摘要
首先确定甲醇芽孢杆菌LB-1蛋白酶(蛋白酶LB-1)最适水解温度和pH值,然后通过持续监测水解pH值和氧化还原电位变化、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析、质谱检测等探究蛋白酶LB-1的蛋白水解特性,并测定其酪蛋白水解产物的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和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)阳离子自由基清除率、α-淀粉酶和α-葡萄糖苷酶抑制率、金属离子螯合率等生物活性。结果表明:蛋白酶LB-1的最适蛋白水解温度为52℃,最适pH 6.0~7.0;该酶对酪蛋白的水解作用显著强于乳清蛋白,水解程度为κ-酪蛋白>α-酪蛋白>β-酪蛋白;对κ-酪蛋白的水解位点主要位于Lys21-Ile22和Lys112-Asn113;酪蛋白水解产物对DPPH自由基和ABTS阳离子自由基最高清除率分别为75.4%、48.45%,对α-淀粉酶和α-葡萄糖苷酶的最高抑制率分别为80.89%、93.12%,对钙离子和锌离子的最高螯合率分别为63.13%、35.31%;表明该酶的酪蛋白水解产物具有一定的抗氧化能力、降血糖作用以及良好的金属离子螯合能力。因此,甲醇芽孢杆菌LB-1蛋白酶在功能性干酪加工和乳源活性肽开发方面具有潜在的应用价值。
In this study, we firstly determined the optimal hydrolysis temperature and pH of the protease from Bacillus methanolicus LB-1(protease LB-1), and then explored the proteolytic characteristics by continuously monitoring the changes in hydrolysis pH and oxidation-reduction potential(ORP) as well as using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and mass spectrometry. Bioactivities of casein hydrolysates obtained with the protease including scavenging effects on 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) cation radical, inhibition effects on α-amylase and α-glucosidase, and metal ion-chelating activity were determined. The results showed that the optimal hydrolysis temperature and pH of protease LB-1 were 52 ℃ and 6.0–7.0, respectively. Casein was more effectively hydrolyzed than whey protein by the protease, and the hydrolysis extent of individual caseins followed the decreasing order: κ-casein > α-casein > β-casein. The hydrolysis sites of κ-casein were mainly Lys21-Ile22 and Lys112-Asn113. The highest scavenging rates of DPPH and ABTS cation radicals by the casein hydrolysate were 75.4% and 48.45%, the highest inhibition rates of α-amylase and α-glucosidase were 80.89% and 93.12%, and the highest chelating rates of calcium and zinc ions were 63.13% and 35.31%, respectively, indicating that the casein hydrolysate had potent antioxidant capacity, hypoglycemic effect and metal ion-chelating ability. Therefore, the protease isolated from B. methanolicus LB-1 could be potentially used in functional cheese processing and the development of bioactive peptides of dairy origin.
作者
杨智杰
郑喆
蔡淼
赵笑
罗天淇
黎润坤
陈超
曹永强
杨贞耐
YANG Zhijie;ZHENG Zhe;CAI Miao;ZHAO Xiao;LUO Tianqi;LI Runkun;CHEN Chao;CAO Yongqiang;YANG Zhennai(Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Technology and Business University(BTBU),Beijing 100048,China;Dongjun Dairy(Yucheng)Co.Ltd.,Yucheng 251200,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2020年第14期153-160,共8页
Food Science
基金
“十三五”国家重点研发计划重点专项(2018YFC1604302-02)
国家自然科学基金面上项目(31871823)。
关键词
微生物蛋白酶
蛋白水解特性
水解位点
抗氧化
降血糖
金属螯合
microbial protease
proteolytic properties
hydrolysis site
antioxidant activity
antidiabetic activity
metalchelating ability
