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 SO₄^2-) 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 SO₄^2- partially alleviated this inhibition. All the ions altered the kinetic parameters. Compared with Cl^- salts, SO₄^2- 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.