Milk substrates influence proteolytic activity of Pseudomonas fluorescens strains

Pseudomonas fluorescens spoiling raw milk produces a heat-stable protease, namely AprX, that may degrade k-casein with a chymosin-like activity thus causing gelation of commercial milk during storage. Four strains of P. fluorescens were selected for both the presence of aprX gene and proteolytic activity in milk agar plate (a negative control was included) and were incubated in various milk substrates, i.e. pasteurized milk, UHT milk and reconstituted milk powder, differing for heat-treatment and presence of fat, in order to evaluate whether the type of milk substrates could affect their growth and proteolytic activity. While bacterial growth was mainly influenced by temperature (4 or 25 degrees C) for all strains, HPLC and CZE patterns of incubated milk samples showed that the extent and trend of proteolysis were highly heterogeneous and not exclusively strain-dependent. Indeed, pasteurized milk was the only substrate where aprX-positive strains led to gelation onset whereas other milk types underwent different destabilization. Ultrastructural features observed by transmission electron microscopy for casein micelles, whey proteins and fat globules, where present, explained how the processing conditions, sometimes including repeated heat-treatments, may have influenced the extent of proteolysis operated by P. fluorescens strains in the tested milk substrates. This study has highlighted that different milk substrates may bring to different conclusions when used in experiments aiming to elucidate the mechanisms of bacterial proteolysis since both ultrastructural and compositional properties may impact on accessibility of cleavage sites to proteases.