Effect of pH, NaCl, CaCl 2 and Temperature on Self-Assembly of β-Lactoglobulin into Nanofibrils: A Central Composite Design Study

The ability of certain globular proteins to self-assemble into amyloid-like fibrils in vitro opens opportunities for the development of new biomaterials with unique functional properties, like highly efficient gelation and viscosity enhancement. This work explored the individual and interacting effects of pH (1 to 3), NaCl (0-100 mM), CaCl(2) (0-80 mM) and heating temperature (80 to 120 °C) on the kinetics of β-lactoglobulin self-assembly and the morphology of resulting nanofibrils. Statistically significant (p < 0.05) interactions included CaCl(2)*temperature, NaCl*pH, CaCl(2)*pH, temperature*pH and NaCl*CaCl(2). Particularly notable was the very rapid self-assembly at pH 3 and the highly nonlinear effect of pH on self-assembly kinetics. Nanofibril morphologies ranged from long and semiflexible or curled and twisted to short and irregular. There did not seem to be a link between the kinetics of fibril formation and the morphology of fibrils, except at pH 3, where self-assembly was very rapid and fibrils were short and irregular, suggesting haphazard, uncontrolled self-assembly.