Limited enzymatic hydrolysis-mediated assembly of whey protein isolate nanofibers: Influence of protease type and hydrolysis time on fiber formation and β-carotene delivery.

The rational design of whey protein-based nanocarriers for hydrophobic bioactives has been hindered by the lack of universal guidelines for limited enzymatic hydrolysis that transcend protease specificity. In this study, whey protein isolate (WPI) was subjected to limited hydrolysis (0-5 h, 5-80% degree of hydrolysis) using four proteases (pepsin, trypsin, papain, and neutral protease), followed by thermal incubation at pH 2.0 and 90 °C for 10 h to induce fibril assembly. The structural evolution of the resulting nanofibers was monitored via ThT fluorescence, TEM, zeta potential, surface hydrophobicity and FTIR, while encapsulation performance was evaluated using β-carotene as a model bioactive. A narrow, enzyme-agnostic hydrolysis window (6-10% degree of hydrolysis, achieved within ∼2 h) was identified as both necessary and sufficient to form β-sheet-rich nanofibers with high aspect ratios (>1 μm). Beyond ∼20% degree of hydrolysis, excessive cleavage led to disordered aggregates, accompanied by decreased β-sheet content and colloidal stability. Among all conditions, pepsin-hydrolyzed WPI (2 h, ∼10% degree of hydrolysis) yielded nanofibers with the highest surface hydrophobicity, ζ-potential (+48 mV), and encapsulation efficiency (93.0%) for β-carotene. Moreover, these complexes also retained 86% of β-carotene after 4 weeks of ambient storage, outperforming other enzymatic treatments. This work establishes a limited-hydrolysis framework centered on a specific degree of hydrolysis (6-10%) as a universal design principle for fabricating WPI nanofibers, decoupling fibril formation propensity from protease choice while demonstrating that enzyme specificity within this window dictates functional performance. This approach offers a scalable strategy for the delivery of sensitive lipophilic bioactives in food and pharmaceutical applications.