The effects of ultrasound pretreatment with different frequencies and working modes, including mono-frequency ultrasound (MFU), dual-frequency ultrasound (DFU) and tri-frequency ultrasound (TFU), on the degree of hydrolysis (DH) of rice protein (RP) and angiotensin-I-converting enzyme (ACE) inhibitory activity of RP hydrolysate were investigated. Ultraviolet-visible (UV) spectroscopy, fourier transform infrared (FTIR) spectroscopy, surface hydrophobicity and scanning electron microscopy (SEM) of RP pretreated with ultrasound were measured. The results showed that ultrasound pretreatment did not increase DH of RP significantly (p>0.05). However, all the ultrasound pretreatment increased the ACE inhibitory activity of RP hydrolysate significantly (p<0.05). The MFU of 20kHz showed higher ACE inhibitory activity compared to that of other MFU. The ACE inhibitory activity of sequential DFU was higher than that of simultaneous with the same frequency combination. Sequential TFU of 20/35/50kHz produced the highest increase in ACE inhibitory activity in contrast with other ultrasound frequencies and working modes. All the results under ultrasound pretreatment showed that ultrasound frequencies and working modes were of great effect on the ACE inhibitory activity of RP. The changes in UV-Vis spectra and surface hydrophobicity indicated the unfolding of protein and exposure of hydrophobic groups by ultrasound. The FTIR analysis showed that all the ultrasound pretreatment with different frequencies and working modes decreased α-helix, β-turn content and increased β-sheet, random coil content of RP. The SEM results indicated that ultrasound pretreatment resulted in the deformation of RP. In conclusion, the frequency selection of ultrasound pretreatment of RP is essential for the preparation of ACE inhibitory peptide.

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Ultrasound pretreatment of protein could improve the angiotensin-I converting enzyme (ACE) inhibitory activity of hydrolysates and by alerting the structure of enzyme substrates. In this research the effects on ACE inhibitory activity and enzymolysis efficiency were explored under the optimum counter flow ultrasound (CFU) pretreatment conditions which obtained from our previous studies. The mechanism was studied by microstructure, nano-mechanical properties and secondary structures of wheat gluten (WG), which measured by scanning electron microscopy (SEM), atomic force microscopy (AFM), and flourier transform infrared spectrum (FTIR). The results showed that the CFU pretreatment resulted in significantly (P<0.05) higher value compared with control, the ACE inhibitory activity was increased by 29.8 % and the value of IC 50 was decreased by 36.92 %. CFU pretreatment increased the initial reaction rate by 9.58-20.27 % at a substrate concentration of 10-50 g/L, the K A was increased by 2.90 % and K M was decreased by 15.83 % compared to control. The microstructure revealed that the meshwork structure of WG was loosened and the surface roughness values of R a and R q were increased by CFU pretreatment. The nanomechanical analyses showed that CFU pretreatment redistributed the adhesion map and decreased the stiffness. The secondary structures were less ordered by CFU pretreatment. Intermolecular β-sheet of protein aggregation and random coil were increased, Intramolecular β-sheet, α-helix and β-turn were decreased. It was concluded that CFU pretreatment can remarkably improve the ACE inhibitory activity of hydrolysate and enzymolysis efficiency of WG confirmed by the changes of microstructure, nano-mechanical properties, and secondary structures.

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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 57W/L, solid-liquid ratio 5.3g/L and enzymolysis time 76min. 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.