Risk of Pseudomonas spp. in raw milk: Biofilm formation and enhanced peptidase production

Pseudomonas spp. represent a major risk for milk spoilage due to the production of heat-resistant peptidases. In this study, the capacity of Pseudomonas strains for biofilm formation and peptidase production on different surfaces was investigated under realistic cold-operating conditions. The differences in the bacterial composition of raw milk-associated biofilms after 6-day incubation at 20 °C and 6 °C were visualized using scanning electron microscopy (SEM). Peptidase production was analysed qualitatively on skimmed milk agar and determined quantitatively using a fluorescamine assay. This assay was also used to compare the peptidase activities between biofilm-associated and corresponding planktonic cells in UHT milk as growth medium. Crystal violet staining revealed comparable biofilm formation capacities on stainless steel und rubber at 6 °C for 6 days, although strain- and surface-specific effects were observed. SEM analysis visualized the ability of raw milk-associated microorganisms to form biofilms on stainless steel and rubber surfaces to be temperature-dependent. Quantification of the peptidase activity of biofilm-associated and planktonic cells also revealed strain-specific effects. Interestingly, it was noted that biofilm-associated cells showed a significant (p ≤ 0.01) almost 50-fold higher average peptidase activity when compared to corresponding activities of planktonic cells. In particular, for P. proteolytica L1-105, the biofilm-associated cells were 190 times more peptidase-active compared to the planktonic growth form. These results indicate that biofilms may represent an important entry route of Pseudomonas spp. into raw milk and may contribute to milk spoilage through increased peptidase production even at refrigeration conditions.