【摘要】 利用一种工业用碱性蛋白酶对新鲜梅花鹿鹿茸进行水解,制备得到生物活性肽.首先,考察了酶用量、底物浓度和反应时间对水解反应的影响,确定了最佳水解条件为酶与底物质量比1:150,底物与溶剂质量比1:13,水解时间60min.其次,利用硫酸胺分级沉淀法制备了鹿茸多肽粗提液,同时采用缓冲液保护其活性,再经SephadexG-25凝胶层析柱进行分离纯化,得到了具有最强抗氧化活性的鹿茸肽组分(VAP-B).最后,利用制备型高效液相色谱柱对VAP-B进一步纯化,得到了分子量在800以内的小肽活性组分(VAP-B1),其蛋白含量为70.45%.在此基础上,对小肽活性组分VAP-B1进行了理化性质分析,检测其抗氧化活性,包括清除超氧阴离子、羟自由基以及抗脂质过氧化的能力和还原能力.实验结果表明,当鹿茸肽VAP-B1浓度为20mg/mL时,对邻苯三酚自氧化的抑制率达到91.9%,有明显的清除超氧阴离子的作用;当浓度为10mg/mL时,对羟基自由基的清除作用达到100%,且在一定的浓度范围内呈剂量依赖关系.此外,鹿茸肽VAP-B1还具有一定的防止脂质过氧化和还原的作用.更多还原

【Abstract】 An industrial alkaline protease alcalase was used to hydrolyze the fresh velvet antler of sika deer(Cervus Nippon Temminck).The conditions of enzymatic hydrolysis were optimized.The effects of several factors,including amount of the enzyme,substrate concentration and hydrolysis time,on the antioxidative activities of the hydrolysis product were examined by orthogonal test design.The optimum hydrolysis conditions were established to be the amount of enzyme,1:150;the substrate concentration,1:13;hydrolysis time,60 min.The crude velvet antlers peptides were prepared by fractionation of ammonium sulphate.An elution peak with high antioxidative activities from Sephadex G-25 column chromatography of the crude velvet antlers peptides was collected and named as VAP-B.Finally,a further purified velvet antlers peptide component(VAP-B1) with molecular weight distribution from 200 to 800 was obtained from VAP-B by preparative HPLC.The protein content of VAP-B1 was 70.45%.An analysis of amino acid composition was also carried out.The antioxidative activities of velvet antlers peptide component(VAP-B1),including removal of superoxide anions,hydroxyl radicals and inhibition of lipid peroxidation were examined.The reducing power of VAP-B1 was also tested.The experimental results indicated that when VAP-B1 concentration was 20 mg/mL,the inhibition ratio to pyrogallic acid autoxidation reached to 91.9%,having a strong ability to scavenge superoxide anion;when VAP-B1 concentration was 10 mg/mL,the clearance rate to hydroxyl radical was 100% and was dosage-dependent in a certain rage of concentration.At the same time,VAP-B1 showed some capability of inhibition of lipid peroxidation and reducing power.更多还原