考马斯亮蓝法测定苹果组织微量可溶性蛋白含量的条件优化

Optimization of Experimental Conditions for the Determination of Water Soluble Protein in Apple Pulp Using Coomassie Brilliant Blue Method

以红星苹果果肉为试材,探讨不同提取缓冲液(磷酸缓冲液、Tris-HCl缓冲液和水)、缓冲液pH值、浓度、外源添加物和料液比等因素对苹果果肉可溶性蛋白提取效率的影响。结果表明:外源添加PVP和EDTA以及料液比是苹果可溶性蛋白提取率的主要影响因素。实验最终确定的提取条件为0.1mol/L pH9.0 Tris-HCl提取缓冲液(内含1mmol/L EDTA和1%PVP)、料液比1:25,此条件下苹果果实可溶性蛋白的有效测定含量为0.544%,为水提组(0.073%)的7.45倍。

The effects of extraction buffer type, pH and concentration, exogenous additive type and solid-to-solvent ratio on the extraction efficiency of soluble protein from Red Star apples were studied with the aim of developing a sample pretreatment method for the determination of water soluble protein in Red Star apples. Exogenous PVP and EDTA and solid-to-solvent ratio were found to be main factors that influence extraction efficiency. The best results were obtained using 0.1 mol/L pH 9.0 Tris- HCl buffer containing 1 mmol/L EDTA and 1% PVP as an extraction solvent at a solid-to-solvent ratio of 1：25 （g/mL）. Under these conditions, the content of water soluble protein in Red Star apples was 0.544% as determined using Coomassie brilliant blue method, a 7.45-fold increase as opposed to 0.073% with pure water extraction.