Abstract
Previous work demonstrates that proteases present in human milk release hundreds of peptides derived from milk proteins. However, the question of whether human milk protein digestion begins within the mammary gland remains incompletely answered. The primary objective of this study was to determine whether proteolytic degradation of human milk proteins into peptides begins within the mammary gland. The secondary objectives were to determine which milk proteases participate in the proteolysis and to predict which released peptides have bioactivity. Lactating mothers (n = 4) expressed their milk directly into a mixture of antiproteases on ice followed by immediate freezing of the milk to limit post-expression protease activity. Samples were analyzed for their peptide profiles via mass spectrometry and database searching. Peptidomics-based protease prediction and bioactivity prediction were each performed with several different approaches. The findings demonstrate that human milk contains more than 1,100 unique peptides derived from milk protein hydrolysis within the mammary gland. These peptides derived from 42 milk proteins and included 306 potential bioactive peptides. Based on the peptidomics data, plasmin was predicted to be the milk protease most active in the hydrolysis of human milk proteins within the mammary gland. Milk proteases actively cleave milk proteins within the mammary gland, initiating the release of functional peptides. Thus, the directly breastfed infant receives partially pre-digested proteins and numerous bioactive peptides.