Study On The Separation And Purification Of Antihypenensive Peptide From Casein Protein

Casein is the most abundant protein in milk and is also essential amino acid for indivi-dual growth. Recently, many researches demonstrated that casein may also be biologically active peptide.ACE inhibitory peptide from casein has received the most attention for its safe and no side effects nature in the therapy of hypertension. In the present study, bovine caseins as the raw material are hydrolysed with protease.The ACE inhibitory activity of hydrolysis products as indicator selected enzyme. Peptides were separated and purified by ultrafiltration, gelfiltration chromatography and reverse phase high performance liquid chromatography (RP-HPLC). Amino acid sequences of the pepitdes with high activity were identified by MS.After screening of several kinds of abundant proteases, alkaline protease was used to hydrolyze casein for the production of antihypertensive peptide.Taking ACE inhibitory activity as index, orthogonal design of four factors three levels was taken to optimize the hydrolysis conditions of casein with alkaline protease.The optimal hydrolysis conditions of casein were:substrate concentration15%, enzyme concentration0.15%, pH9.5,50℃. After digested with alkaline protease for six hours, the casein hydroly-sates showed an IC50value of0.76mg/mL.The mixture of peptides was separated by ultrafiltration membrane (PBS) under the con-dition:10℃,0.12MPa. The mixture of peptides was divided into three parts, MW (molecular weight)>10kDa,3kDa<MW<10kDa, MW<3kDa.The ACE inhibitory activity was analyzed, and the part of MW<kDa was the best.Seven fractions from the sparation of the antihypertensive peptides were obtained with a RP-HPLC column. The most active antihypertensive fraction was the three.Upon further on a RP-HPLC high resolution column, this fraction was separated into two fractions, and the second (3-2) was found to have most of the activity with aninhibitory ratio of93.56%. After amino acid sequence analysis by MS, the MW of antihypertensive fraction (3-2) was956Da and the analysis of their amino acid sequences may containing a dipeptide:Gly-Ala.