Structural changes induced by ultrasound in proteases and their consequences on the hydrolysis of pumpkin seed proteins and the multifunctional properties of hydrolysates

The effects of ultrasound (US) (40 kHz, 23.8 W/L) on commercial protease structures and their impact on pumpkin seed protein (PSP) hydrolysis were evaluated, as well as the solubility and antioxidant activity of hydrolysates. The ultrasonic pre-treatment activated Neutrase (R) (22.7 %), Brauzyn (R) (20.8 %) and Flavourzyme (R) (22.4 %), with more significant changes in the tertiary structure of Flavourzyme (R), which also showed the highest increases in particle size (-21 %) and polydispersity index (PDI) (-60 %) compared to its native form. On the other hand, US pre-treatment under inactivation conditions resulted in substantial structural changes, indicating protein denaturation with the formation of heterogeneous and strongly electronegative aggregates. Proteolysis of PSP with US-activated enzymes showed a higher degree of hydrolysis (DH) (up to 75 %) and the obtained peptides showed higher solubility (up to 53 %) and antioxidant activity (up to 40 %). The findings emphasize that applying ultrasound under specific conditions can induce moderate structural changes in proteases, enhancing their hydrolytic efficiency.