Molecular Analytical Assessment of Thermally Precipitated α-Lactalbumin after Resolubilization

Abstract

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 Ca2+ 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.

World Allergy Organization (WAO) Diagnosis and Rationale for Action against Cow's Milk Allergy (DRACMA) Guidelines update - III - Cow's milk allergens and mechanisms triggering immune activation

“…When ALA is thermally precipitated during fractionation of whey proteins, its fold can almost be reconstituted by pH adjustment, while BLG irreversibly aggregates upon heat treatment, but not upon acidification. 58 However, extensive cooking above >72 C also destroys ALA.…”