Role of Anion Identity in the Assembly and Morphology of Whey Protein Isolate Nanofibril Aggregates

To investigate the respective contributions of cations (Na+, K+, Zn2+, Fe3+) and anions (Cl− and SO42−) to the formation of whey protein isolate (WPI)-based nanofibrils, eight salts with a 4 × 2 factorial design were added to WPI solutions. The morphology and aggregation process of the fibril aggregates were examined under fixed low salt concentration (10 mmol/L) to isolate ion-specific effects. The salts altered the pH, conductivity, and fibril yield. Notably, the Na+, K+, and Zn2+ salts increased fibril production, whereas Fe3+ salts reduced it. Mechanistically, Fe3+ strongly suppressed fibrillation via strong electrostatic interaction and accelerated protein hydrolysis, while SO42− partially alleviated this inhibition. All the ions altered the kinetic parameters. Compared with Cl− salts, SO42− salts induced shorter, clustered fibrils and stronger kinetic suppression, preserving elongated fibrils. X-ray photoelectron spectroscopy (XPS) confirmed anion incorporation, and X-ray diffraction (XRD) revealed secondary structural changes. These results demonstrate that while cations contribute to fibril formation, anions play a deterministic role in regulating assembly kinetics and morphological outcomes, independent of cation valence. In this study, we establish a mechanistic basis for tailoring WPI fibril aggregation states through anion-specific salt selection.