Molecular properties and bioactivity of phosphopeptides obtained from controlled proteolysis of milk proteins in an enzymatic bioreactor

A family of casein-derived peptides, produced in vivo or in vitro from proteolysis of alpha and beta casein, enriched in phosphoseryl groups, and named casein phosphopeptides (CPP), is able to bind and solubilize calcium. A commercial CPP mixture (CPP DMV) and a single synthetic CPP, corresponding to fragment 1-25 of bovine beta casein, were found to elicit a marked and transient rise of intracellular free calcium concentration in human intestinal tumor HT-29 cells, differentiated in culture [1]. Here we report on the properties of phosphopeptides produced by controlled milk hydrolysis with food-grade proteases (TPC-CPP), and on a comparison of their bioactivity with that of CPP DMV. Large-sized peptides were obtained by controlled and limited proteolysis of milk in a custom-designed ultrafiltration pilot-scale plant [2-4]. Several heat-resistant, food-grade proteases were tested. Phosphopeptides were isolated by selective precipitation [5], followed by diafiltration. The effects of CPPs on cellular calcium uptake were assessed by video-imaging microscopy on single cells, by using fura2 as fluorescent free-calcium indicator. The molecular properties of TPC-CPPs were studied by chromatographic and mass spectroscopy techniques, and compared to those of CPP DMV. MS spectroscopy [5] indicated that CPP DMV mainly derived from the N-terminus region of beta-casein, whereas TPC-CPPs included significant amounts of phosphopeptides deriving from other portions of beta-casein, and from alpha-S1-casein. Both CPP mixtures promote calcium uptake in HT-29 cells in a dose- and extracellular calcium concentration- dependent fashion, but TPC-CPPs appear more bioactive than CPP DMV, inducing higher cellular calcium increments. The CPP bioactivity is related to their molecular structure as well as to their ability to bind and solubilize calcium. Studies are in progress to identify specific CPPs as possible nutraceuticals and/or functional food ingredients.