Studies On Preparation ACE Inhibitory Peptides From Rapeseed Meal By Enzymes

Hypertension is one of the largest epidemics in the world today, the incidence ofhypertension in China increased year by year. Only3%of patients with hypertension in ourcountry can effectively control their blood pressure. Modern research has shown that animportant way to countrol blood pressure is to inhibite the activity of angiotensinconverting enzyme （ACE）. ACE inhibitory peptide has become a hot reaserch due to itsnatural, efficient, and non-toxic.In this study, rapeseed protein was made from rapeseed meal, and take rapeseedprotien as raw material for the follow-up experiments. Hydrolyzing, decolorization andseparating were optimized, and then, the components of high activity were analyzed for thestructure primitively. The main experimental results are as follows.（1） Alcalase and neutral protease were selected to hydrolyze rapeseed ptotien forpreparing ACE inhibitory peptides. Box-Behnken experimental design combining withresponse surface methodology and quadratic programming was employed for maximizingthe ACEI and a quadratic regression model was obtained using the Design-Expert software.The regression equation was ACEI=＋50.22600－0.29250×T＋0.47750×S+0.70250×[E]/[S]－0.11750×T×S－1.11250×T×[E]/[S]＋1.41750×S×[E]/[S]－9.41175×T2－2.45675×S2－3.83175×[E]/[S]2, the determination of optimum conditions： substrateconcentration5.65%, ratio of enzyme to substrate （[E]/[S]）6.5%，temperature54.9℃,The theoretical value of ACEI is50.303%,and the actual value is50.12%. The peptidecontent of rapeseed ACE inhibitory peptides is72.77%, the IC50is1.78mg/mL, so theresults can be used in practice.（2） The peptides were decoloured by activated carbon The result shows that theoptimum decoloration conditions are as follows: the quantity of activated carbon is1%,pH₃.0, temperature is80℃, the time is50minutes.Under these conditions, the actualdecolouring eficiency of hydrolyzate, ACEI, peptide loss, were85.52%,57.3%and5.69%．（3） Ultrafitration technology was adopted for the separation of the ACE inhibitorypeptides and the influence of the separation parameters was investigated.For the first stageof ultrafiltration with the membrane of10kDa molecular weight,the optimal was peptideconcentration of2%, peptide solution temperature of40℃, pressure of0.3kg/cm². Forthe second stage of ultrafiltration with the membrane of3kDa molecular weight, theoptimal parameter was peptide concentration of2%, peptide solution temperature of30℃, pressure of0.7kg/cm².After ultrafiltration the original liquid is divided into3components, The IC50valuesand the activity results demonstrate that ultrafiltration technology can raise the activity of the ACE inhibitory peptides from rapeseed markedly with the IC50value of1.33mg/mLwhich was decreased by25%.HPLC chromatography of the peptides point out that thereare4components of high activity peak in the hydrolyzate.（4） HLPC, Infrared spectroscopy and UV spectra were adopted for amino acidanalysis and preliminary structural analysis of the ACE inhibitory peptides ultrafiltrated bythe membrane of3kDa molecular weight.By amino acid analysis results, the ACE inhibitory peptides and the protein fromrapeseed both consist of17kinds of amino acids （Tryptophan without taking into account）.Arginine （18.19mg/100mg）, glutamate （12.58mg/100mg）, aspartate （10.05mg/100mg）are the major amino acid component of the ACE inhibitory peptides. Lysine can helpincrease appetite and promote calcium absorption and accumulation.UV scanning spectra show that the absorption peaks in the216.0nm. Infraredscanning spectra show that the peaks may represent the amide bond, O-H, N-H, sugar ring,C=O, C-N,-CH₃.（5）The determinations of the peptides properties result that the ACE inhibitorypeptide has a proper solubility which has not be impacted by the pH; the ACE inhibitorypeptide has a proper viscosity which has little change in the concentration range of5%to20%; the activity of the peptide was stable under hot or strict pH condition.It is lower thanrapeseed protein of the peptides for oil absorption, foaming and emulsion stability, while itis higher than rapeseed protein of the peptides for water absorption and emulsifying.