Molecular Analytical Assessment of Thermally Precipitated alpha-Lactalbumin after Resolubilization

Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, -lactalbumin (-la) and -lactoglobulin (-lg). By choosing appropriate environmental conditions the thermal precipitation of either -la or -lg can be induced. Whereas -lg irreversibly aggregates, the precipitated -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 -la compared to its native counterpart as a reference in order to assess whether the resolubilized -la can be considered close to `native'. Turbidity and quantification by RP-HPLC of the resolubilized -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 -la. FTIR spectroscopy provided insights in the secondary structure. The refolding of -la achieved best results using pH 8.0 and a 3-fold stoichiometric amount of Ca2+ per -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.     «

Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, -lactalbumin (-la) and -lactoglobulin (-lg). By choosing appropriate environmental conditions the thermal precipitation of either -la or -lg can be induced. Whereas -lg irreversibly aggregates, the precipitated -la can be resolubilized by a subsequent adjustment of the solution's pH and the ionic composition. This study reports on the analytical charact...     »