Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk

Abstract

The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli . The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca +2 . The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca +2 , on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.

Biochemical Characterization of an Extracellular Heat‐Stable Protease from Serratia liquefaciens Isolated from Raw Milk

“…These results showed that Mn 2+ is not required but can influence positively the Ser2 activity. Similar results were found with heat‐stable enzyme of P. fluorescens Rm12 (Mu and others ) and the protease AprX of P. fluorescens 041 (Martins and others ).…”

Lipolytic psychrotrophic bacteria and lipase heat-resistant property in bovine raw milk of North China

“…Although the isolate were dead, the lipase which was produced by P. putida and P. fluorescens appeared to be heat stable. Lipase of isolates 7, 11 and 38 presented the highest heat stability compared to the ones of the other isolates as already mentioned in the literature (Lee & Rhee, ; Martins, Pinto, Riedel, & Vanetti, ). The study demonstrated that several lipolytic psychrotrophic bacteria key species were to be explored due to their action on the lipolytic degradation of milk products.…”

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review

“…At a pH higher than the isoelectric point, the proteolytic activity of AprX remains high across the pH range of 4 to 10 (Table ), because the ionization and deprotonation generated by increasing the pH do not disrupt the flexible enzyme structure of AprX (Alves et al., ). It is noteworthy that even though AprX is most active at neutral pH, it still exhibits a relatively high activity at low pH, which means that AprX can also influence the quality of fermented products, like yogurt and cheese (Martins et al., ), as further discussed in section Effects of AprX on the Quality of Other Dairy Products.…”

“…Martins et al. () found that AprX lost 96% of its activity when it was incubated in a buffer at low temperature long time pasteurization conditions (65 °C for 30 min), while it lost only 30% of its activity when treated at high temperature short time pasteurization conditions (75 °C for 20 s).…”