Preparation, Binding Behavior and Molecular Simulation of Binary Complexes of Phloridzin with Whey Protein Isolate

Whey protein isolate (WPI) can assemble into supramolecular complexes with flavonoids via non-covalent interactions, although the underlying binding mechanisms remain not fully understood. In this work, the formation mechanism of the WPI–phloridzin (PHL) complex was systematically investigated using an integrated experimental and computational approach. High-performance liquid chromatography quantified the binding content of PHL as 1.3% (w/w). Isothermal titration calorimetry indicated that the process was entropy-driven and governed predominantly by hydrophobic and electrostatic interactions. Complementary circular dichroism spectroscopy and molecular dynamics simulations revealed that complexation induces modest conformational adjustments in the protein’s secondary structure. Collectively, this multi-scale analysis provides mechanistic insights into the dynamic formation of the WPI–PHL complex, offering theoretical insights into protein–flavonoid recognition.