Preparation And Bacteriostasic Activity Of Antimicrobial Peptides From Egg White Lysozyme

Lysozyme is a widespread alkaline protease in organisms with high safety and antibacterial activity.It is commonly used as a food preservative and antistaling agent.However,lysozyme belongs to non broad-spectrum antibacterial agent,which can effectively kill G+,but the effect of G-is not significant.As a protein,lysozyme has immunogenicity and its thermal stability is extremely poor under non acidic conditions.Enzymatic hydrolysis is an effective way to solve the above problems.This subject adopts the pepsin and papain enzyme to hydrolyze egg white lysozyme,and measured the antibacterial activity and thermal stability of hydrolysates.Then,separated and purified an antimicrobial peptide with highest antibacterial activity,and studied on antibacterial mechanism of the antibacterial peptide,the main results are summarized as follows:First,under the optimum conditions of enzyme reaction,egg white lysozyme were hydrolyzed by pepsin and papain,and the double hydrolysate Lyz-pp was obtained.Lyz-pp lost all enzyme activity,and its molecular weight was mainly distributed within 1000-5000 Da,which initially reduced its immunogenicity.The antibacterial spectrum of Lyz-pp was basically consistent with that of egg white lysozyme,all of which had good bacteriostasis effect on G+,the effect on G-was not significant.The MIC for Staphylococcus aureus and Bacillus subtilis were 1.25 mg/mL and 2.5 mg/mL respectively.Under different pH conditions,the thermal stability of Lyz-pp was stronger than that of lysozyme and Nisin,but it decreased with the increase of pH.Secondly,the Sephadex G-25 gel column,the HiTrap SP HP cation exchange column and the prepared RP-HPLC three steps were used to separate and purify Lyz-pp,and the antibacterial peptide F-2-3-1 with the most antibacterial activity was obtained.The single peak of F-2-3-1 was obtained by MALDI-TOF-MS,and the molecular weight was 1418.2 Da.The amino acid sequence was KKIVSDGNGMNAW by LC-MC/MC,which corresponded to the 96-108 amino acid residue at the N end of the egg white lysozyme.F-2-3-1 had better thermal stability than Lyz-pp,and had broad spectrum bacteriostasis.It had good antibacterial activity to both G+and G-.The MIC for Staphylococcus aureus and Bacillus subtilis was 0.312 mg/mL and 0.615 mg/mL respectively.The MIC for Escherichia coli and Salmonella was 1.25 mg/mL.Finally,the bacteriostatic mechanism of antibacterial peptide F-2-3-1 was preliminarily explored.After F-2-3-1 treatment,the morphology of Staphylococcus aureus and Escherichia coli was seriously deformed,the surface of the cell membrane was depressed and appeared some holes.The surface hydrophobicity of Escherichia coli decreased with the increase of the concentration of antibacterial peptide,the outer and inter membrane permeability became larger and the K+appeared leak,which all indicated that the antibacterial peptide F-2-3-1 exert bacteriostasis by the mechanism of membrane damage.