Preparation Of Antioxidant Peptides From Scallop Skirt By Enzymatic Hydrolysis

Scallop (Argopecten irradians) is an economically important edible shellfish, widelycultured in East of China. Every year, a considerable amount of by-products is generatedby the scallop processing industries. The scallop adductor is the role part of the scallopbody and other parts would be discarded as wastes. However, the inadequate treatment ofindustrial residues can cause environmental problems. Simultaneously, as naturalantioxidants, antioxidant peptides gains from proteins have attracted increasing interestsbecause of their safety and wide distribution properties in recent years. This study tookscallop skirt as raw materials, and prepared scallop skirt peptides which have antioxidantactivities through enzymatic hydrolysis, ultrafiltration, gel chromatography, reversed-phasehigh-performance liquid chromatography (RP-HPLC) and so on. This study is aim toprovide reasonable and scientific basis for the developing and utilizing of scallop skirt.In this dissertation, six kinds of enzymes, neutral protease, trypsin, alkaline protease,pepsin, flavor protease and papain were used for hydrolysis. The hydrolysates by neutralprotease exhibited higher degree of hydrolysis and superoxide radical scavenging activitythan the others. Effects of hydrolysis temperature, pH value, enzyme dose, substrateconcentration and hydrolysis time on preparation of scallop skirt peptide with neutralprotease were studied by single factor analysis. The regression models were establishedwith factors and the indexes, including degree of hydrolysis (DH) and antioxidant activities.The results showed that the optimum values of temperature, pH, enzyme dosage, reactiontime and substrate concentration were45℃,8,12.9%,6h and4%. Under this condition,the scavenging rate of superoxide anion reached25.63%, and the degree of hydrolysis isup to30.67%.Enzyme solution usually contains many impurities, such as unhydrolysis scallop skirtprotein and degenerated protease. Ultrafiltration is a commonly used separation method inenzyme solution. The enzyme solution was first filtered through0.45μm membrane andthen with membrane flux as an index. The hydrolysate was lyophilized, prepared forfractionation using UF membrane with MWCO (10kDa,5kDa,3kDa, and1kDa). Allfractions recovered were lyophilized and named as SSPH-Ⅰ（parent hydrolysates）,SSPH-Ⅱ(over10kDa), SSPH-Ⅲ(10kDa~5kDa), SSPH-Ⅳ(5kDa~3kDa),SSPH-Ⅴ(3kDa~1kDa), SSPH-Ⅵ(below1kDa). The antioxidant activities of these peptide fractions were well investigated by assaying the activities of the DPPH radical scavenging,the reducing power and superoxide radical scavenging. Results showed that fractionSSPH-Ⅲ demonstrated the most significant antioxidant actives.The fraction SSPH-Ⅲ was separated using Sephadex G-50gel chromatographyseparation. The best Sephadex G-50gel chromatography separation conditions ofantioxidant peptides were determined: eluted with deionized water,2ml/min elution speed,75mg/mL of sample concentration, and0.50mL sample. Each fraction collected at avolume of4ml was monitored at280nm. The scallop skirt peptides could be separatedinto two peaks, among which the second peak (F2) had the highest antioxidant actives. Andits DPPH radical (DPPH) scavenging activity reached37.16%.The peak F2was isolation by reversed-phase high performance liquid chromatography(RP-HPLC). The mobile phases for the analysis of chromatography were:(A)0.05%trifluoroacetic acid (TFA) in acetonitrile; and (B)0.05%TFA in water. Gradient elutionwas as follows:0–25min, linear gradient10-30%B;25-30min, linear gradient30-10%B;the flow rate:1.0mL/min; the detection wavelength280nm.The molecular weight (MW) of antioxidant peptide was measured by LTQ-Orbitrap(Thermo Fisher). Ion source conditions were optimized using the tuning and calibrationsolutions recommended by the instrument provider. LTQ-Orbitrap has the precise effect ofvarying resolution as a parameter on peptide identification. The fragmentation mass spectracontained a major ion m/z at1206.28. And detected molecular mass calculated4101.09Daby LC/LTQ-Orbitrap.