酶解鸭蛋清制备抗氧化肽的研究

Preparation on antioxidant peptides from duck egg white by proteases

为制备鸭蛋清抗氧化活性肽,采用中性蛋白酶、碱性蛋白酶、木瓜蛋白酶和风味蛋白酶4种酶对新鲜鸭蛋蛋清进行水解,并用葡聚糖凝胶柱(Sephadex G)进行初步分离纯化。以水解度和抗氧化能力为指标,筛选得出木瓜蛋白酶为水解鸭蛋清制备抗氧化肽的最适用酶,其水解最适条件为pH9.5、温度50℃、底物浓度25%、酶底比(w/w)8%、时间4h,水解度达到29.01%,其水解液对·OH清除率为52.44%,对O2-·清除率为72.46%。木瓜蛋白酶水解液经阴、阳离子树脂脱盐后,脱盐率为57.45%;后采用Sephadex G-25凝胶过滤层析,通过测定各洗脱峰的抗氧化活性,分离得出在洗脱时间为42min时的峰组分(峰2)为抗氧化活性最高的肽,其对·OH清除率为77.46%,对O2-·清除率为70.18%;此活性肽分子量集中在1800u,其相对含量达到93.8%,而分子量在4000u以上的肽含量相对较少仅为6.2%,氨基酸组成分析表明,其中Glu和Leu两种氨基酸含量最为丰富,摩尔分数分别为12.11%和11.82%,且人体必需的氨基酸(Lys、Phe、Met、Thr、Ile、Leu、Val)含量占抗氧化肽的31.23%,表明所分离制备的抗氧化肽不仅具有较高的生物学活性,同时也具有较高的营养价值。

To preparation on the antioxidant activity of peptide from duck egg white, four proteases （neutrase, alkaline, papain, flavourzyme protease） were used to hydrolyze duck egg white and had preliminary filtration chromatography with Sephadex G. As an indicator of the degree of hydrolysis （DH） and antioxidant capacity, the results shows that the papain was the most suitable enzymatic preparation of the antioxidant activity of peptide. The best reaction conditions was as follows： pH9.5, reaction temperature 50 %, substrate concentration 25%, enzyme/substrate （w/w） 8%, time 4 h and DH was 29.01%. The clearance rate of hydrolyzate to the .OH was 52.44% and O2 was 72.46%. After desalting of papain hydrolyzate by anion and cation resin, the desalination rate was 57.45%. Using Sephadex G-25 gel filtration chromatography and measuring the elution peak antioxidant activity, the peak component （peak 2） at 42 min was the highest activity of antioxidant peptide which the clearance rate to the .OH was77.46% and O2 was 70.18%, the molecular weight of the active peptide is concentrated in about 1800 u, the relative content of 93.8%, and the molecular weight peptide content over 4000 u relatively small only 6.2%. Amino acid composition analysis showed that the most abundant in the two amino acids Glu and Leu mole fraction were 12.11% and 11.82%, and content of essential amino acids （Lys, Phe, Met, Thr, lie, Leu, Val） accounted for 31.23% of the antioxidant peptide. These showed that the antioxidant peptide which isolated prepared not only has a higher biological activity, but also has a higher nutritional value.