Optimization of ultrasound-assisted enzymatic hydrolysis Zophobas morio protein and its protective effects against H2O2-induced oxidative stress in RAW264.7 cells.

Abstract

Zophobas morio protein (ZMP) is a promising protein resource with notable biological properties, and its hydrolysis could unlock enhanced bioactivities. This study investigated ultrasound-assisted enzymatic hydrolysis (UAEH) of ZMP using different enzymes (Alcalase, Neutrase, and Protamex) to determine its effect on the degree of hydrolysis (DH) compared to enzymatic hydrolysis (EH). UAEH showed greater hydrolysis efficiency than EH, with Alcalase exhibiting the highest DH. Response surface methodology (RSM) was applied to optimize UAEH conditions for Zophobas morio protein hydrolysate (ZMPH). Optimal conditions for producing ZMPH with the maximum DH were a substrate concentration of 3.52 % (w/v), enzyme to substrate ratio of 7.64 % (v/v), and pH of 8.35. Under the optimal condition, the maximum DH was 25.03 %. In addition, significant structural changes in the optimized ZMPH compared to ZMP were identified, showing decreased α-helix and β-sheet content, with increased β-turn and unordered coil. Moreover, the optimized ZMPH demonstrated significantly improved ABTS antioxidant activity and attenuated H₂O₂-induced cell death in RAW264.7 cells compared to ZMP, which was attributed to better mitigation of ROS production. These findings provide an effective enzymatic hydrolysis method for producing ZMPH with significant antioxidant activity, demonstrating the potential of ultrasound-assisted hydrolysis in enhancing the bioactivity of insect proteins.