Preparation Of Antioxidant Peptides From Scallop Skirt Of Argopecten Irradians By Bromelain Hydrolysis

China has vast sea area with rich marine resources. Scallop play an important position in ourmarine resources.They have certain values in development and application with their delicious,nutritional and healthy. Scallop farming of Hebei province is mainly concentrated in the Bohai region,the annual yield is considerable, but the utilization of the by-products is low, It has not only leaded tothe waste of the scallop resources, but also caused environmental pollution in the zone of the breedingand fishing. A series of studies found that scallop skirt rich in protein, can be used as raw materials forprotein products.The traditional aquatic product processing in China is simple. On account of low utilization of thewaste,in this study,the by-products from processing of scallop skirt was as the research object, preparedscallop skirt peptides which have antioxidant activities through enzymatic hydrolysis, ultrafiltration, gelchromatography, reversed-phase high-performance liquid chromatography (RP-HPLC) and so on. Thisstudy was aim to provide reasonable and scientific basis for the developing and utilizing of scallop skirt.The main contents and results of this study are as follows:1. Study on process for preparing antioxidant peptides from scallop skirt by bromelainhydrolysis.Based on previous studies, in this paper, the DPPH clearance was investigated as indicators.In this dissertation, bromelain was used for hydrolysis. Effects of hydrolysis temperature, pH value,enzyme dose, substrate concentration and hydrolysis time on preparation of scallop skirt peptide withbromelain protease were studied by single factor analysis. And then response surface was used tooptimize hydrolysis conditions. The results showed that the optimum values of temperature, pH,enzyme dosage, substrate concentration and reaction time were68.37℃,9.35,6000U/g,6.25%and6h.Under this condition, the scavenging rate of DPPH reached70.32.%.2. Study on separation and purification of scallop skirt antioxidant peptides. Enzyme solutionusually contains many impurities, such as unhydrolysed scallop skirt protein and degenerated protease.Ultrafiltration is a commonly used separation method in enzyme solution. The enzyme solution was firstfiltered through0.45μm membrane and then with membrane flux as an index. The hydrolysate waslyophilized, prepared for fractionation using UF membrane (10kDa,5kDa,3kDa, and1kDa). Allfractions recovered were lyophilized and named as SSPH-Ⅰ(<1kDa),SSPH-Ⅱ(1kDa~3kDa),SSPH-Ⅲ(3kDa~5kDa),SSPH-Ⅳ(5kDa~10kDa),SSPH-Ⅴ(>10kDa) and SSPH-Ⅵ(parenthydrolysates). The antioxidant activities of these peptide fractions were well investigated by assaying the activities of the DPPH radical scavenging, the hydroxyl radical scavenging. Results showed thatfraction SSPH-Ⅱ(1kDa~3kDa)demonstrated the most significant antioxidant actives, its DPPH radicalscavenging activity reached94.16%, the hydroxyl radical scavenging activity reached31.20%.The second, the fraction SSPH-Ⅱ(1kDa~3kDa)was separated using Sephadex G-25gelchromatography separation. The best Sephadex G-25gel chromatography separation conditions ofantioxidant peptides were determined: eluted with deionized water,1mL/min elution speed,0.25mLsample,50mg/mL of sample concentration. Each fraction collected at a volume of2ml was monitoredat280nm. The scallop skirt peptides could be separated into two peaks, among which the frist peak (F1)had the highest antioxidant actives. And its DPPH radical scavenging activity reached34.10%, DPPHradical scavenging activity of F2reached16.85%.The third, the peak F1was isolated 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. elution was as follows:A:B=1:9; Column SyncronisaQ (250mm×4.6mm,5μm); column temperature:30℃; the flow rate:0.6.mL/min; injection volume:15μL; the detection wavelength280nm. a series peaks of SSPH-Ⅱ-F1-P were gived. After separationand purification, SSPH-Ⅱ-F1-P7was analyzed by mass spectrometry.The fourth, SSPH-Ⅱ-F1-P7was measured by online purification liquidchromatography-quadrupole Orbitrap tandem mass spectrometer (Q Exative). SSPH-Ⅱ-F1-P7was amixture of seven kinds of polypeptides, the weight of these polypeptides between1000~1700Da,consistent with the molecular weight of SSPH-Ⅱ (1kDa~3kDa).