Study On Isolation, Purification Of Apple Snail (Ampullaria Gigas) Antianicrobial Peptides And Their Antibacterial Activities

Wild apple snails were collected and used for study.The extraction,isolation and purification techniques of antimicrobial peptides from the intestine,liver,muscle and tissue fluid of the snail were investigated.There were four experiments included in the study.Experiment 1:Extraction of antimicrobiai peptides from Apple snail.Four extraction methods including extraction by(NH₄)₂SO₄,extraction by methanol, extraction by Tris-HCL and extraction by modified acetic acid method were compared. The results showed that the the method of acetic acid extraction seemed to be the best one for the extraction and isolation.Two species of bacterium,Escherichia coli and staphylococcus aureus were used as indicative bacteria,and the antibacterial activities were evaluated by agarose diffusion test.The test indicated that the extractions from both intestine and tissue fluid had obvious antibacterial effects.Contrarily,extraction from muscle or liver showed much less antibacterial activity.Experiment 2:Purification of antimicrobial peptides from Apple snail.One antimicrobial peptide was purified from snail intestine and tissue fluid by Sephadex G-100 gel filtration.The filtration column with specification of 2,5cm×60cm and flow rate of 0.6ml/min was selected for use.The efficiency peak of antimicrobial peptide came from 27^thto 32^thtube and the strongest antibacterial activity was found at 28^thtube.Experiment 3:Characterization of antimicrobial peptides from apple snail.Methods of PAGE and SDS-PAGE were used for characterization.The results showed that the electrophoretogram appeared to be a single elcctrophoretic band,so the antimicrobial peptide was thought to be electrophoretically pure.Its molecular weights were estimated to be about 5000～7000Da.In addition,another two characteristics of this antimicrobial peptide have been found,i.e.the stability of its antibacterial activity still remained eventhough after being bathed at 40℃～80℃for 30min and the optimal range of pH for its antibacterial activity was between 6.5～7.5. Experiment 4:Determination of the antibacterial spectrum of this antibacterial peptide(ABP)and study on its antibacterial mechanism.The minimal inhibitory concentrations(MIC)of E.Coli,Staphylacoccus aureus, Aeromonas hydrophilia and Bacillus subtilis were measured to be 12.5μg/ml, 25μg/ml,100μg/ml and 25μg/ml respectively by agarose diffusion test.After test,the structure and shape of E.Coli was observed under a scanning electron microscope (SEM).It was revealed that the treated E.Coli had become crimpled,shrivelled and deformed.This phenomenon indicated some bacteria were actually inhibited by this ABP.