Low- and intermediate-frequency ultrasound modification of millet protein: Implications for emulsifying and gelling...

Ultrasonic processing is increasingly being explored as a green strategy to tailor protein functionality, yet the mechanisms underlying frequency-dependent effects remain insufficiently understood. In this study, millet protein (MP) was subjected to low- and intermediate-frequency ultrasound (20 kHz and 207 kHz) and controlled power densities (48.70 W/L and 80.29 W/L) to elucidate structure-function-application relationships. Ultrasonic treatment induced pronounced conformational rearrangements, characterized by increased surface hydrophobicity and β-sheet content, accompanied by reduced free sulfhydryl groups and α-helix content. Molecular dynamics simulations supported these observations, revealing intensified residue-level fluctuations under intermediate-frequency ultrasound, indicative of enhanced structural flexibility. These molecular changes translated into markedly improved functional performance, including enhanced emulsifying stability and optimized rheological behavior, with ultrasound power identified as the dominant governing parameter. Notably, LUS-80.29 W/L emulsion gel exhibited superior 3D printability and effectively encapsulated curcumin, enabling intestinal-targeted release and improved micellization. This study provides mechanistic insight into frequency-regulated ultrasonic modification of MP and demonstrates its potential for designing functional, printable protein-based delivery systems in advanced food applications.