Haller Nicole, Maier Isabel, Kulozik Ulrich
Chair of Food and Bioprocess Engineering, TUM School of Life Sciences, Technical University Munich, Weihenstephaner Berg 1, 85354 Freising, Germany.
Foods. 2021 Sep 20;10(9):2231. doi: 10.3390/foods10092231.
Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, α-lactalbumin (α-la) and β-lactoglobulin (β-lg). By choosing appropriate environmental conditions the thermal precipitation of either α-la or β-lg can be induced. Whereas β-lg irreversibly aggregates, the precipitated α-la can be resolubilized by a subsequent adjustment of the solution's pH and the ionic composition. This study reports on the analytical characterization of resolubilized α-la compared to its native counterpart as a reference in order to assess whether the resolubilized α-la can be considered close to 'native'. Turbidity and quantification by RP-HPLC of the resolubilized α-la solutions were used as a measure of solubility in aqueous environment. RP-HPLC was also applied to determine the elution time as a measure for protein's hydrophobicity. DSC measurement was performed to determine the denaturation peak temperature of resolubilized α-la. FTIR spectroscopy provided insights in the secondary structure. The refolding of α-la achieved best results using pH 8.0 and a 3-fold stoichiometric amount of Ca per α-la molecule. The results showed that the mechanism of aggregation induced by gentle thermal treatment under acidic conditions with subsequent mechanical separation is reversible to a certain extent, however, the exact native conformation was not restored.
选择性热沉淀后接机械分离步骤是一种已被充分描述的用于分离主要乳清蛋白α-乳白蛋白（α-la）和β-乳球蛋白（β-lg）的方法。通过选择合适的环境条件，可以诱导α-la或β-lg发生热沉淀。β-lg会不可逆地聚集，而沉淀的α-la可以通过随后调节溶液的pH值和离子组成来重新溶解。本研究报告了与天然α-la作为参考相比，重新溶解的α-la的分析表征，以评估重新溶解的α-la是否可被视为接近“天然”状态。重新溶解的α-la溶液的浊度和RP-HPLC定量被用作衡量其在水性环境中溶解度的指标。RP-HPLC还用于确定洗脱时间，作为蛋白质疏水性的衡量指标。进行DSC测量以确定重新溶解的α-la的变性峰温度。FTIR光谱提供了二级结构方面的见解。使用pH 8.0和每个α-la分子3倍化学计量的钙，α-la的重折叠取得了最佳结果。结果表明，在酸性条件下进行温和热处理并随后进行机械分离所诱导的聚集机制在一定程度上是可逆的，然而，并未恢复到确切的天然构象。
