Effect of calcium on the reversible and irreversible thermal denaturation pathway of β-lactoglobulin

Thermal denaturation of β-lactoglobulin (BLG), boosted in the presence of calcium ions, is responsible for the dairy fouling issue in milk processing. However, the reported kinetic model concerning BLG denaturation was generally based on a simplified one-step reaction model where the unfolding of the protein was considered as a kinetically driven reaction. In such a model, the presence of break-slope phenomenon in the Arrhenius plot was justified by the fact that unfolding step constitutes a rate-limited step compared to aggregation at low temperatures. Nevertheless, there is accumulated experimental evidence implying that the unfolding step is much faster than aggregation regardless of the temperature. Therefore, in this paper, it is proposed that the thermal unfolding of BLG is thermodynamically controlled such that a chemical equilibrium between native and unfolded BLG species can be instantaneously established, followed by an irreversible aggregation of unfolded BLG. This novel approach allows to mathematically interprets the break-slope behavior in the Arrhenius plot. The developed model was applied to investigate the effect of Ca2+ on the unfolding equilibrium and subsequent aggregation process of BLG in whey protein solutions. Ca2+ was confirmed to have a protective role on the unfolding equilibrium of BLG by increasing the enthalpy ΔHu and decreasing the equilibrium constant Kc, while it facilitated the aggregation of BLG by showing a reduction of the corresponding enthalpy and entropy which cannot be seen as reported using one-step reaction model. This approach allows us to be closer to reality when mimicking the heat-induced appearance/disappearance of different BLG species, including native, unfolded and aggregated ones. Accurate population balance of these foulant species during thermal processing could lead to new perspectives on fouling prediction.