Optimizing double-enzymatic hydrolysis conditions of alfalfa protein for the preparation of ACE inhibitory peptides

In order to enhance the economic value and develop the utilization potential of alfalfa, in this study, protein was extracted from fresh alfalfa using alkali-soluble acid precipitation and was hydrolyzed to prepare angiotensin-converting enzyme (ACE) inhibitory peptides using papain, neutral protease, trypsin, alkaline protease, flavor protease, and pepsin. The results indicated that neutral protease and papain were the appropriate proteases and had higher ACE inhibition rates of 71.48% and 69.99%, respectively, for preparing ACE inhibitory peptides from alfalfa protein. The effects of the combined-enzyme (neutral protease and papain) hydrolysis temperature, time, pH, and ratio on the ACE inhibition rates and hydrolysis degrees of alfalfa protein hydrolysate were investigated. Three variables of pH (X₁), temperature (X₂), and time (X₃) were selected and designed for a response surface test, which used the central combination design principle of Box-Behnken and utilized the ACE inhibition rates as response values to optimize the process conditions of enzymatic hydrolysis of alfalfa protein for preparing ACE inhibitory peptides on the basis of a single factor. The results showed that the optimal conditions for preparing the ACE inhibitory peptides were neutral protease∶ papain = 3∶1, pH = 8.12, temperature = 60 ℃, and time = 6 h, and the ACE inhibition rate was 76.12% when the ACE inhibitory peptides was prepared from alfalfa protein by double-enzymatic hydrolysis. Therefore, we conclude that the alfalfa protein has the potential to be used for preparing ACE inhibitory peptides. These results may be used for further improving the utilization efficiency and economic value of alfalfa.