Identification of peptides reflecting the storage of UHT milk by MALDI-TOF-MS peptide profiling

Proteolysis during the storage of UHT milk is associated with major technological problems, particularly bitter off-flavors and age gelation limiting the shelf life of milk. In this study, untargeted peptide profiling by MALDI-TOF-MS identified peptides that were formed by proteolysis and reflected the storage of UHT milk. Analysis of nine different commercial UHT samples recorded peptide profiles during and at the end of their shelf life. Relative quantification and sequencing of the peptides revealed that the concentrations of 22 peptides increased significantly during the storage of UHT milk due to the activity of endogenous milk proteases and microbial proteases as well as other unidentified proteolytic mechanisms. Based on highly discriminative AUC values from receiver operator characteristic (ROC) curve analysis, we selected ten peptides as marker candidates. Among those, the peptide β-casein_192-206 (m/z 1668.9) was the most suitable marker differentiating expired-UHT from regular-UHT samples with 100% accuracy. Additionally, β-casein_191-206 (m/z 1782.0) showed 100% specificity and β-casein_139-161 (m/z 2696.4) 100% sensitivity. Thus, β-casein_192-206, either by itself or in combination with β-casein_191-206 and β-casein_139-161, presents a reliable marker to monitor the storage of UHT milk based on proteolytic mechanisms. SIGNIFICANCE: Enzymatic hydrolysis is the main reason why processed milk spoils during storage. The present study recorded peptide profiles to monitor the release or degradation of peptides in stored UHT milk. Among the detected peptides, statistical analysis revealed that the relative concentration of β-casein_192-206 reflected those proteolytic processes most precisely. Food authorities can now refer to β-casein_192-206 as a reliable marker to differentiate between freshly processed milk and products at the end of their shelf life. Furthermore, the food industry can use this marker peptide to improve production processes by monitoring the proteolysis during storage. The recorded peptide profile helps to explain the basic mechanisms leading to storage-induced proteolysis.