CN116375828A - Antibacterial peptide KTA with high antibacterial activity and application thereof - Google Patents
Antibacterial peptide KTA with high antibacterial activity and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses an antibacterial peptide KTA with high antibacterial activity and application thereof, belonging to the technical field of biology, wherein the amino acid sequence of the antibacterial peptide KTA is RIKTATWRLALRWLKL, mainly is an alpha-helix structure, comprises 16 amino acid residues, has a theoretical molecular weight of 2025.52Da, an isoelectric point of 12.31 and a net charge of +5. The antibacterial experiment result shows that KTA has broad-spectrum and efficient antibacterial activity and has good inhibition effects on escherichia coli, salmonella typhimurium, klebsiella pneumoniae, staphylococcus aureus and the like. The safety evaluation experiment result shows that the antibacterial peptide KTA has lower hemolytic activity and cytotoxicity. In general, the antibacterial peptide KTA provided by the invention has the characteristics of small molecular weight, strong antibacterial activity, low biotoxicity and the like, and provides a certain reference basis for subsequent researches on green and safe antibiotic substitutes.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibacterial peptide KTA with high antibacterial activity and application thereof.
Background
In recent years, due to the fact that the abuse of traditional antibiotics by people leads a plurality of pathogenic bacteria to generate resistance to the antibiotics, the antibacterial effect of the antibiotics is reduced, the life health of people is seriously threatened, and meanwhile, the global public medical and health are also subjected to great examination. Therefore, the development of new antibiotic alternatives is particularly important. The natural antibacterial peptide is widely focused by people due to the characteristics of high antibacterial activity, wide antibacterial range, low molecular weight, simple secondary structure and the like, and is considered as the most potential antibiotic substitute due to the fact that the antibacterial mechanism is different from that of antibiotics and drug resistance is not easy to generate. Therefore, the development and research of the antibacterial peptide have very important significance.
As a new generation of "antibiotics", most conventional antibiotics act mainly on intracellular targets, while antibacterial peptides destroy bacterial cell membranes mainly by electrostatic interactions or physical effects, thereby achieving sterilization. Meanwhile, the unique sterilization mechanism ensures that the antibacterial peptide is not easy to generate drug resistance and can effectively improve the sterilization capability, so that the antibacterial peptide has wide development prospect and important research value.
Although the antimicrobial peptides are widely available and easily obtained, they have certain drawbacks such as large differences in antimicrobial activity of different natural antimicrobial peptides, high biotoxicity to mammalian cells, low in vivo bioavailability, and poor stability, which are major problems limiting their practical application. In recent years, researchers have begun to attempt to modify antibacterial peptides isolated from natural sources to obtain antibacterial peptides with more excellent properties.
At present, the research in the field of antibacterial peptides focuses on exploring the relationship between the physicochemical parameters (i.e., net charge number, hydrophobicity, amphiphilicity, structural propensity, etc.) and the antibacterial efficacy of antibacterial peptides. Based on the modified natural antibacterial peptide, the natural antibacterial peptide is reasonably modified and designed. The results show that the antibacterial activity of the antibacterial peptide is gradually enhanced with the increase of the hydrophobicity of the antibacterial peptide, and the decrease of the antibacterial activity and the increase of the hemolytic toxicity are caused by the excessive hydrophobic structure. In addition, the bacterial cell membrane contains negatively charged teichoic acid, phosphatidylglycerol (PG) and other phospholipid components, so that the increase of the positive charge number of the antibacterial peptide is beneficial to enhancing the electrostatic interaction between the bacterial cell membranes, thereby improving the sterilization capability. The cell membrane of the mammal is mainly composed of phospholipid components such as electrically neutral Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the like, the antibacterial peptide has weak binding capacity with the phospholipid components, and the antibacterial peptide has small toxicity to the cells of the mammal. Therefore, the regulation of the hydrophobicity and the number of positive charges of natural antibacterial peptides has become a major means of developing novel antibacterial peptides.
Disclosure of Invention
The invention aims to provide a novel antibacterial peptide KTA with high-efficiency antibacterial activity and an application thereof, which are constructed based on a rational molecular design strategy aiming at the defects of natural antibacterial peptides.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides a novel antibacterial peptide KTA, and the amino acid sequence (shown as SEQ ID NO. 1) of the novel antibacterial peptide KTA is RIKTATWRLALRWLKL.
Specifically, the antibacterial peptide KTA is obtained by rational molecular design based on natural antibacterial peptide Leg2 of chickpea Legumin source. Wherein the amino acid sequence of the natural antibacterial peptide (shown as SEQ ID NO. 2) is as follows: RIKTVTSFDLPALRWLKL.
Specifically, the novel antibacterial peptide KTA comprises 16 amino acid residues, the theoretical molecular weight is 2025.52Da, the isoelectric point is 12.31, the net charge is +5, and the proportion of hydrophobic amino acids is 56%.
The application of the antimicrobial peptide KTA in inhibiting escherichia coli, staphylococcus aureus, salmonella typhimurium and klebsiella pneumoniae.
Specifically, the novel antibacterial peptide KTA has high-efficiency antibacterial activity and has excellent antibacterial effects on 6 strains such as escherichia coli ATCC 35150, escherichia coli ATCC 8739, salmonella typhimurium ATCC 14028, klebsiella pneumoniae ATCC 14028, staphylococcus aureus ATCC 25923 and methicillin-resistant staphylococcus aureus ATCC 43300.
The minimum inhibitory concentrations of the antimicrobial peptide KTA on E.coli ATCC 35150 and E.coli ATCC 8739 were 2.5. Mu.M and 4.9. Mu.M, respectively.
The minimum inhibitory concentrations of the antimicrobial peptide KTA on staphylococcus aureus ATCC 25923 and methicillin-resistant staphylococcus aureus ATCC 43300 are 9.9 mu M and 7.4 mu M respectively.
The minimum inhibitory concentration of the antimicrobial peptide KTA on salmonella typhimurium ATCC 14028 strain is 9.9 mu M.
The minimum inhibitory concentration of the antimicrobial peptide KTA on klebsiella pneumoniae ATCC 13883 strain is 12.3 mu M.
The concentration range of the antimicrobial peptide KTA is 2.5-160 mu M, and the hemolysis rate of the antimicrobial peptide KTA on rat red blood cells is less than 20%; the cell survival rate of the LO2 and the NIH3T3 is more than 80% within the concentration range of 19.8-316.0 mu M of the antimicrobial peptide KTA.
An antibacterial agent contains the antibacterial peptide KTA as its main active component.
Compared with the prior art, the invention has the advantages and positive effects that:
according to the invention, natural antibacterial peptide Leg2 from chickpea globulin is used as a template, and rational molecular design is carried out based on an antibacterial peptide modification theory, so that a brand new antibacterial peptide KTA is obtained. The antibacterial peptide KTA is mainly of an alpha-spiral structure, has a theoretical molecular weight of 2025.52Da, an isoelectric point of 12.31 and a net charge of +5, and an antibacterial activity experiment result of the novel antibacterial peptide KTA shows that the novel antibacterial peptide KTA can remarkably improve the antibacterial effect on escherichia coli, staphylococcus aureus, salmonella typhimurium and klebsiella pneumoniae to a certain extent. Meanwhile, the novel antibacterial peptide provided by the invention has lower hemolytic activity and cytotoxicity, and can be used as a green and safe antibacterial substance to be applied to the related microorganism control fields of food, medicine, agricultural production and the like.
Drawings
FIG. 1 is a secondary structure prediction diagram of antibacterial peptide Leg2 and KTA;
FIG. 2 haemolysis of rat erythrocytes by the antimicrobial peptide KTA at different MIC concentrations;
FIG. 3 effect of antimicrobial peptide KTA on LO2 and NIH3T3 cell viability;
FIG. 4 effect of the antimicrobial peptide Leg2 on LO2 and NIH3T3 cell viability.
Detailed Description
The following detailed description of the present invention will be presented with reference to specific examples and drawings, but the following embodiments do not represent the scope of the invention defined by the claims.
Example 1
According to the invention, the chickpea antibacterial peptide Leg2 is subjected to molecular modification according to the antibacterial peptide modification theory, so that the antibacterial activity of the chickpea antibacterial peptide Leg2 is improved, and the hemolytic activity and cytotoxicity of the antibacterial peptide are reduced.
The natural antibacterial peptide Leg2 separated from the chickpea legumain is a small molecular polypeptide consisting of 18 amino acid residues, the molecular weight is 2157.63Da, the net charge is +3, the proportion of hydrophobic amino acid is 52.6%, and the amino acid sequence is: RIKTVTSFDLPALRWLKL serine and (S) proline (P) residues in the amino acid sequence of the antibacterial peptide have a large influence on the antibacterial activity of the antibacterial peptide, mainly due to the presence of larger side chain groups, i.e. the bulky CH of serine side chains 2 N-CH with bulky OH and proline side chains 2 The groups have a limited conformation of amino acid residues, which is detrimental to the formation of an alpha-helical structure of the antimicrobial peptide. Furthermore, the acidic amino acid aspartic acid (D) in this sequence affects the net charge of the antimicrobial peptide. Therefore, the rational molecular design provided by the invention for the natural antibacterial peptide Leg2 comprises the specific formulas of replacing, deleting neutral or acidic amino acid, increasing the number of electropositive amino acid, changing the hydrophobicity of the molecule, improving the proportion of alpha-helix and the likeThe scheme is as follows:
the amino acid arginine (R) with positive charges is adopted to replace amino acid aspartic acid (D) with negative charges at the 9 th position in the sequence of the natural antibacterial peptide Leg2, so that the positive charge quantity of the antibacterial peptide is improved; tryptophan (W) is adopted to replace phenylalanine (F) at position 8 in the sequence of the antibacterial peptide Leg2, so that the interaction between the antibacterial peptide and bacterial cell membrane is improved; alanine (A) is adopted to replace valine (V) at position 5 in the sequence of the antibacterial peptide Leg2, so that the hydrophobicity of amino acid is changed, and the cytotoxicity is reduced; in addition, by deleting serine (S) and proline (P) in the sequence of the antibacterial peptide Leg2, the structural proportion of alpha-helix can be effectively improved. According to the technical scheme, a novel antibacterial peptide KTA with high antibacterial activity potential is designed, and physicochemical properties of the antibacterial peptide such as molecular weight, charge number, isoelectric point, hydrophobicity ratio, average hydrophilicity value, instability index and possible secondary structure formed by the antibacterial peptide are predicted through analysis software Expasy and I-TASSER.
The predicted physical and chemical properties are shown in table 1, and compared with the natural antibacterial peptide Leg2, the novel antibacterial peptide KTA obtained through rational molecular design has improved ratio of net charge to hydrophobicity, which is beneficial to enhancing interaction between the antibacterial peptide and bacterial cell membrane, improving antibacterial activity, and reducing unstable index of KTA, so that the novel antibacterial peptide KTA can play an antibacterial role more stably. In addition, as shown in fig. 1, the secondary structure of the antibacterial peptide Leg2 is mainly a random coil structure, a small amount of alpha-helix structure is formed at the C end of the antibacterial peptide, and in the novel antibacterial peptide KTA, the proportion of the alpha-helix structure is obviously increased, which is likely to help promote the interaction of the antibacterial peptide and bacteria and improve the antibacterial activity of the antibacterial peptide.
TABLE 1 prediction of physicochemical Properties of antibacterial peptides Leg2 and KTA
Example 2
This example was used to determine the antimicrobial activity of the antimicrobial peptide KTA.
Determining minimum antibacterial concentration of antibacterial peptide KTA by micro dilution methodMIC): 6 strains of escherichia coli ATCC 35150, escherichia coli ATCC 8739, salmonella typhimurium ATCC 14028, klebsiella pneumoniae ATCC 14028, staphylococcus aureus ATCC 25923, methicillin-resistant staphylococcus aureus ATCC 43300 and the like are selected as test bacteria, the strains are respectively inoculated into Muller-Hinton broth (MHB) liquid culture medium for activation culture, the culture is carried out in a constant temperature shaking table with the temperature of 37 ℃ and the rotating speed of 180r/min for overnight culture until logarithmic growth phase, and then the bacterial liquid is diluted to about 1 multiplied by 10 by fresh MHB liquid culture medium 6 CFU/mL for use.
Firstly, adding 100 mu L of the bacterial suspension into a sterile 96-well plate, secondly, adding 100 mu L of antibacterial peptide diluents with different concentrations into the 96-well plate one by one, and finally, placing the 96-well plate into a constant temperature incubator at 37 ℃ for static culture for 24 hours. Detection of absorbance values (OD) of each well at 600nm wavelength by using a microplate reader 600 ) The minimum inhibitory concentration was determined as absorbance value (OD 600 )<Minimum antimicrobial peptide concentration of 0.1.
Minimum Bactericidal Concentration (MBC) assay: according to the MIC measurement method, 100 mu L of bacteria are respectively dripped into PCA agar plates of corresponding bacteria from each hole with absorbance value of <0.1 (after the bacteria liquid of each hole is uniformly mixed, the PCA agar plates are sucked), uniformly coated, inverted and cultured in a constant temperature incubator for 18-24 hours, and colony growth conditions on the plates are observed. The lowest concentration at which colony growth does not occur is the minimum bactericidal concentration of the antimicrobial peptide against the bacteria.
The minimum inhibitory concentration of the antibacterial peptide on 6 test bacteria was measured by a microdilution method. As shown in Table 2, the novel antimicrobial peptide KTA provided by the invention has high-efficiency antibacterial activity, the MIC of the novel antimicrobial peptide KTA for different escherichia coli is 2.5-4.9 mu M, the MIC for different staphylococcus aureus is 7.4-9.9 mu M, the MIC for salmonella typhimurium is 9.9 mu M, the MIC for klebsiella pneumoniae is 12.3 mu M, and compared with chickpea natural antimicrobial peptide Leg2, the MIC value of the novel antimicrobial peptide KTA for various bacteria is reduced, so that the antibacterial activity of the novel antimicrobial peptide KTA is obviously improved.
TABLE 2 antibacterial peptide Leg2 and KTA antibacterial Activity determination results
Meanwhile, MBC results show that the Leg2 can not kill bacteria at the concentration of 370.8 mu M, and the KTA can play a role in sterilization at a lower concentration, wherein the MBC of the MBC for different escherichia coli is 9.9-24.7 mu M, the MBC for different staphylococcus aureus is 12.3-49.4 mu M, and the MBC for salmonella typhimurium and klebsiella pneumoniae is 49.4 mu M. In conclusion, the antimicrobial peptide KTA has high-efficiency inhibitory/bactericidal activity and is a novel antimicrobial substance with great potential.
Example 3
This example was used to determine the haemolytic activity of the antimicrobial peptide KTA. The specific scheme is as follows: SD rat blood cells were collected, mixed with PBS repeatedly by pipetting, centrifuged at 3000g for 10min at 4℃and the supernatant removed, and repeated three times until no significant red color was present in the supernatant. The resulting erythrocytes were diluted with PBS to a 5% erythrocyte solution, and then 0.5mL of solutions of the antimicrobial peptide KTA at different concentrations (1, 2,4,8,16,32 and 64 times MIC) were added to 0.5mL of the above erythrocyte solution, respectively. Meanwhile, 1% Triton X-100 and PBS were used as positive and negative controls, respectively. After 1h incubation at 37℃the erythrocytes were centrifuged at 3000g for 10min. The absorbance value of hemoglobin released in the supernatant was measured with a microplate reader at a wavelength of 570nm, and the antibacterial peptide hemolysis rate was calculated by the following formula.
The hemolysis experiment result shows that: in the range of 1-64 times MIC concentration, along with the increase of the concentration of the antimicrobial peptide KTA, the hemolysis rate of rat erythrocytes gradually rises, but in the range of the test concentration, the hemolysis rate of the antimicrobial peptide KTA to the rat erythrocytes is lower than 20%, which indicates that the novel antimicrobial peptide KTA provided by the invention has smaller permeability to erythrocytes and lower hemolytic activity, and can be used in the fields of foods, medicine and the like applied as safe biological antimicrobial medicaments.
Example 4
This example was used to determine the cytotoxicity of the antimicrobial peptide KTA. The invention adopts an MTT method to measure the cytotoxicity of the antibacterial peptide KTA, and specifically comprises the following steps:
and (3) paving: taking out cell culture bottle, sucking culture medium, adding 1mL PBS, washing for 2 times (washing serum), digesting logarithmic phase liver cell LO2 and mouse embryo fibroblast NIH3T3 with 1mL 0.25% pancreatin, centrifuging 1000g for 3min, collecting cells, regulating cell suspension concentration, inoculating into 96-well plate with 100 μl per well, maintaining cell number at 3000-5000 cells per well, placing at 37deg.C and 5% CO 2 The cells were allowed to adhere to the wall by incubator culture overnight (16-24 h).
Sample adding: taking out 96-well plate with cells, sucking old cell culture medium, adding fresh culture medium containing antimicrobial peptide KTA with different concentrations (19.8-316.0 μm), setting 3-5 parallel per concentration, 100 μl per well volume, adding fresh cell culture medium with the same volume and without antimicrobial peptide as control, placing at 37deg.C, 5% CO 2 The incubator is kept at constant temperature for continuous cultivation for 24 hours.
Adding MTT: after 24 hours of incubation, 20. Mu.L of MTT solution (5 mg/mL, i.e., 0.5% MTT) was added to each well, and the incubation was continued in a constant temperature incubator in the dark for 4 hours.
The culture was terminated and crystals were dissolved: the medium in the wells was aspirated, 100. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, and the wells were placed in a shaking table at 37℃for 10min with low-speed shaking, after which the crystals were sufficiently dissolved, and absorbance at 490nm was measured using an ELISA.
As shown in figures 3 and 4, compared with the natural antibacterial peptide Leg2, in the concentration range of 19.8-316.0 mu M, KTA does not have obvious inhibition effect on proliferation and activity of LO2 and NIH3T3 cells, namely, the cell survival rate is more than 80%, which indicates that the novel antibacterial peptide KTA provided by the invention has lower biotoxicity on cells and is reliable in biosafety.
The embodiments of the present invention are not limited thereto, and the present invention may be modified, replaced or altered in various other ways by using the general knowledge and conventional means in the art according to the above-mentioned aspects of the present invention without departing from the basic technical idea of the present invention, and all the modifications and alterations fall within the scope of the present invention.
Claims (10)
1. An antibacterial peptide KTA with high antibacterial activity is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. The antibacterial peptide KTA with high antibacterial activity according to claim 1, which is obtained by taking a natural antibacterial peptide Leg2 of a chickpea legummin source as a template and adopting rational molecular design, wherein the amino acid sequence of the natural antibacterial peptide Leg2 is shown as SEQ ID No. 2.
3. Use of the antibacterial peptide KTA with high antibacterial activity according to claim 1 for inhibiting escherichia coli, staphylococcus aureus, salmonella typhimurium and/or klebsiella pneumoniae.
4. Use according to claim 3, characterized in that the strain type of escherichia coli is standard strain ATCC 35150 and/or ATCC 8739.
5. Use according to claim 3, characterized in that the strain type of staphylococcus aureus is the standard strain ATCC 25923 and/or the methicillin-resistant staphylococcus aureus strain ATCC 43300.
6. The use according to claim 4, wherein the minimum inhibitory concentration of said antimicrobial peptide KTA against e.coli ATCC 35150 and e.coli ATCC 8739 is 2.5 μm and 4.9 μm, respectively.
7. The use according to claim 5, wherein the minimum inhibitory concentration of said antimicrobial peptide KTA against staphylococcus aureus ATCC 25923 and methicillin-resistant staphylococcus aureus ATCC 43300 is 9.9 μΜ and 7.4 μΜ, respectively.
8. The use according to claim 3, characterized in that the minimum inhibitory concentration of said antimicrobial peptide KTA against salmonella typhimurium ATCC 14028 strain is 9.9 μm.
9. The use according to claim 3, wherein said antimicrobial peptide KTA has a minimum inhibitory concentration of 12.3 μm against klebsiella pneumoniae ATCC 13883 strain.
10. An antibacterial agent comprising the antibacterial peptide KTA having a high antibacterial activity as claimed in claim 1 as a main active ingredient.
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