Antibacterial spectrum analysis results of antimicrobial peptide ATMP23

[0029] The antibacterial activity test results in Table 1 show that the antimicrobial peptide ATMP23 of the present invention has a very broad antibacterial spectrum, and effectively inhibits Gram-positive bacteria (Staphylococcus aureus, Streptococcus, etc.) and Gram-negative bacteria (Escherichia coli, Vibrio parahaemolyticus, Vibrio splendens and Vibrio alginolyticus, etc.), livestock and poultry, and aquatic pathogens, and has good application prospects in the prevention of livestock and poultry and aquatic animal diseases.

[0030] Example 3: Random mutagenesis and mutant screening of antimicrobial peptide ATMP23 The optimized ATMP23 sequence was used as a template to randomly introduce mutations using the GeneMorph II Random Mutation PCR Kit (Stratagene) to construct a mutant library.

[0031] The antibacterial activity of the antimicrobial peptide ATMP23 mutant was determined by agar plate diffusion method, and the tested microbial strain was Staphylococcus aureus ACCC01337.

[0032] The above-mentioned test microorganisms (OD600=0.2~0.3) were mixed with 25 mL of LB solid culture medium at 55°C and spread on a plate (bacterial suspension: LB culture medium = 1:100). After solidification, holes were punched with a sterilized hole puncher (diameter 7 mm), and 70 μL of antimicrobial peptide ATMP23 mutant fermentation liquid was added to the holes. The cells were cultured at 30°C for 8~12 h, and the diameter of the transparent circle around the holes was recorded. The measurement was repeated three times for each strain, and the average value was calculated.

[0033] Antibacterial potency test: While measuring the size of the antibacterial zone of the antimicrobial peptide product by agar diffusion method, add chlortetracycline samples of different concentrations as a control. Linear fitting is performed with the diameter of the antibacterial zone (mm) as the X-axis and the logarithmic value of the chlortetracycline potency (ppm) as the Y-axis, and then the corresponding chlortetracycline potency of the antimicrobial peptide product is calculated based on the diameter of the antibacterial zone.

[0034] After antibacterial activity test, Figure 1As shown in the figure, after sequencing analysis, the antibacterial activity of the fermentation broth of mutants A23-M1, A23-M2, and A23-M3 was further increased from 3550ppm to 4150ppm, 5300ppm, and 6230ppm, which is equivalent to the potency of chlortetracycline. The amino acid sequences of mutants A23-M1, A23-M2, and A23-M3 are as follows: SEQ ID NO:2: LVQRGRFGRFLRK V RRFRPKVTITIQGSARF; SEQ ID NO:3: LVQRGRFGRFLRK V RRFRP R VTITIQGSARF; SEQ ID NO:4: LVQRGRFGRFL I K V RRFRP R VTITIQGSARF.

[0035] Example 4: Analysis of the heat resistance of antimicrobial peptide ATMP23 mutants The antimicrobial peptide ATMP23 and its mutant solutions with a concentration of 0.2 g / mL were placed at 0°C, 80°C, and 100°C for 5 minutes; then the antibacterial activity of the antimicrobial peptide ATMP23 and its mutant solutions treated at four different temperatures was analyzed according to the antibacterial activity analysis method in Example 2 (the antibacterial activity was analyzed by the above-mentioned agar plate diffusion method), and the tested microbial strain was Staphylococcus aureus 01337.

[0036] The results are as follows Figure 2 As shown in the figure, with the increase of temperature, the antibacterial activity of antimicrobial peptide ATMP23 and its mutants against Staphylococcus aureus ACCC01337 gradually decreased, but after high temperature (80℃ and 100℃) treatment, the mutant of antimicrobial peptide ATMP23 still had good antibacterial activity, and its antibacterial activity was much higher than that of ATMP23. This shows that the antimicrobial peptide mutants A23-M1, A23-M2, and A23-M3 all have good heat resistance.

[0037] Example 5: Antibacterial effect of antimicrobial peptide ATMP23 mutant on Vibrio splendens The antimicrobial peptide ATMP23 and its mutant solutions with a concentration of 0.2 g / mL were then subjected to the antibacterial activity analysis in Example 2, with Vibrio splendens as the indicator bacteria, and verified on agarose plates.

[0038] The results are as follows Figure 3As shown, the diameter of the inhibition zone of the antimicrobial peptide mutants was measured to be larger than that of ATMP23, indicating that the antibacterial activity of the antimicrobial peptide mutants A23-M1, A23-M2, and A23-M3 against Vibrio splendens was better than that of the original gene.

[0039] MIC detection of antimicrobial peptide mutant A23-M3: (1) Preparation of 200,000 ppm antimicrobial peptide (A23-M3) Weigh 2.00 g of antimicrobial peptide spray-dried powder, add water to make up to 10 ml, stir magnetically for 30 min, centrifuge at 10000 rpm for 10 min, and filter the supernatant with a 0.22 μm filter membrane for sterilization.

[0040] (2) Preparation of indicator bacteria After inoculating the indicator bacteria at a 1% inoculum, culture the culture in a shaker at 37°C and 200 rpm until the late stable phase (about 16-20 hours), and dilute the bacterial solution to less than 500 CFU / ml with blank culture medium (the concentration of indicator bacteria used in actual operation is shown in the table).

[0041] (3) Add antimicrobial peptides using the 2-fold dilution method Add 100 μl of LB medium to each well of a 96-well plate, add 100 μl of sterile 200,000 ppm antimicrobial peptide to the medium in the first column, mix well, take out 100 μl and add it to the medium in the second column, mix well again, take out 100 μl and add it to the medium in the third column, and so on until 100 μl is taken out and discarded in the 11th column. There is no antimicrobial peptide in the 12th column.

[0042] At this time, the concentration of antimicrobial peptides in each well (ppm) is as follows:

[0043] (4) Add indicator bacteria The indicator bacteria cultured to the late stable phase were diluted 10 5 , 10 6 , 10 7 , 10 8 100 μl blank LB medium was added to rows AB (only one negative control group without indicator bacteria is needed for the same antimicrobial peptide), and 100 μl diluted 10 was added to rows CD. 8 Add 100 μl of the bacterial solution to the EF row and dilute it by 10 7 100 μl of bacterial solution was added to the GH line for dilution 10 6 times the bacterial solution (or 10 5 and other appropriate dilutions).

[0044] At this time, the dilution multiples of indicator bacteria in each well are as follows:

[0045] At this time, the concentration of antimicrobial peptides in each well (ppm) is as follows:

[0046] (5) Cultivation and testing After mixing the liquid in the 96-well plate, let it stand at room temperature for 4 hours, and then culture it with shaking at 100 rpm at 30℃ for 8-12 hours. Use an enzyme reader to detect the absorbance value at 620 nm (cultivate until the negative control group containing only indicator bacteria shows significant growth), or directly observe whether there is turbidity such as bacterial growth.

[0047] (6) The results are as follows Figure 4 As shown, the minimum inhibitory concentration of antimicrobial peptide A23-M3 is about 195 ppm.

[0048] Example 6: Effects of antimicrobial peptide A23-M3 on the growth performance of Litopenaeus vannamei (1) Experimental design A total of 270 Penaeus vannamei with a body weight of 4-5 g / tail were selected and randomly divided into three treatment groups according to body weight using a single-factor experimental design, including a control group and two experimental groups. The control group: no antimicrobial peptides were added to the basal diet, experimental group 1: 100 mg / kg of antimicrobial peptide A23-M3 was added to the basal diet; experimental group 2: 200 mg / kg of antimicrobial peptide A23-M3 was added to the basal diet. Each group had 3 replicates, and each replicate had 30 shrimps.

[0049] Three meals were fed daily at 8:30, 12:30, and 16:30. The feeding amount was based on 1-hour satiation, ensuring that a small amount of bait remained in the tank after 1 hour. The remaining bait was then collected by siphon in each tank, dried at 105 °C to constant weight, weighed, and recorded. The breeding experiment lasted for 30 days.

[0050] (2) Determination of growth performance indicators After 30 days of the breeding experiment, the vannamei shrimp were fasted for 24 h, and then the shrimp were taken out and the surface moisture was gently absorbed with filter paper and weighed to calculate the weight gain rate, specific growth rate and feed coefficient.

[0051] The formula for calculating weight gain rate is: WGR (%) = 100 × [(Wt-W0) / W0]; The specific growth rate is calculated as follows: SGR (%·day -1 )=100×[(lnWt-lnW0) / t]; The formula for calculating the feed coefficient is: FCR=F / (Wt-W0); Where W0 is the initial average body weight (g), Wt is the final average body weight (g), t is the number of experimental days (d), and F is the feed intake (g, feed amount minus residual feed amount).

[0052] (3) Test results The effects of antimicrobial peptide A23-M3 on the growth performance of Penaeus vannamei are shown in Table 2. The experimental Penaeus vannamei was reared for 30 days, and the results showed that antimicrobial peptide A23-M3 could significantly increase the weight gain rate and specific growth rate of Penaeus vannamei, and also reduce its feed conversion rate to a certain extent.

[0053] Table 2 Effects of different doses of A23-M3 on weight gain rate, specific growth rate and feed coefficient of Penaeus vannamei

[0054] Example 7: Effect of dietary antimicrobial peptide A23-M3 on growth performance of weaned piglets (1) Experimental design 120 healthy weaned piglets of similar age, litter size and parity were selected. A single factor experimental design was used and they were randomly divided into 3 groups, each with 4 replicates and 10 pigs in each replicate, namely the control group (fed with a basic diet), the experimental group 1 (basic diet supplemented with antimicrobial peptide A23-M3 100mg / kg), and the experimental group 2 (basic diet supplemented with antimicrobial peptide A23-M3 200mg / kg). The pre-test period was 3 days and the main test period was 28 days.

[0055] (2) Index determination At 8:00 a.m. on the first day of the experiment and the 29th day of the experiment, the experimental pigs in each pen were weighed on an empty stomach, the feed consumption was recorded, and the daily weight gain, daily feed intake and feed-to-weight ratio were calculated. During the experiment, the defecation of each piglet was observed every day, and the diarrhea individuals and diarrhea days were recorded. Finally, the diarrhea frequency was calculated in repetition units.

[0056] (3) Test results Table 3 Effect of antimicrobial peptide A23-M3 on growth performance of weaned piglets

[0057] As shown in Table 3, compared with the control group, the daily weight gain of the experiment group increased by 13.57g / day and 23.21g / day, the feed intake increased by 7.71g / day and 11.04g / day, the feed-to-meat ratio decreased by 0.06 and 0.11, and the diarrhea rate decreased by 1.23% and 2.65%. This shows that the antimicrobial peptide A23-M3 has a significant effect on reducing the diarrhea rate and feed-to-meat ratio of piglets, increasing daily weight gain and daily feed intake, and can significantly improve the production performance of piglets.

[0058] The above results show that the antimicrobial peptide ATMP23 and its mutants A23-M1, A23-M2, and A23-M3 in the present invention have a broad antibacterial spectrum, and have significant antibacterial effects on Gram-positive bacteria including Staphylococcus aureus, Streptococcus agalactiae and Streptococcus iniae; Gram-negative bacteria including pathogens such as Vibrio splendens, Escherichia coli, Vibrio parahaemolyticus and Vibrio alginolyticus, and have good thermal stability. It can be widely used in feed, food, medicine and other industries, especially as a feed additive, and applied to livestock and poultry breeding and aquaculture such as fish, shrimp, and sea cucumber.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An antimicrobial peptide ATMP23 with improved antibacterial activity, characterized in that: The amino acid sequence of the antimicrobial peptide ATMP23 is shown in SEQ ID NO:

1.

2. An antimicrobial peptide ATMP23 mutant A23-M1 with improved antibacterial activity, characterized in that: The amino acid sequence of the mutant A23-M1 is shown in SEQ ID NO: 2, which is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine.

3. An antimicrobial peptide ATMP23 mutant A23-M2 with improved antibacterial activity, characterized in that: The amino acid sequence of the mutant A23-M2 is shown in SEQ ID NO: 3, which is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine, and the 20th amino acid from lysine to arginine.

4. An antimicrobial peptide ATMP23 mutant A23-M3 with improved antibacterial activity, characterized in that: The amino acid sequence of the mutant A23-M3 is shown in SEQ ID NO: 4, which is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine, the 20th amino acid from lysine to arginine, and the 12th amino acid from arginine to isoleucine.

5. Use of the antimicrobial peptide ATMP23 according to claim 1 and / or the antimicrobial peptide ATMP23 mutant according to any one of claims 2 to 4 in the preparation of an antimicrobial agent.

6. The use according to claim 5, characterized in that: The antibacterial agent is a preparation that inhibits Gram-positive bacteria and Gram-negative bacteria.

7. The use according to claim 6, characterized in that: The Gram-positive bacteria include Staphylococcus aureus, Streptococcus agalactiae and Streptococcus iniae; the Gram-negative bacteria include Vibrio splendens, Escherichia coli, Vibrio parahaemolyticus and Vibrio alginolyticus.

8. Use of the antimicrobial peptide ATMP23 according to claim 1 and / or the antimicrobial peptide ATMP23 mutant according to any one of claims 2 to 4 in the preparation of feed additives for livestock, poultry and aquatic animals.

9. The use according to claim 8, characterized in that: The livestock and poultry animals include chickens, ducks, pigs, geese, cattle and sheep.

10. The use according to claim 8, characterized in that: The aquatic animals include crucian carp, carp, tilapia, turbot, grass carp, silver carp, sea bass, bighead carp, black carp, dace, Atlantic salmon, grouper, rainbow trout, bighead bream, bream, eel, large yellow croaker, yellow catfish, mandarin fish, prawns, crayfish, green shrimp, sea cucumber and crab.

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