Proteolysis and volatile compounds in Mediterranean buffalo milk blue cheese: Quality determinants and functional peptides

This study explores the proteolytic changes and volatile organic compound (VOC) profiles occurring during the ripening of Mediterranean buffalo milk blue cheese. The main objective was to characterize bioactive peptides, casein phosphopeptides, and volatile compounds and to assess their potential implications for functional food development. Peptide profiling was conducted using nanoLC-ESI-MS/MS and MALDI-TOF-MS, while VOCs were analyzed via headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME)-GC-MS. β-casein emerged as the predominant source of bioactive peptides, including sequences with predicted ACE-inhibitory, antimicrobial, and antioxidant activities. It also generated opioid peptide precursors such as β-casomorphin-8 (BCM-8) and β-casomorphin-9 (BCM-9); however, in silico analyses suggest that these sequences may lack significant opioid activity. Importantly, β-casomorphin-7 (BCM-7) was not detected at any stages of ripening, likely due to the presence of lysine at position 68, which inhibits its formation. Casein phosphopeptides displayed ripening-dependent dephosphorylation patterns, driven by fungal phosphatase activity. VOC profiling revealed 55 key compounds, mainly methyl ketones, esters, and short-chain acids, that contributed to the evolving sensory profile of the cheese. These findings highlight the complex biochemical transformations that define both the functional and sensory qualities of buffalo blue cheese. The identification of health-promoting peptides reinforces its potential as a functional food. Future studies should investigate the bioavailability and systemic activity of these peptides in vivo.