核桃球蛋白的分离纯化、鉴定及抗氧化性分析

Isolation, Purification, Identification and Oxidation Analysis of Walnut Globulin

为揭示核桃球蛋白主要组成及体外抗氧化活性,以脱脂核桃粕为原料,对其中的球蛋白进行分离纯化并通过2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS)阳离子自由基清除能力对其抗氧化能力进行评价,即经进一步脱脂后,根据Osborne分级法提取得到核桃球蛋白粗提物,再通过HiTrapCaptoQ阴离子交换柱和Sephacryl TM S 100HR凝胶过滤柱两步分离纯化,得到主要蛋白组分。结果表明,制备的球蛋白蛋白含量为51.53%,占核桃总蛋白的15.3%,等电点为3.8。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示,纯化得到主要蛋白组分分子量为10.1 kDa。经高效液相色谱-质谱联用分析表明,该主要蛋白组分在活性状态下由一种7S球蛋白vicilin Car i 2.0101和2S清蛋白组成。研究发现,球蛋白粗提物和纯化蛋白组分均对ABTS+自由基有较好的清除能力,其IC50值分别为0.302和0.075 mg/mL,表明纯化蛋白组分的清除能力提高了4倍以上。研究结果可为核桃球蛋白开发利用提供理论基础。

In order to reveal the main composition of walnut globulin and its antioxidant activity in vitro, defatted walnut meal was used as raw material to separate and purify the globulin. And 2,2’-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS) cationic radical scavenging ability was used to evaluate its antioxidant capacity, after further defatting, the crude walnut globulin extract was extracted according to the Osborne fractionation method, and then the main protein components were separated and purified by HiTrapCaptoQ anion exchange column and SephacrylS 100HR gel filtration column. The results showed that the prepared globulin protein content was 51.53%, accounting for 15.3% of the walnut protein, and the isoelectric point was 3.8. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the purified main protein component was 10.1 kDa. The major protein component in the active state consists of a 7S globulin vicilin Car i 2.0101 and2S albumin. It was found that both crude globulin extract and purified protein component had good scavenging ability against ABTS+·, with IC50 values of 0.302 and 0.075 mg/mL, respectively, indicating that the scavenging ability of purified protein component was increased by more than 4 times. The research results could provide a theoretical basis for the development and utilization of walnut globulin.