Beyond the gut: Investigating the mechanism of formation of β-casomorphins in human blood

: To evaluate the potential differences in the propensity of β-casein A1 (β-CNA1) and A2 (β-CNA2) from bovine milk to release health-relevant β-casomorphins (BCMs), food-derived peptides were monitored over time in the blood of eight human volunteers who consumed milk containing both protein variants. Liquid chromatography coupled with high resolution tandem mass spectrometry revealed interindividual variability of milk peptidomic profiles in human blood. BCMs were not detected, whereas BCM precursors originating from both β-CNA1 and β-CNA2 were ascertained, with β-CNA2-derived peptides showing a slightly greater susceptibility to proteolysis. Ten synthetic peptides mimicking circulating BCM precursors from β-CNA1 and β-CNA2, which were incubated ex vivo with the blood of two volunteers, showed comparable potential to generate BCMs. The formation of BCMs seemed to depend mainly on the size of the BCM precursors and less on the presence of His67 or Pro67. These findings challenge the belief that BCMs are released exclusively from β-CNA1 and support the nutritional safety of conventional milk, informing health policies regarding milk consumption.