Localized enzymolysis and dual-frequency ultrasound modification of underutilized fava bean protein: Techno-functional, structural, in vitro digestibility, molecular docking, and interrelationship characteristics

Abstract

This study examined the effect of using dual-frequency ultrasonic treatment (DFUT) and targeted enzymolysis on the physiochemical, structural, and techno-functional properties of the underutilized fava bean protein (FBP) and its hydrolysate (FBPH). FBP was enzymatically hydrolyzed (5–15% DH) to produce FBPH, employing alcalase as the catalyst. The enzymolysis process declined the color brightness, turbidity, and particle size for both non-sonicated and DFUT-treated FBP. Additionally, the enzymatic treatment resulted in an increase (P < 0.05) in the absolute values of zeta potential. Further, DFUT decreased water holding capacity for both FBP and FBPH compared to the untreated ones. DFUT, on the other hand, increased oil absorption capacity compared to non-sonicated ones. Additionally, XRD, FTIR, and intrinsic fluorescence spectral analyses demonstrated that FBPH exhibited a greater degree of flexibility and mobility in its secondary structures compared to FBP. Moreover, SEM and CLSM investigations provide convincing evidence that DFUT improves and speeds up the breakdown of proteins' structures, especially at higher DH. The molecular weight of DFUT-treated FBP showed a notable decrease in the SDS-PAGE profile, but there were no discernible differences between DFUT-treated and non-sonicated FBPH. The DFUT-treated FBP-10DH had significantly better in vitro digestibility (85.70%) compared to the non-sonicated FBP-15 (76.63%) and control FBP-0DH (68.57%). This indicates that the DFUT modification effectively improved the digestibility of the hydrolysate. Correlation and molecular docking research suggest that DFUT has different impacts on specific FBP and its FBPH characteristics. Taken together, localized enzymolysis and DFUT can alter the conformation of proteins and their intermolecular interactions with FBP and FBPH, potentially enhancing their functionality and expanding their utilization in the food industry.