Development of an Integrative Process for the Production of Bioactive Peptides from Whey by Proteolytic Commercial Mixtures

Whey proteins are a rich source of angiotensin converting enzyme I (ACE) inhibitory peptides. The ACE inhibition is one of the key mechanisms by which reduction in blood pressure is exerted in the body so some of these peptides have also shown a blood pressure reduction effect ie, antihypertensive activity. They can therefore be part of a dietary intervention to treat this health condition. The aim of this work is to develop a simplified integrative process using a combination of adsorption and microfiltration steps for the production of ACE inhibitory (ACEi) peptides by selectively separating and immobilizing these two proteins, βlactoglobulin and casein derived peptides from rennet whey. Unlike the conventional methods, this approach integrates the selective separation of the target proteins and their in-situ hydrolysis which results in the production of partially pure, less complex peptides with higher bioactivity, free from the non hydrolysed proteins. In this work protease N Amano was added directly to the adsorbed proteins at different enzyme to substrate ratios (E: S) in a thermostatically controlled membrane reactor operated in a batch mode. The hydrolysates were analysed by HPLC, total protein was measured using the BCA method and ACE activity was determined applying the widely used enzyme assay which is based on the quantification of hipuric acid by HPLC; the bioactivity was measured in terms %ACE inhibition and as IC50 which is the peptide concentration required to reduce the ACE activity to 50%. One of the most important outcomes of this research is that applying the immobilisation method we can produce two different hydrolysates with high ACE inhibitory activity starting from the same feedstock: (i) mainly composed of casein derived peptides with IC50=280μg/ml and (ii) mainly composed of β-lactoglobulin derived peptides with IC50=128μg/ml. Furthermore separation of the smaller peptides from the enzyme and larger peptides was achieved with a 1 kDa molecular weight cut off (MWCO) ultrafiltration membrane which allows also recycling of the larger peptides and enzyme.