Preparation And Evaluation Of Bioactive Peptide Fraction Of Mustelus Muscle Protein

In the present experiment, the antioxidative hydrolysates from Mytilus edulis and Mytiluscoruscus muscle proteins were prepared by proteinase using single-factor and orthogonal tests. Thehydrolysates subsequently separated and purified by ultrafiltration (UF) memebrane, DEAE-52celluloseanion-exchange chromatography, Sephadex G-25gel filtration column and Reversed Phase HighPerformance Liquid Chromatography (RP-HPLC), and one new antioxidative peptide (MNH) wasisolated from the hydrolysate and and its molecular mass and N-terminal amino-acid sequencewas determined by using ESI-MS and Procise Protein/Peptide Sequencer. The antioxidantand angiogenesis inhibitory activities of hydrolysates, their fractions and purified peptidewere evaluated.Inspired by OH scavenging assay, the efficient methods had been developed toacquire protein hydrolysates (MCNH and MEAH) from M. coruscus and M. edulis bysingle-factor and orthogonal tests, respectively. Under optimal hydrolysis parameters ofneutral protease (hydrolysis time3h, the hydrolysis temperature60℃, the solid-liquidratio1:2, enzyme dose3%), the OH scavenging rate of MCNH reached66.82%at theconcentration of10mg/mL. The OH scavenging rate of MEAH reached80.97%at theconcentration of10mg/mL under the optimum conditions of temperature45℃, hydrolysistime3h, pH8.0, the enzyme dose3%, solid-liquid ratio1:1for alcalase.Based on the molecular weight (MW), MCNH, MEAH, MEPH (the hydrolysate fromM. edulis by papain) and MENH (the hydrolysate from M. edulis by neutral protease) werefractionated by ultrafiltration membrane system. The·OH scavenging rate of fractions atconcentration of10mg/mL were measured, and the selected fractions (MCNH-1,MEAH-1,MEPH-1, MENH-2) with higher antioxidant activities were separated through DEAE-52 cellulose anion-exchange chromatography and gel filtration chromatography, and the antioxidantactivities of resulted subfractions were evaluated. MENH-3was purified through aSephadex G-15gel filtration and RP-HPLC on Zorbax C18, and a new peptide (MNH) withmolecular weight of574.68Da was obtained. The hydroxyl radical scavenging ratesshowed dose dependency and reached23.49%at the concentration of5mg/mL.The fractions with higher antioxidative activities, including crude protein (MCNH-1,MEAH-1, MEPH-1, MENH-2, MENH-3), fractions through ion chromatography(MCNH-1-1, MEAH-1-4, MEPH-1-6-2-1), fractions through gel chromatography(MCNH-1-1-2the MEAH-1-4-1, MEPH-1-6-1, MENH-2-1-1MENH-3-3) and MNH, werecarried on inhibiting formation of the blood vessels of the chick embryo chorioallantoicmembrane (CAM). The results indicated that all of the samples showed strength activitieson angiogenesis inhibitory, especially the fractions obtained from MEAH, which wasslightly better than the other protease hydrolysates.