Optimization of low-abundance protein extraction and abundant protein removal from defatted soybean meal

Abstract

The aim of this study was to optimize the conditions for the extraction of low-abundance proteins (LAPs) and the removal of abundant proteins (APs; β-conglycinin and glycinin) from soybean meal. Single factor and orthogonal experiments were designed to determine the effects of four factors (isopropanol concentration, total extraction time, ultrasonic power, and ultrasonic time) on protein concentration in isopropanol extracts. Proteins in the isopropanol supernatant and the cold acetone precipitate of isopropanol were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The results showed that the optimal conditions were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W, and a total extraction time of 1 h. Under these conditions, the protein concentration in the isopropanol extracts reached 0.8081 g/L. Many LAPs were detected, including β-amylase, soybean agglutinin, soybean trypsin inhibitor, fumarylacetoacetase-like, phospholipase D alpha 1-like, oleosin, and even some unknown soybean proteins. The soybean APs (β-conglycinin and glycinin) were not found. The method may be useful for discovering new soybean proteins and extracting enough LAPs of soybean to allow further studies of their physiological effects on animals without the influence of APs.

摘要

目 的

优化低温脱脂大豆粕中的低丰度蛋白提取和高丰度蛋白去除条件, 为进一步探讨大豆低丰度蛋白对动物的生理功能影响提供试验材料。

创新点

结合提取时间和异丙醇浓度, 将超声波 (超声时间、 功率) 用于辅助异丙醇去除大豆高丰度蛋白和富集低丰度蛋白的研究。

方 法

在单因素试验基础上, 设计了超声时间、 超声功率、 提取时间和异丙醇浓度等四因素三水平的正交试验。 并通过测定提取液中的蛋白浓度和通过聚丙烯酰胺凝胶电泳 (SDS-PAGE) 、 质谱分析 (MS) 等蛋白质组学手段鉴定提取液或丙酮沉淀物中的蛋白质含量, 实现了大豆高丰度蛋白去除和低丰度蛋白提取条件的优化。

结 论

50% 异丙醇, 350 W 超声 15 min, 提取1 h 是大豆低丰度蛋白提取的最佳条件。 提取物含多种低丰度蛋白 (油质蛋白、 大豆未知蛋白、 磷酸酯D和无特征大豆蛋白) , 且无大豆高丰度蛋白 (大豆球蛋白和β-伴大豆球蛋白) 。