Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Abstract

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC50 value of 47.1 μg mL−1. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w−1) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g−1 proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g−1 proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g−1 proteins, the IC50 value of the casein plasteins was lowered to 0.2 μg mL−1.