In Vitro ACE-inhibitory And Antioxidant Activities Of Casein Hydrolysates Subjected To Enzymatic Modification In The Presence Of Three Extrinsic Amino Acids

Hypertension is a serious risk factor for cardiovascular disease such as heart failure, stroke, coronary heart disease and myocardial infarction. It is especially dangerous because it often has no warning signs or symptoms. It was reported that more than 12000 thousands of peoples died from the cardio- and cerebro-vascular diseases resulted from hypertension every year in the world. Hypertension has now become the first killer to people's health and is the great public problem. Recently, many researches demonstrated that casein is not only sources of nutrients, but may also be ones of biologieally active peptides, among which ACE-inhibitory peptides have received the most attention for its safe and no side effects nature in the therapy of hypertension. These pepetides show great promise in the development of a novel physiologically functional food for preventing hypertension as well as for therapeutic purposes. In order to prepare bioactivie peptides with high activity from milk proteins, casein hydrolysates were modified by plastein reaction with Alcalase in the presence of extrinsic amino acids in the present work.The bioactive peptides were prepared by hydrolyzing casein with Alcalase, an alkaline protease from Alcalase 2.4L FG. Casein hydrolysates were prepared by incubating casein with Alcalase for 6 h, and exhibited the highest ACE-inhibitory activity (IC₅₀=42.2μg/mL) with a DH 12.4% and a DPPH radical scavenging activity of 41.8%. The ACE-inhibitory activity of the casein hydrolysates prepapred changed as the DH of casein hydrolysates increased during hydorlysis. When the DH of casein hydrolysates was too low, the hydrolysates only had little ACE-inhibitory activity because the ACE inhibitory peptides were not fully released from the casein. When the DH of casein hydrolysates was too high, the ACE-inhibitory peptides generated were destroyed by protease, which made them lost their ability to inhibit the ACE.Four reaction conditions including addition level of amino acids, enzyme addition, reaction temperature and the types of amino acids for the Plastein reaction of the casein hydrolysates were optimized by a central composite design and response surface methodology analysis with the decrease of free amino groups in the reaction mixture as the response. When the concentration of casein hydrolysates and reaction time were fixed at 35% (w/w) and 6 h, the practical results indicated that the addition level of amino acids, reaction temperature and the types of amino acid added had significant impacts on the plastein reaction of the casein hydrolysates, while the addition level of Alcalase only had little influence. The effects of these reaction condictions on the Plastein reaction of the casein hydrolysates behaved similar profiles when added amino acid was phenylalanine, leucine or valine, respectively. The optimal conditions were thus selected to be an addition level of amino acid of 0.6 mol/mol free amino groups (hydrolysates), enzyme addition of 3 kU/g proteins and reaction temperature of 40℃. The factors impacted Plastein reaction of the casein hydrolysates significantly was in the order of addition level of amino acids and reaction temperature.Three modified casein hydrolysates were prepared with the selected suitable conditions in the presence of phenylalanine, leucine and valine, respectively. The analysis results indicated that the ACE-inhibitory or antioxidant activities of the modified casein hydrolysates increased as the extent of modification increased. The IC₅₀ values of the modified hydrolysates were in range of 21.0 to 25.1μg/mL, which indicates that the ACE-inhibitory activities of the modified hydrolysates were improved by the Plastein reaction and addition of amino acids. The types of amino acids were not important to the activity of the modified hydrolysates. Antioxidant activity of the modified casein hydrolysates was also significantly improved. The antioxidant activity of the modified casein hydrolysates was different when different extrinsic amino acids were added to the casein hydrolysates. DPPH radical scavenging and reducing activity of the modified casein hydrolysates had the highest value when phenylalanine was added to the casein hydrolysates, while hydroxyl radical scavenging of the modified casein hydrolysates had the highest value when leucine was added to the casein hydrolysates.