中国农业科学 ›› 2016, Vol. 49 ›› Issue (12): 2379-2388.doi: 10.3864/j.issn.0578-1752.2016.12.013

• 贮藏·保鲜·加工 • 上一篇    下一篇

从麦胚清蛋白分离制备高活性抗氧化肽

陈思远,刘永祥,曹小舟,张逸婧,陈海娟,沈新春   

  1. 南京财经大学食品科学与工程学院/江苏省现代粮食流通与安全协同创新中心/江苏高校粮油质量安全控制及深加工重点实验室,南京 210023
  • 收稿日期:2015-11-03 出版日期:2016-06-16 发布日期:2016-06-16
  • 通讯作者: 沈新春,Tel:13675121836;E-mail:shenxinchun@njue.edu.cn
  • 作者简介:陈思远,E-mail:824170843@qq.com
  • 基金资助:
    国家自然科学基金(31271983)、江苏省高校自然科学研究重大项目(14KJA550002)、2014年度江苏省高校第四期“333工程”资助科研项目、2015年度江苏省高校优秀科技创新团队项目、江苏高校优势学科建设工程资助项目

The Preparation Process for Isolation of a Highly Active Antioxidant Peptide Derived from Wheat Germ Albumin

CHEN Si-yuan, LIU Yong-xiang, CAO Xiao-zhou, ZHANG Yi-jing, CHEN Hai-juan, SHEN Xin-chun   

  1. College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing 210023
  • Received:2015-11-03 Online:2016-06-16 Published:2016-06-16

摘要: 【目的】探究能有效地从小麦胚芽清蛋白二步双酶酶解物中分离得到高活性抗氧化肽的工艺。【方法】以小麦胚芽清蛋白为原料,DPPH自由基清除率和Fe2+螯合能力为抗氧化活性的评价指标,通过胰蛋白酶进行酶解,依次采用超滤膜和凝胶过滤色谱(Sephadex G-75)对酶解产物进行分离纯化,筛选出活性较高的组分;采用碱性蛋白酶对获得的活性较高组分进行酶解,通过凝胶过滤色谱(Sephadex G-15)和反相高效液相色谱(RP-HPLC)分离纯化其酶解产物;采用质谱技术(ESI-TOF MS/MS)对抗氧化活性最高的组分进行结构鉴定。【结果】分离得到3个活性组分(峰)PaPb、Pc,采用DPPH法和Fe2+螯合能力测定法测定其抗氧化活性的结果都显示,在3个组分中,提取率为23.1%的组分Pb抗氧化活性最强(P0.05),因此选取组分Pb进行下一步碱性蛋白酶酶解。该酶解物经Sephadex G-15分离后得到Pd和Pe两个组分(峰),经抗氧化活性测定,选取提取率为52.1%的高抗氧化活性组分PdP0.05)进一步通过RP-HPLC进行疏水性分离,得到P1、P2、P3、P4、P5五个活性组分(峰),其中提取率为7.3%的组分P3的抗氧化活性最强(P0.05),其DPPH自由基清除率和Fe2+螯合能力的EC50值分别为1 mg·mL-1、0.6 mg·mL-1。最后通过质谱技术(ESI-TOF MS/MS)鉴定组分P3的结构,得到其氨基酸序列为AREGETVVPG,分子量为1 013.51 Da,纯度约为85%,与用化学方法合成的多肽AREGETVVPG(纯度95%以上)的抗氧化活性相比,结果没有显著差异(P>0.05)。【结论】二步双酶酶解结合超滤膜、凝胶过滤色谱和RP-HPLC等蛋白分离纯化技术为直接得到单一的高活性抗氧化肽提供了技术参考。

关键词: 小麦胚芽, 清蛋白, 二步双酶酶解, 分离纯化, 抗氧化肽

Abstract: 【Objective】 The aim of this study is to develop an effective preparation process for obtaining highly active antioxidant peptide derived from wheat germ albumin using two-step dual-enzymatic hydrolysis. 【Method】 A two-step dual-enzymatic hydrolysis and separation were employed to screen the antioxidant peptide (AOP) from wheat germ albumin. The in vitro AOP’s antioxidant activity was evaluated by DPPH radical scavenging activity and Fe2+ chelating ability assays. The hydrolysate derived from tryptic hydrolysis of wheat germ albumin was fractionated by ultrafiltration according to molecular weight; then the fractions with high antioxidant capacity were further isolated by gel filtration chromatography (Sephadex G-75). After that, the fraction with higher antioxidant activity was further hydrolyzed by an alcalase, and the hydrolysate was subjected to purification by Sephadex G-15 gel filtration chromatography and reversed-phase HPLC (RP-HPLC), respectively. The highest antioxidant fraction was achieved and the structure was identified by ESI-TOF MS/MS. 【Result】 Hydrolysate derived from tryptic hydrolysis of wheat germ albumin was fractionated by ultrafiltration according to molecular weight. Three fractions, Pa, Pb and Pc were obtained from the fraction with high antioxidant capacity. The fraction Pb showed the strongest antioxidant activity evaluated by DPPH radical scavenging activity and Fe2+ chelating ability assays and its extraction yield was 23.1%. The fraction Pb which had the highest antioxidant activity was further hydrolyzed by an alcalase, and the hydrolysate was subjected to gel filtration chromatography (Sephadex G-15) and two more groups, Pd and Pe were obtained. The extraction yield of fraction Pd which had higher antioxidant capacity was 52.1%. After further purification by RP-HPLC, five fractions, P1, P2, P3, P4 and P5 were separated from the fraction Pd, and the fraction P3 exhibited the highest antioxidant activity with the in vitro EC50 values of DPPH radical scavenging activity at 1 mg/ml and Fe2+ chelating ability at 0.6 mg/ml, respectively. The extraction yield of the fraction P3 was 7.3% in RP-HPLC step. Finally, employing the electrospray ionization-time-of-flight mass spectrometer (ESI-TOF MS/MS), the structure of the fraction P3 was identified. Its amino acids sequence, molecular weight and purity were AREGETVVPG, 1013.51Da and 85%, respectively. Consistently, the antioxidant capacity of the synthetic peptide with the same sequences ( purity ≥95%) showed the similar activity to the fraction P3 (P>0.05). 【Conclusion】 The preparation process of a two-step dual-enzymatic hydrolysis in combination with ultrafiltration, gel filtration chromatography, and RP-HPLC was effective in separation, screen and purification of a highly active antioxidant peptide.

Key words: wheat germ, albumin, a two-step dual-enzymatic hydrolysis, separation and purification, antioxidant peptide (AOP)