In vivo endogenous proteolysis yielding beta-casein derived bioactive beta-casomorphin peptides in human breast milk for infant nutrition

•;Beta-casein derived beta-casomorphin (BCM) peptides are released in human milk before digestion by the infant.•Naturally occurring endogenous BCM-8,-9,-10, -11, precursors and truncated peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the human milk.•The identified BCM-8,-9,-10, -11 and BCM precursor peptides meet the structural requirements to elicit the biological function in the infants. Objective Beta-casein is a major protein in breast milk and an important source for several bioactive peptides that are encrypted within the sequence. Beta-casomorphins (BCMs) are short-chain proteolytic peptides that are derived from the beta-casein protein and have opioid effects in newborns. Human milk is known to contain naturally occurring milk-protein-derived bioactive peptides but the identification of naturally occurring beta-casein-derived BCMs in human breast milk has been limited due to difficulties in the detection of BCM peptides, which are small and circulate in low concentrations. Methods The present study aimed to identify the naturally occurring BCM peptides from beta-casein in human breast milk using liquid chromatography-tandem mass spectrometry. The BCM peptides identified in the breast milk were analysed to predict the milk proteases responsible for the cleavage patterns using a computational tool EnzymePredictor. Results In-depth peptidomics analysis of breast milk samples that were collected at different lactation stages during human lactation revealed the presence of BCMs including BCM-8, -9, -10, and -11 as well as precursors and truncated forms of the original peptide, which suggests that milk protease activity in the mammary gland generates biologically relevant BCMs. Conclusions To our knowledge, this is the first report to describe the presence of naturally occurring human BCM-10 and -11 in breast milk. Our study provides evidence of beta-casein-derived BCM peptides in human milk before infant digestion. Proteases that are present in milk are likely specific in their proteolysis of beta-casein. The identified bioactive BCM-8, -9, -10, and -11 as well as the precursor peptides meet the structural requirements to elicit opioid, immunomodulatory, antioxidative, and satiety functions in newborns. [Display omitted]