Mechanistic insights and chain-length-related effects of polyphosphate-mediated suppression of heat-induced ovalbumin aggregation.

Protein aggregation is a major challenge that compromises the quality and stability of food science, biopharmaceuticals, and other biotechnological products. Although various additives have been explored, current aggregation-suppressor technologies remain insufficient, and their molecular mechanisms remain unclear. The aims of this study were to clarify the aggregation-suppressing effect of inorganic polyphosphate (PolyP) on ovalbumin (OVA) and provide mechanistic insights and chain-length related effects. We quantified the PolyP-OVA interactions, thermal stability, and colloidal behavior using isothermal titration calorimetry, differential scanning fluorimetry, differential scanning calorimetry, zeta potential analysis, second virial coefficient, and diffusion interaction parameter measurements. Isothermal titration calorimetry demonstrated that PolyP binds to OVA with favorable thermodynamic contributions dominated by entropy. Differential scanning calorimetry and differential scanning fluorimetry showed that PolyP increased the apparent melting temperature of OVA by approximately 10 °C, indicating enhanced resistance to thermal unfolding. Analyses of the zeta potential, second virial coefficient, and diffusion interaction parameters suggested a shift toward more repulsion-dominant intermolecular interactions, accompanied by reduced heat-induced turbidity and suppression of micron-sized aggregate formation. PolyP suppressed turbidity and micron-sized aggregate formation, likely through the combined effects of enhanced resistance to unfolding and modulation of intermolecular interactions, with longer-chain PolyP exhibiting stronger effects within the tested concentration range. This study provides mechanistic insights into PolyP-mediated stabilization of a model protein system.