Study On Antibacterial Mechanism Of Defensin And Def-a3 And Their Preservation Effect On Chilled Pork

Antimicrobial peptides(AMPs)are a class of peptides with low molecular weight,which were encoded by specific genes of organisms and are important effectors of natural immunity of organisms.Defensin is a important member among AMPs of house fly Musca domestica,which plays a important role in house defending against microorganisms infection.However,the molecular mechanism of this peptide is poorly reported.Moreover,few studies have investigated the feasibility of using M.domestica Defensin as potential antimicrobials for the preservation of chilled meat.In regard to these,we identified AMPs from immunized pupae of M.domestica and synthesized Defensin and its analogs.MIC of these peptides were determined by a microdilution assay.The molecular mechanism of Defensin and Def-a3 were explored systematically.We also assessed the feasibility of using Defensin and Def-a3 to control microbial growth in chilled pork.The main findings are as follows:(1)Seven AMPs were identified from immunized pupae of M.domestica by liquid chromatography–tandem mass spectrometry,which included Domesticin,Antifungal peptide-1,Attacin,Cecropin,Lysozyme,Sarcotoxin II and Defensin.M.domestica Defensin belongs to Defensin 2 family and mature peptide contains 40 amino acids.Defensin and its analogs(Def-a1,Def-a2 and Def-a3)were designed and synthesized.All of them are basic AMPs and p I of Def-a3 is 12.48.(2)Defensin at concentrations from 23.67 to 65.67 ?g/ml exhibited antibacterial activity against four tested bacteria strains,Staphylococcus aureus,Listeria monocytogenes,Escherichia coli and Salmonella enterica.Def-a3 showed higher activity against tested bacteria than that of Defensin.MIC of Def-a3 was between 7.30 and 41.00 ?g/ml.The combination of Defensin and Nisin synergistically improved activity against S.aureus and L.monocytogenes.Co-toxicity coefficient(CTC)of Defensin and Nisin against L.monocytogenes was 534.87.CTC of Def-a3 and Nisin against L.monocytogenes was 212.53.(3)Results of bacterial cell membrane permeability and phosphorus leakage showed that both of Defensin and Def-a3 increased bacterial membrane permeability of four tested strains,resulting in the leakage of phosphorus-containing materials.Transmission electron microscopy analyses demonstrated that Defensin and Def-a3 induced morphological changes and led to the leakage of inner materials.Def-a3 induced pore-formation on the cell surface of L.monocytogenes.Confocal fluorescence microscopic observations expressed that both peptides could enter the bacteria cells rapidly and no peptide was accumulated on the cell surface.Flow cytometry analysis showed that both peptides could penetrate the cell membrane of S.aureus and E.coli.(4)Def-a3 rapidly bound bacteria DNA and inhibited its migration in a concentrationdependent manner.Defensin could not bind with four tested bacteria DNA.Results of q RTPCR showed that Defensin and Def-a3 induced a increased expression in the genes dna B and rec A.Both peptides down-regulated expression genes ssb encoding DNA replication.(5)Based on the parameters measured,compared with the control samples,treatment with Defensin and Def-a3 alone or compound mixture of Defensin and Def-a3 with Nisin were the most effective treatments for controlling bacterial growth in chilled pork.Treatment with Def-a3 alone extended the shelf life up to 6 days.Treatment with Defensin and Nisin synergistically improved the level of inhibition.Both peptides showed negligible hemolytic activity against human erythrocytes.The results of this study provided important information for further exploring understanding the molecular mechanism of Defensin and its analogs against bacteria,and also will help to improve its application in food preservation.