Mechanism study of dual-frequency ultrasound assisted enzymolysis on rapeseed protein by immobilized Alcalase

• Immobilized enzyme was used to investigate the mechanisms of ultrasonic assisted enzymolysis.
• DH significantly increased after DFU assisted-enzymolysis.
• Yield of soluble solids content increased after DFU assisted-enzymolysis.
• Content of α-helix and random coil in rapeseed protein decreased, β-chain increased after DFU assisted-enzymolysis.
• Surface roughness of immobilized Alcalase increased after DFU assisted-enzymolysis.

The mechanism of ultrasound field promoting enzymolysis efficiency is difficult to study, because the reaction system mixes with enzymes, proteins and hydrolysates. Immobilized enzyme is a good option that can be used to investigate the mechanism by separating enzymes out from the system after enzymolysis. The objective of this study was by using immobilized Alcalase to investigate the effects and mechanisms of the promotion of dual-frequency ultrasound (DFU) assisted-enzymolysis on rapeseed protein. Based on single factor experiments, response surface methodology model with three factors – hydrolysis time, power density and solid–liquid ratio at three levels was utilized to optimize the degree of hydrolysis (DH). Circular dichroism (CD) was used to analyze the secondary structure change of the protein, scanning electron microscopy (SEM) was used to analyze the surface microstructure change of the enzyme. The results showed that with DFU assisted-enzymolysis, the DH increased by 74.38% at the optimal levels for power density 57 W/L, solid–liquid ratio 5.3 g/L and enzymolysis time 76 min. After DFU assisted-enzymolysis, the yield of soluble solids content, including protein, peptides and total sugar in hydrolysate increased by 64.61%, 40.88% and 23.60%, respectively. CD analysis showed that after DFU assisted-enzymolysis, the number of α-helix and random coil decreased by 10.7% and 4.5%, β-chain increased by 2.4%. SEM showed that the degree of surface roughness of immobilized Alcalase increased. The above results indicated that the improvement of hydrolysis by DFU assisted-enzymolysis was achieved by enhancing the solid solubility, changing the molecular structure of protein and increased the surface area of immobilized enzyme.