Lopes Aline Soriano, Garcia Jerusa Simone, Catharino Rodrigo Ramos, Santos Leonardo Silva, Eberlin Marcos Nogueira, Arruda Marco Aurélio Zezzi
Universidade Estadual de Campinas, Institute of Chemistry, Spectrometry, Sample Preparation and Mechanization Group, P.O. Box 6154, 13084-862 Campinas, São Paulo, Brazil.
Anal Chim Acta. 2007 May 8;590(2):166-72. doi: 10.1016/j.aca.2007.03.043. Epub 2007 Mar 25.
This work describes the optimization of a cloud point extraction (CPE) method for casein proteins from cow milk samples. To promote phase separation, polyoxyethylene(8) isooctylphenyl ether (Triton X-114) and sodium chloride (NaCl) were used as nonionic surfactant and electrolyte, respectively. Using multivariate studies, four major CPE variables were evaluated: Triton X-114 concentration, sample volume, NaCl concentration, and pH. The results show that surfactant concentration and sample volume were the main variable affecting the CPE process, with the following optimized parameters: 1% (w/v) Triton X-114 concentration, 50 microL of sample volume, 6% (w/v) NaCl concentration and extractions carried out at pH 7.0. At these conditions, 923+/-66 and 67+/-2 microg mL(-1) of total protein were found in the surfactant-rich and surfactant-poor phases, respectively. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was then used to evaluate those target proteins (alpha(s1)-casein, alpha(s2)-casein and beta-casein) separation as well as to check the efficiency of the extraction procedure, making a fingerprint of those target proteins possible.
本研究描述了一种从牛奶样品中提取酪蛋白的浊点萃取（CPE）方法的优化。为促进相分离，分别使用聚氧乙烯（8）异辛基苯基醚（Triton X - 114）和氯化钠（NaCl）作为非离子表面活性剂和电解质。通过多变量研究，评估了四个主要的CPE变量：Triton X - 114浓度、样品体积、NaCl浓度和pH值。结果表明，表面活性剂浓度和样品体积是影响CPE过程的主要变量，优化参数如下：Triton X - 114浓度为1%（w/v），样品体积为50 μL，NaCl浓度为6%（w/v），在pH 7.0条件下进行萃取。在这些条件下，富含表面活性剂相和贫表面活性剂相中总蛋白含量分别为923±66和67±2 μg mL⁻¹。最后，使用基质辅助激光解吸电离飞行时间质谱（MALDI - TOF MS）评估那些目标蛋白（αs1 - 酪蛋白、αs2 - 酪蛋白和β - 酪蛋白）的分离情况，并检查萃取程序的效率，从而获得那些目标蛋白的指纹图谱成为可能。
