Preparation Of ACE Inhibitory Peptides By Coupling Of Enzymatic Hydrolysis And Membrane Separation From Casein Non-Phosphopeptides

Casein phosphopeptides(CPPs)have been reported as the typical functional peptides for mineral binding and extensive researches have already been carried out. Casein non-phosphopeptides (CNPPs), by-products for industrial production of CPPs, however, have not been paid enough attention. Extensive studies showed that, CNPPs have high content of hydrophobic amino acids and are good sources of ACE inhibitory peptides, which could help modulate blood pressure. ACE inhibitory activity will be more significant when the peptide molecular weight is small and the structure consists of certain hydrophobic amino acids. Therefore, protease was screened and controlled hydrolysis of CNPPs and separation of the hydrolysates were carried out in an enzymatic membrane reactor to obtain high ACE inhibitory peptides.First, the amino acid composition and molecular weight distribution of CNPPs were determined. Results show that, molar percentage of hydrophobic amino acids and hydrophobicity values of CNPPs were 54.67% and 7.47 respectively. A big portion of peptides with molecular weight greater than 1500 Da exist in the CNPPs. CNPPs were separated by 1K membrane ultrafiltration, and ACE inhibition rate of peptides with molecular weight less than 1K Da was 50.40%. Meanwhile, peptides with molecular weight greater than 1K Da possessed ACE inhibition rate of 18.82%, which is much lower than that of small peptides. Thus, CNPPs could be adopted as abundant sources for have ACE inhibitory peptides due to its strong hydrophobic features.Several proteases were screened for enzymatic hydrolysis of CNPPs. Considering the ACE inhibitory activity, the yield and enzyme activity retention rate after hydrolysis. Protease N was choosen for preparation of ACE inhibitory peptides, which provided ACE inhibition rate more than 55% and enzyme activity retention rate more than 90%. Both are better than the other several proteases. AS1398 neutral protease also brought very high ACE inhibitory activity, but lower than Protease N. However, the yield of its ACE inhibitory peptides is higher than Protease N. Therefore, Protease N was adopted to prepare the ACE inhibitory peptides.The conditions of reaction coupling of enzymatic hydrolysis and membrane separation on CNPPs had been studied. ACE inhibitory peptides yield and the enzyme activity retention rate were considered when optimize the reaction and separation conditions. The significance of different factors were disclosed as: Pressure> feed speed> temperature> enzyme concentration. And optimized conditions of reaction were: pressure of 12psi, feed rate of 145mL/min, temperature of 47.5℃, enzyme concentration of 6000 U/g protein. Under the optimal conditions, the filtrate peptide concentration was 2.5mg/mL, membrane flux 50mL.min^-1.m^-2, reactor peptide concentration 5.7 mg/mL, enzyme activity retention rate decreased about 8% per hour.Desalting of ACE inhibitory peptides was carried out and the properties of the products were determined. The existence of a large amount of inorganic salts deteriorated the quality of ACE inhibitory peptides, so DA201-C macroporous resin was adopted for salt removal from crude ACE inhibitory peptides. With 85% (v/v) ethanol static desorption, the protein recovery was more than 85% and more than 95% salt could be removed. IC50 value was reduced from 0.86mg/mL to 0.12mg/mL after desalting. In vitro digestion stability of ACE inhibitory peptides was reliable.