Preparation Of Grub Antimicrobial Peptide Microparticles And Reseach On Its Antimicrobial Properties

In this paper,grub protein was hydrolyzed by trypsin to prepare small-molecule antimicrobial peptides with inhibitory activity against Staphylococcus aureus(Gram-positive bacteria)and Escherichia coli(Gram-negative bacteria).The optimum conditions for the preparation of antimicrobial peptides by trypsin digestion were determined by single factor analysis and orthogonal experiment with four factors and four levels.The results showed that the optimal conditions was enzyme dosage 50.0 ku/g,enzymolysis time 90 min,solid-liquid ratio 1:10(g/mL)and pH 7.5,under these conditions,the hydrolyzate showed strongest inhibition activity against Escherichia coli and Staphylococcus aureus,the inhibition zone diameter was8.7 mm and11.5 mm respectively.Using Escherichia coli and Staphylococcus aureus being as indicator bacteria,the hydrolyzate of grub protein was separated and purified by cation exchange chromatography,and the environmental stability of purified peptide was studied.The resultshowed that: the purified antimicrobial peptides exhibitted best antibacterial activity when pH was at 6.0;antibacterial peptides had excellent temperature stability,when the treatment temperature was 100 °C,it still possessedgood antibacterial activity;when the ambient salt concentration exceeded 80.0 mmol/L,the antibacterial activity began to decline;it was resistant to trypsin but not pepsin.In the experiment,The W/O/W double emulsion method was adopted to prepare antimicrobial peptides microparticles with the polylactic acid glycolic acid being used as the polypeptide carrier,and the encapsulation efficiency of the polypeptide was taken as the main index.The effect of the concentration of the polypeptide in the inner aqueous phase,the concentration of the polylactic acid glycolic acid in the oil phase and the volume of inner phase and oil phase on encapsulation efficiency was stuied.The results showed that when the peptide concentration was 2.5 mg/mL,the concentration of the polymer was 2.5 mg/mL,the volume of the inner and the oil phase were 4.0 mL and 2.0 mL respectively.The highest entrapment efficiency of the prepared nanoparticles hit the highest point which was 79.80%.At this point,the microparticle size was 1.5?m and the polypeptide loading was 10.83%.Finally,the in vitro release process of microparticle in different simulated liquids werestudied,and the pepsin hydrolysis resistance and effect on cell intergrity of bacteria were studied.The results showed that the release kinetic of antibacterial peptide microparticles in water was in accordance with Higuchi equation and the release kinetic in simulated gastric liquid wasin accordance with first-order kinetic equation.The microparticles had good Pepsin hydrolytic resistance ability;SEM showed antimicrobial peptide microparticles may acted on E.coli with bucket-barrel model and Staphylococcus aureus with cappet model.