Extraction And Purification Of Antimicrobial Peptides From Avian Leukocytes And Comparison Of Their Antimicrobial Activities

To extract and study avian neutrophil peptides, the representative avian species—Yingtaogu duck, Luoman Layer chick, Sanhuang and Luosi308 Broiler chicks were selected. After the antimicrobial activities being tested, the minimal inhibition concentrations (MIC)of the extracts were tested and their bactericidal activities were compared among the above species. In addition, extraction and purification of antimicrobial peptides from duck have been not reported in china. Here are two series in this articleSeries I Purification and extraction of avian antimicrobial peptides. Fresh anticoagulant blood was collected and treated with 0.83% ammonium chloride to lyse the red blood cells whereas keep the leukocytes intact. Then the white blood cells were disrupted by sonication and centrifugated. Pellets were resuspended in 10% acetic acid and stirred to extract peptides, followed by centrifugation and freeze-drying. The crude extract was collected and tresuspended in 0.01% acetic acid.Antimicrobial activity was checked and the results showed that there were natural antimicrobial substances in avian leukocytes, which were active against Gram-positive, Gram-negative becteria and fungi.The crude extract was run by ion-exange chromatography to separate the anionic and cationic fractions, and the latter showed antimicrobial activities. HPLC method was utilized to purify the active ion-exchange components.The results of SDS-PAGE and Mass Spectrometry showed that the molecular weights of the active peaks was 3800-4300Da.Series II Comparison of antimicrobial activities.The MICs of the cationic ion-exchange fraction were tested against Gram-positive and Gram-negative becteria, and fungi, and from low to high were respectively products from Yingtaogu duck, Luoman Layer chick, Sanhuang and Luosi308 Broiler chicks, with antimicrobial activities of Luosi308 Broiler chick significantly lower than Sanhuang counterpart. This implied there were probably differences in both quantity and quality of antimicrobial peptides among the selected avian species.Samely, the MICs of active HPLC peaks were correlated with that of the...