Impact of ultrasound–enzyme pretreatment sequence on recovery and functionality of proteins from an oat fiber-rich side stream

The residual fiber-rich fraction after oat protein extraction, rich in residual protein and insoluble fiber, is a promising resource for sustainable protein recovery, though its rigid protein–fiber matrix hinders extraction. This study assessed mechanical (wet milling, ultrasound) and biochemical (Viscozyme®) pretreatments, individually and in sequence, before alkaline extraction and isoelectric precipitation. Wet milling slightly improved yields via particle size reduction, while ultrasound alone enhanced protein purity. The combination of ultrasound and enzymatic treatment doubled protein recovery and mass yield, also boosting gelation and viscoelastic strength. Pretreatment order was crucial: ultrasound followed by enzyme treatment yielded higher surface charge and gel strength, whereas enzyme-first produced finer protein particles with moderate gel strength. Ultrasound-first treatments showed lowest phytate retention, enzyme-first the highest. These results reveal the synergistic, sequence-dependent effects of ultrasound and enzymatic pretreatments in releasing protein from fiber-rich oat side stream, supporting their upcycling into functional, plant-based food ingredients. • Ultrasound followed by carbohydrase pretreatment doubled protein yield from oat press cake • Sequence of treatments critically affected protein structure and gel properties • Ultrasound enhanced purity, unfolding proteins and reducing phytate retention • Enzyme-first led to finer particles but increased phytate–protein complexation • Combined methods offer sustainable route to functional oat protein ingredients