Study On Antimicrobial Peptide From Blue Mussel (Mytilus Edulis)

In this paper the purification and antimicrobial activity of the peptide from blue mussel(Mytilus edulis) enzymic hydrolysates are systematically studied, the effect of the peptide on the preservation of strawberry and weever are also investigated.The four parts of content in this study are as follows:1. The technology of preparation of blue mussel enzymic hydrolysates with antimicrobial activity is studied. Based on the antibacterial clear zones in the hydrolysates,the optimal scheme of preparation is determined through orthogonal test of single enzyme and compound enzyme hydrolytic experiment.The optimal compound enzymic hydrolysis conditions are:substrate concentration 1:3,the original pH 8.5,hydrolyzed by Alcalase with the dosage of 300U/g for 4 h at 55℃;then adjust pH to 5.5,and add Bromelin with the dosage of 900U/g to the system and hydrolyzed for another 3 h at 50℃.2. The purification and the antimicrobial activity of blue mussel enzymic hydrolysates are investigated.Taking the antibacterial clear zones as the index, the enzymatic hydrolysate of blue mussel is purified by activity-guided isolation orderly through Cellulose CM-52,which is equilibrated with 20mmol/l NaOAc buffer(pH 4.5)and eluted with a linear gradient of NaCl concentration (0～1.0mol/l) at a volume flow rate of 0.6ml/min. The active fraction C3 has antimicrobial on Bacillus subtills, Escherichia coli,Micrococcus lysodeikticus.The diameters of antibacterial clear zones are larger than 10mm.C3 is collected and added onto Sephadex G-25 column,eluted with distilled water at a volume flow rate of 0.6ml/min. The diameters of antibacterial clear zones of C3S1 on Bacillus subtills, Escherichia coli,Micrococcus lysodeikticus are 13.2mm,10.0mm and 14.43mm. C3S1 is further purified three times on RP-HPLC.The operation conditions for the first time are as follows:C18 semi-prep column (9.2mm×25cm),eluted with a linear gradient of acetonitrile 0 to 40% at a volume flow rate of 0.8ml/min. The chromatogram was monitored by the absorbance at 225nm with the column temperature of 30℃. The antimicrobial activity of C3S1H3 is much stronger than other fractions. At the second time the analysis column(4.6mm×200mm)is eluted with a linear gradient of acetonitrile 0 to 30% ,the volume flow rate is 0.8ml/min.The other operation conditions are the same as above.A1 has antimicrobial activity. A1 is eluted with a linear gradient of acetonitrile 0 to 20%,other operation conditions are the same as the second time. As a result,the antimicrobial peptide A1A1 is proved to be a single peak.It is named MAMP.3. The characters of the antimicrobial peptide were studied.The peptide is with perfect pH and thermal stabilities,its MIC is between 15.312 and 61.25μg/ml.The antimicrobial activity on G+ were stronger than that on G-.The peptide has abroad antibacterial activity spectrum. The relative molecular weight of MAMP was measured by HPSEC on TSK-GEL3000 column.As a result its molecular weight was about 2490Da. The amino acid compositions was analyzed by the automatic amino acid analyzer.By comparison,the content of Cys was the highest,and the content of Pro and Ser was also very high ,they occupy 54.26% of all the amino acids.4. The applications of the antimicrobial peptide on the preservation of strawberry and weever are investigated. The results showed that the sensory analysis, total acid,Vc of strawberries treated with hyaluronic acid chitosan, potassium sorbate, antimicrobial peptide are better than that of the control.By measuring the sensory analysis,pH,T-VBN,K,the results show that hyaluronic acid chitosan, potassium sorbate, antimicrobial peptide are able to antisepticisis of weever differently, the effect of hyaluronic acid chitosan is the strongest, and the effect of antimicrobial peptide is similar as that of potassium sorbate.