Peptidomic Fingerprints of Stored UHT Milk Inoculated with Protease Extracts from Different Pseudomonas Strains Relative to aprX Expression and Visible Spoilage

Abstract

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.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics

“…are mainly driven by variances in the expression and secretion of AprX rather than qualitative differences at enzyme level between strains (Dufour et al, 2008;Zhang et al, 2019). A relationship between the expression level of aprX produced by Pseudomonas and the hydrolysis of milk proteins during storage has been previously observed (Aguilera-Toro et al, 2023). However, by alignment of the AprX amino acid sequence between the different strains, structural differences within the catalytic domain were observed, with each alignment group showing a distinct amino acid residue in the sequence HEIGHTLGLxHP of the Zn 2+ -binding motif.…”

“…The collection of extracellular media was performed as described by Aguilera-Toro et al (2023). Briefly, TSB cultures from the 56 strains (prepared as described in Section 2.1) were centrifuged for 5 min at 20 • C at 8000 ×g to separate the supernatant and the pellet containing the cells.…”

“…The combined influence of the environmental and genetic factors leads to a highly heterogeneous proteolytic activity of Pseudomonas spp. in UHT milk and accurate methods to predict spoilage potential are currently lacking in the dairy industry (Aguilera-Toro et al, 2023;Maier et al, 2021;Marchand et al, 2009).…”