Walnut angiotensin Ⅰ converting enzyme (ACE) inhibitory peptide was prepared by two-step hydrolysis and modified by Plastein reaction, and the effects of substrate concentration, reaction temperature, reaction time, and addition of exogenous amino acids on the reaction were investigated. After modification by the Plastein reaction, the highest ACE inhibition rate of 90.67% was achieved for the Plastein reaction product without the addition of exogenous amino acids. Stability studies of the Plastein reaction product showed that the product had good structure stability at lower temperatures (0-60 ℃) and lower ionic concentrations (0-2 mol/L NaCl solution), high concentrations of denaturant (urea, sodium dodecyl sulfate) may destabilize the product. Circular dichroism spectroscopy was used to study the structural changes of the Plastein reaction products, and the results showed that the Plastein reaction promoted the re-twisted folding of the denatured peptide segment, leading to changes in its α-helix and β-fold. X-ray diffraction analysis showed that the intensity of the crystalline peaks became stronger at 31.7 and 45.5, and new crystalline peaks appeared at 56.4, 66.1, 75.3, and 84.1, where the spatial arrangement of the atoms changed, indicating that the reaction changed the structure of the ACE inhibitory peptide, thus increasing its activity.
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