In Vitro Angiotensin I-Converting Enzyme Inhibition of Casein Hydrolysate Responsible for Plastein Reaction in Ethanol-Water Medium, Solvent Fractionation, and Protease Digestion

Abstract

A casein hydrolysate generated by Alcalase had in vitro ACE-inhibitory activity of 44.4%, and was treated by Alcalase-catalyzed plastein reaction in ethanol-water medium. Alcalase addition, ethanol, substrate concentration, and reaction temperature optimized from experimental design were 8.36 kU/g peptides, 56.8 (v/v), 56.8% (w/v), and 37.5°C, respectively, when reaction time was fixed at 6 h. Two treated casein hydrolysates, namely TCH4 and TCH8, were obtained with reaction time of 4 and 8 h, and exhibited the highest ACE-inhibitory activity of 62.5% or the greatest reaction extent but an activity of 35.6%, respectively. Fractionation of TCH4 and TCH8 by applying ethanol-water of 7:3 (v/v) conferred the obtained supernatant (precipitate) fractionates higher (lower) activity than the parent substrate, while applying ethanol-water of 3:7 (v/v) or water led to an opposite result in activity for the fractionates. In vitro digestion of TCH4, TCH8, and their fractionates revealed that they had resistance in activity towards the investigated four proteases, as the resulted 47 out of 48 digests had higher activities than casein hydrolysate. TCH8 exhibited better protease resistance than TCH4. It is concluded that the applied plastein reaction can enhance ACE inhibition and protease resistance of casein hydrolysate.