Department of Food Science, University of Guelph, Ontario, Canada.
J Dairy Sci. 2010 Mar;93(3):893-900. doi: 10.3168/jds.2009-2820.
The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).
本工作旨在改进一种现有的方法，使用离子交换色谱法从含有乳清蛋白的牛奶样品中分离和定量 4 种主要的酪蛋白。分离过程使用迷你制备型阳离子交换柱（柱体积为 1 或 5mL），在 pH 3.5 时使用乙酸盐作为洗脱缓冲液，并用 NaCl 梯度洗脱。所有 4 种主要的酪蛋白均被分离，使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）评估每个峰的纯度。还将纯化的酪蛋白级分添加到生牛乳中，以确认它们的洗脱体积。使用缓冲液中的纯化酪蛋白以及直接添加到新鲜脱脂乳中进行定量。该方法也可用于确定凝乳酶酶解过程中κ-酪蛋白的减少和酪蛋白-大分子肽的释放。在这种情况下，与传统的大分子肽分离方法（使用反相高效液相色谱法（RP-HPLC））相比，使用该方法的主要优点是无需有机溶剂。
