双酶直接酶解米糠制备短肽的工艺优化

Study on Optimal Hydrolysis Process for Preparing Rice Bran Short Peptides with Two Enzymes

【目的】建立在中低温度下直接酶解米糠制备短肽的优化工艺。【方法】采用二次回归正交旋转组合设计优化直接酶解米糠制备短肽的工艺条件,其中总糖含量测定采用蒽酮比色法,水解度(degree of hydrolysis,DH)采用pH-stat法,蛋白质回收率采用凯氏定氮法。【结果】确定直接酶解米糠制备短肽最佳工艺条件为:米糠先经糖酶(viscozyme)反应2h去除糖类杂质,然后用碱性蛋白酶(alcalase)和胰蛋白酶双酶水解,最适pH8.2,温度45℃,alcalase与胰蛋白酶酶活比59﹕41,总酶活5750U/g底物,水解时间3h。在此条件下,DH为23.04%,蛋白质回收率为84.33%,酸溶性多肽(TCA-SN)为68.40%,短肽分子量主要集中在1000D以下。【结论】在中低温和偏中性(pH8.2)条件下,采用碱性蛋白酶和胰蛋白酶双酶直接酶解米糠制备短肽,与先提取蛋白后酶解制备短肽的方法相比,具有操作步骤简单、蛋白质利用率高等特点,是一种制备米糠肽的新途径。

[ Objective] To establish an optimal enzymatic hydrolysis process for short peptides directly from rice bran under low and medium temperature. [Method] Quadratic regression orthogonal rotational combing design was used to determine the optimal enzymatic hydrolysis process. Anthrone colorimetry＇s method was used to measure the carbohydrates content, pH-stat method to measure the degree of hydrolysis and Kjeldahl＇s method to measure the recovery of protein. [Result] The optimum enzymatic hydrolysis conditions for short peptides were： reaction time of viscozyme for removing carbohydrate was 2 hours firstly, then protease hydrolysis with alcalase and trypsin was carried out at pH 8.2, temperature 45℃, the activity ratio between alcalase and trypsin was 59 ： 41, the total enzyme activity was 5 750 U/g substrate, and 3 hours. Under such conditions the DH was 23.04%, the recovery of protein could reach up to 84.33%, TCA-SN was 68.40%, and the molecular weight of peptides was mainly concentrated at below 1 000 D. [ Conclusion ] It is a better approach with convenient operation and high recovery of protein for preparing rice bran short peptides compared to traditional ways of extracting protein first and then preparing short peptides with enzymatic hydrolysis.