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Lately, concern about the protease AprX produced by Pseudomonas has increased in the dairy industry due to its ability to survive UHT treatment and spoil UHT milk. Efficient prediction methods for UHT milk spoilage are currently lacking, mainly due to high diversity in proteolytic potential between Pseudomonas strains. The present study aimed to gain more insight into the variability between Pseudomonas strains regarding proteolytic potential by comparing their proteolytic capability with their aprX expression levels and differences in peptide formation. The variability in aprX expression levels in four Pseudomonas strains were related to physical stability, milk proteolysis and peptidomic cleavage patterns of milk proteins in a storage experiment of UHT milk inoculated with protease extracellular extracts and stored for 45 days at 20 °C. A positive relationship was observed between the relative expression of aprX and milk proteolysis during storage, with the strain Pseudomonas panacis DSM 18529 showing the highest level in both parameters. This strain was the only strain to show visual gelation, which occurred after 21 days. The peptide formation analysis showed a similar protein hydrolysis pattern between strains and high hydrolysis of αs1-caseins during long-term spoilage putatively due to the activity of AprX was observed.

Ultra-high temperature (UHT) processing of milk can result in protein changes during storage; however, the progress of dehydroalanine (DHA) mediated protein cross-linking and Maillard reactions in relation to the sediment formation have not been investigated previously. Liquid chromatography–mass spectrometry, based on multiple reaction monitoring (MRM), was used to absolutely quantify concentrations of furosine, N-ε-(carboxyethyl)lysine (CEL), N-ε-(carboxymethyl)lysine (CML), lanthionine (LAN) and lysinoalanine (LAL) in skim milk and sediment of UHT milk produced from raw milk with either small or large casein micelles. The results showed a higher molar proportion of the advanced stage Maillard reaction products CEL and CML in the sediment, compared to early stage Maillard reaction product furosine, whereas furosine was predominant in the skim milk. Both LAL and LAN increased during storage in the skim milk phase, however only LAL was identified in the sediment. The milk pool with large native casein micelles, known to have a higher percentage of sedimentation, contained higher proportions of furosine, CEL, CML and LAL in the sediment compared to milk with smaller native casein micelles. The study demonstrates the potential contribution of processing-induced protein-protein interactions to sedimentation in UHT milk during storage.

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