Angiotensin I Converting Enzyme(ACE) Inhibitory Peptides Derived From Casein

Casein is the main protein in milk, it is accounted for 80% of the total protein. Recently, many rsearches demonsrtated that casein was not only soucre of nutrients such as essential amino acids for individual gorwth, it also contains many functional and biological activity of peptides. with the growing incidence of hypertension in recent years, people's health consciousness is growing, blood pressure peptides which derived from casein has received the most attention for its safe,wide variety of sources and no side effects advantages in the therapy of hypertension.In the present study, optimal hydrolysis conditions of casein of casein hydrolysates were determined, and it can obtained higher activity hydrolyzates.Then,we used some separation and purification methods to achieve higer ACE inhibitory peptide activity. The following were the results:First, High performance liquid chromatography method was established for the anlysis of angiotensin converting enzyme(ACE) inhibitory activity of antihypertensive peptides fast. With this method, the activity of the inhibitory peptides was determined with the amount of hippuric acid which was released from substrate Hippuryl-His-leu (HHL) by ACE.The chromatographic conditions were as follows:column temperature:25℃,Methanol+0.02mo 1/L acetic acid solution (15+85(V/V)) was identified as the flow of mobile phase,the detection wavelength was 228nm,velocity was 1.0 ml/min;At this time, an excellent lineari ty(R2=0.9999) was observed. The hippuric acid reovery efficieney is 99.17%-100.26%. The samples were treated with HPLC,the qualitative and the quantitative analy ses were carried out by chromatography based on the retention time and peak area respectively. This method is simple, rapid and reproducible. This reliable method can be used for analysis of ACE activityof all kinds of antihypertensive peptide.In this article, we would required a large use of ACE in the determin-ation of ACE inhibition process, Therefore, in this study, ACE was fracti-onated from pig's lung organism, the results the optimum extraction con-ditions:pH 7.6;the ammonium sulfate saturation which remove impurities is 26%;the ammonium sulfate saturation which precitipiate is 45%;The ratio bewteen quality and solution is 1:2.5. Under these conditions,the to-tal enzyme activty was 132.2U, the specific activity was 0.362U/mg pro-tein.The enzyme solution was purified by filtration on Sephadex G-100 column,300g feash pig's lung can obtained ACE which the total ACE ac-tivity is 1027.68 U, the specific activity was 1.085, enzyme activity reco- very was 60.8%.ACE inhibitory peptides were prepared from casein protein by four commercial proteases (alcalase, trypsin, neutrase, alcalase(Novozymes)), and their ACE inhibitory activity were determined in vitro by high-performance liquid chromatography. Finally alcalase was used to hydrolyze casein for the production of ACE inhibitory peptides. The optimum hydrolysis conditions of casein of alcalase were studied by the orthogonal design. The result showed that the ACE inhibitory activity of the alcalase's hydrolysates of casein can reach 95.60% under hydrolytic temperature 50℃, E/S is 6.0% and hydrolytic pH10.0 conditions after hydrolyzed 6 hour.DA201-C Macroporous were used for desalting of ACE inhibitory and antioxidative peptides from the hydrolysis by gradient ethanol elution. The results indicated that the optimum condition for desalting was obtained as follows:loading sample concentration of 20 mg/ml,sampling flow rate of 0.5 BV/h and 75% alcohol aseluent. Under this condition,the ash content was decreased to 1.34% and ACE inhibitory activity was improved, desalination ratio reached to 92.78% and nitrogen recovery ratio was 84.22%.The Alcalase hydrolyzate of casein were separated by molecular sieve chromatographic column Sephadex G-25. The best separation condition was charaters:concentration of sample,30 mg/ml;injection volume,3 ml;flow,0.6ml/min;eluting reagent,0.05 mol/L HAc-NaAc buffer(pH 4.0). The ACE peptides obtained from the Sephadex G-25 process is the fraction D which its inhibitory activity is 92.3%,component protein concentration is 0.828mg/ml.This fraction were further fractionated with ultrafiltration Sephadex G-15,in order to obtain the fraction F with higher ACE inhibitory activity. The ACE inhibitory activity was 96.49% when the component protein concentration is 0.567mg/ml. The ACE inhibitory activity of this fraction was susceptible to pH. Overall, The ACE inhibitory activity under acid environment was prone to loss than that of alkaline,and it was shown high stability against different temperature and gastrointestinal proteases systerm.