Study On Preparation Of Antibacterial Peptides From Scallop Skirt By Enzymatic Hydrolysis

Scallop (Argopecten irradians) is the seafood on larger scale of China’s coastalaquaculture, with great nutritional and economic value. The scallop adductor （adductormuscle）is the main edible part, as the main processing products. However, by-products ofscallops (scallop skirt) would be discarded as wastes, cause a lot of waste of resources andenvironmental pollution. Scallop skirt is rich in protein, which is the high quality source ofmarine protein, it could be perpara antibacterial peptide by enzymatic hydrolysis. Becauseof its unique antibacterial mechanism, with wide antibacterial spectrum, no resistance,safety and no pollution, antibacterial peptides have attracted increasing interests scholars athome and abroad. This study took scallop skirt as raw materials, then separate and purifiedscallop active peptides through enzymatic hydrolysis and ultrafiltration. At the end,antibacterial peptide were screened by turbidimetry. This study is aim to providereasonable and scientific basis for the developing and utilizing of scallop skirt. Preparationof antibacterial peptide from scallop skirt by hydrolysis expand the types and the quantitiesof antibacterial peptide from marine organism. This study has positive significance andbroad market prospects.In this paper, six kinds of commercial protease (alkaline protease, neutral protease,pepsin, papain, trypsin and flavourzyme) were used for hydrolysis. In order to maximizeaccess to sources of antibacterial peptide from marine organism, the study was assessed thedegree of hydrolysis. Alkaline protease and neutral protease were selected from six kindsof protease as applicable protease because of their high rates of hydrolysis. In this paper,four single factors (temperature, pH,[E]/[S]%, S%) which affect the rate of hydrolysiswere selected to designing the orthogonal tset which optimize the process of enzymatichydrolysis. The alkaline protease result showed that the optimum hydrolysis was obtainedunder the conditions of pH11，9%material/liquid ratio，40℃，12%enzyme/substrate ratioand4h hydrolysis，which displayed the highest degree of hydrolysis of36.04%。Andneutral protease result showed that the optimum hydrolysis was obtained under theconditions of pH9，4%material/liquid ratio，40℃，13%enzyme/substrate ratio and4hhydrolysis，which displayed the highest degree of hydrolysis of28.83%。This paper separate and purified enzyme solution through ultrafiltration. The enzymesolution was first centrifuged (4℃,10000r/s,20min), then filtered through0.45μm membrane and then with membrane flux as an index. The hydrolysate was segmented byUF membrane with MWCO (1kDa,3kDa,5kDa,10kDa). Five fraction was segmentedfrom enzymolysis liquid: SSPH-Ⅰ（<1kDa）、SSPH-Ⅱ（1kDa～3kDa）、SSPH-Ⅲ（3kDa～5kDa）、 SSPH-Ⅳ（5kDa～10kDa）、SSPH-Ⅴ（>10kDa）. Freeze-drying thedifferent fraction.The antibacterial peptide was screening from different fraction by cylinder platemethod and turbidimetry. Turbidimetry was established the way that screen antibacterialpeptide by comparing the two methods. The results showed that these fraction hasantibacterial effects: alkaline peotease hydrolyzate SSPH-Ⅱ（1kDa～3kDa）, neutralpeotease hydrolyzate SSPH-Ⅱ（1kDa～3kDa）, neutral peotease hydrolyzate SSPH-Ⅲ（3kDa～5kDa）. The MIC value of those antibacterial peptide was measured bymicro-dilution. The results showed the MIC value of alkaline peotease hydrolyzateSSPH-Ⅱ（1kDa～3kDa） against E.coli was40μg/ml，which has the MIC value againstStaphylococcus aureus was80μg/ml；the MIC value of neutral peotease hydrolyzateSSPH-Ⅱ（1kDa～3kDa） against E.coli and Staphylococcus aureus was both80μg/ml；the MIC value of neutral peotease hydrolyzate SSPH-Ⅲ（3kDa～5kDa）against E.coli andStaphylococcus aureus was both40μg/ml.This paper provide a theoretical basis and datasupport for preparing antibacterial peptide from scallop skirt by enzymatic hydrolysis.