Impact of β-casein variants on the formation of β-casomorphins in blue cheeses: Investigating key drivers through peptidomic analysis

[Display omitted] •β-Casomorphin release in four blue cheeses was assessed via peptidomics.•Diverse exopeptidases of Penicillium roqueforti drive extensive proteolysis.•Both β-casein A1 and A2 variants are susceptible to proteolysis.•Comparable levels of β-casomorphin-7 are released from β-CN A1 and A2.•The kinetics of β-casomorphins’ appearance in cheese varies with ripening stages. β-Casomorphins (BCMs), food-associated peptides resulting from the proteolytic cleavage of β-casein (β-CN), have been widely investigated for their opioid-like activity. This research aimed to identify the presence of BCM7, BCM6, and BCM5 in different bovine milk-deriving blue cheese types and to describe the intricate mechanisms behind their formation, focusing on their origin from cheese with β-CN A1 and A2 variants. Using nanoLC-ESI-Q-Orbitrap-MS/MS and advanced computational tools, we explored the peptidomes of Bleu d’Auvergne, Gorgonzola, Stilton, and Bergader blue cheeses from milk containing both β-CN A1 and A2 variants. This integrated approach ascertained the occurrence of BCM7 in all cheese samples, although at different concentrations. Evidence demonstrated that all cheeses containing β-CN A1 and A2 variants exhibited proteolysis, resulting in a pronounced BCM7 release because of the concerted activity of multiple Penicillium roqueforti exopeptidases. This study provides insights into the processes underpinning the formation of BCM7, BCM6 and BCM5 in various blue cheese types, identifying putative molecular determinants that might govern the proteolytic release of BCMs from both β-CN A1 and A2 variants as well as the complex interplay between mold peptidases and cheese polypeptide substrates.