Development Of ACE Inhibitory Peptides From Casein Hydrolysates

Casein, with essential amino acid for individual growth, is the most abundant protein in milk. Recently, many researches demonstrated that casein was not only sources of nutrients, but may also be ones of biologically active peptides, among which ACE inhibitory peptide has received the most attention for its safe and no side effects nature in the therapy of hypertension. In China, there was no study on the production of casein-derived ACE inhibitory peptides thus far. In the present study, optimal hydrolysis conditions of casein, ultrafiltration and desalinisation of casein hydrolysates were studied, and the manufacturing technique of ACE inhibitory peptide was established.A reliable assay method of ACE inhibitory peptides with high accuracy and repeatability was modified and established, and it can be used to determine the ACE inhibitory activities of all kinds of samples.After screening of five kinds of abundant proteases, protease C was used to hydrolyze casein for the production of ACE inhibitory peptide.Taking ACE inhibitory activity as index, orthogonal design of four factors three levels was taken to optimize the hydrolysis conditions of casein with protease C. The optimal hydrolysis conditions of casein were: temperature 45 , enzyme concentration 5%, pH 7.5, substrate concentration 5%. After digested with protease C for six hours, the casein hydrolysates showed an IC50 value of 0.68mg/mL.Ultrafiltration (with membranes of MW cut-off 10kDa and 3kDa ) can increase the ACE inhibitory activities of casein hydrolysates. However, permeates of 3kDa membrane showed higher ACE inhibitory activities with IC50 of 0.46mg/mL. The optimal ultrafiltration condition for 3kDa membrane was at 35 with pressure 0.09Mpa.Salt was removed by ion exchange resin at a flow rate of 10 column volume /hour with desalinization rate of approximate 80% and nitrogen recovery rate of 83%. NO differences was observed on the ACE inhibitory activities of samples.An optimum processing technology of ACE inhibitory peptides was established as: fresh milk-casein separation-hydrolysis of casein-centrifugation-supernatant-desalinization by ion exchange resin - freeze drying - ACE inhibitory peptides. In this processing technology, high ACE inhibitory activities and low nitrogen loss were obtained with direct desalinization of casein hydrolysates.Molecular weight distribution of ACE inhibitory peptides was measured by gelchromatography and it showed less than 1500. Further analysis of amino acid compositions indicated that ultraflltration and desalinization had little effect on the amino acid compositions of hydrolysate, permeate, desalted product. The principal amino acids of this product were Val, Asp, Leu, Lys, Pro, Glu.