Antioxidative And Antibacterial Activities Of TP1 And MS1 Antimicrobial Peptides And Their Mechanism Of Action

Antimicrobial peptides(AMPs)are the earliest immunologically active molecules produced by organisms in the long-term evolution process to adapt to the environment and survive,and are important mediators of natural immunity.In this study,Temporin-TP1 and Temporin-MS1 were used as the research objects,and various online tools were used to predict the biological information such as the biological activity score,isoelectric point,hydrophilic index,water solubility and three-dimensional structure of AMPs,and to measure their own free radical scavenging.The antibacterial mechanism of Temporin-TP1 and Temporin-MS1 was revealed by scanning electron microscope observation,electrophoretic mobility change and membrane permeability change.The main research contents of this paper are as follows:1.Bioinformatics analysis of AMPsThe sequences of AMPs were retrieved from the AMPs database,and the solid-phase synthesis method was used to synthesize them to obtain AMPs with high purity and consistent with the theoretical molecular weight,and the next experiment could be carried out.The biological activity score,hydrophilicity,water solubility,isoelectric point,toxicity and three-dimensional structure of peptides are predicted by various online tools such as Expasy,PeptideRanker,Innovagen,DPL,ToxinPred,and Pymol.The prediction results show that the AMPs obtained in this study are not toxic,and Temporin-TP1 and Temporin-MS1 have good water solubility,high biological activity score and hydrophilic index,and their structures have LEU,VAL,TRP,MET,ALA,ILE,and PHE are hydrophobic residues,and most of the secondary structures are random coils,without α-helix and β-sheet.2.Antioxidative activity of AMPsIn order to test the antioxidant capacity of AMPs,the DPPH free radical scavenging ability method,ABTS free radical scavenging ability method,rhodamine B-Fe2+-H2O2 photometric method and pyrogallol auto-oxidation method were used to measure the effect of AMPs on DPPH,ABTS,·OH,O2-· scavenging ability.The results showed that Temporin-TP1 had good scavenging ability to DPPH(33.46%),ABTS(33.73%),·OH(44.77%),O2-·(33.34%)free radicals,Temporin-MS1 had good scavenging ability to DPPH(37.12%),ABTS(15.55%),·OH(52.39%),O2-·(33.31%)free radicals had a certain scavenging ability,and the antioxidant capacity of AMPs at the same concentration was lower than that of VC.3.Antibacterial activity and mechanism of action of antimicrobial peptidesThe bacteriostatic activities of AMPs against Escherichia coli,Staphylococcus aureus and Bacillus subtilis were determined by the zone of inhibition method,and their MIC concentrations were determined.It can be seen from the results that only kanamycin has bacteriostatic effect on Escherichia coli,while Temporin-TP1 and Temporin-MS 1 have no bacteriostatic effect on Escherichia coli at the concentration of 1024 μM.Their MIC concentrations against Escherichia coli were all greater than 1024 μM.The MICs of Temporin-TP1 against Staphylococcus aureus and Bacillus subtilis were 256 μM and 16 μM respectively,and the MIC of Temporin-MS1 against Bacillus subtilis was 256 μM.From the electron microscope results,cell membrane damage such as membrane pores,deformation and dissolution were observed in bacterial cells treated with Temporin-TP1 and Temporin-MS1,and the damage to bacteria by Temporin-MS 1 was mild.The results of DNA electrophoretic migration distance change showed that Temporin-TP1 and Temporin-MS1 could completely inhibit the electrophoretic migration of bacterial genomic DNA only at high concentrations.It shows that the binding ability of AMPs and bacterial DNA is concentration-dependent.The results of permeability measurement of bacterial cell membrane showed that compared with the control group,Bacillus subtilis and Staphylococcus aureus cells were treated with Temporin-TP1 at 0.5×MIC and MIC for 4 h,the nucleic acid and protein contents of bacterial culture medium were significantly increased.After the Bacillus subtilis cells were exposed to Temporin-MS1 at 0.5×MIC and 1×MIC for 6 h,the nucleic acid content in the medium began to increase significantly,and the protein content began to increase significantly after 4 h of exposure.This paper aims to study the antioxidant activity and bacteriostatic activity of AMPs and explore the possible antibacterial mechanism,which will theoretically help to strengthen the understanding of the biological activity of AMPs and its mechanism of action,and it will contribute to the new research and development of food antioxidants and antibacterial agents.