Strain-specific effects of bacterial proteolytic activity on acid and rennet coagulation of milk

Spoilage of dairy products by psychrotrophic bacteria poses a significant challenge to the food industry, compromising product quality and increasing food waste. Among these microorganisms, Pseudomonas species are particularly problematic due to their production of heat-resistant extracellular enzymes that degrade milk proteins and alter milk functionality. However, the mechanisms underlying strain-specific differences in AprX production and activity, and how these differences influence casein degradation and acid-induced gelation, remain insufficiently understood. In this study, we investigated how diverse Pseudomonas strains with varying proteolytic capabilities impact milk quality, specifically through their effects on casein degradation and coagulation properties. During growth in milk, the strains displayed distinct preferences for degrading either κ-casein or β-casein. κ-Casein degradation correlated with earlier and stronger acid gelation, accompanied by rapid gel development, while β-casein degradation was associated with earlier rennet coagulation. Genomic analysis of the aprX-lipA2 operon revealed variability in operon structure and gene expression that did not consistently predict proteolytic activity across strains. Proteomic and AprX sequence analyses indicated that factors such as gene regulation and secretion system efficiency critically influence strain-specific spoilage potential. Overall, these findings clarify how differences in AprX expression and proteolytic behaviour among Pseudomonas strains influence both acid and rennet coagulation, highlighting the broader impact of strain-level variability on milk functionality during refrigerated storage.