Linking enzymatic hydrolysis to structural, volumetric, and hydrodynamic evolution of β-lactoglobulin in solution

This paper presents an integrated real-time framework linking α-chymotrypsin hydrolysis of β-lactoglobulin to simultaneous changes in structural, volumetric and hydrodynamic properties in buffered media. High-resolution ultrasonic spectroscopy, complemented with densitometry, enabled real-time monitoring of peptide bond cleavage and changes in protein volume and compressibility. Multi-frequency ultrasonic relaxation analysis yielded molar adiabatic relaxation compressibility values of 2-4 × 10^-14 m³ mol^-1 Pa^-1, previously not reported for the β-lactoglobulin system. Circular dichroism and intrinsic tryptophan fluorescence spectroscopy confirmed progressive disruption of secondary structure and exposure of the hydrophobic core, respectively. Light scattering method and viscometry revealed corresponding changes in hydrodynamic properties. Proteolysis in phosphate-buffered media produced a slight viscosity increase despite extensive fragmentation, consistent with an expansion from compact globules to coil-like fragments, potentially involving phosphate-protein interactions. This integrated approach offers a quantitative basis for characterizing enzymatic hydrolysis and guiding the design of functional protein ingredients.