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

Abstract

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra‐high‐temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat‐resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf‐life.

The Predominance of Psychrotrophic Pseudomonads on Aerobically Stored Chilled Red Meat

“…The genes encoding protease (aprX) and lipase (lipA) are encoded in an operon conserved in pseudomonads (Zhang et al, 2019) and located at opposite ends of the same operon (Woods, Burger, Beven, & Beacham, 2001). Several factors, such as temperature, iron content, and quorum sensing, affect the regulation of lipase and peptidase production in psychrotrophic Pseudomonas (Machado et al, 2017;Woods et al, 2001).…”

Application of LC‐HRMS identified marker peptides in an LC‐MS/MS method for detection and quantification of heat‐resistant proteolytic activity in raw milk

“…The LC-MS/MS method is based on detection of marker peptides produced by pseudomonads which are the most important psychrotrophs in cooled raw milk worldwide. These bacteria produce an extracellular heat-resistant proteolytic enzyme AprX which survives the UHT treatment and causes shelf-life problems occurring mainly in UHT milk and in other dairy products to a lesser degree (Zhang et al 2019). This problem has increased in recent years because of the increasing export of UHT milk with long transport times.…”

“…Matta et al 1997), whereas multiple antibodies would be needed to cover the AprX diversity, with no guarantee of adequate sensitivity. Direct detection methods for AprX enzymatic activity such as the spectrophotometric TNBS, the OPA or azocasein method or the fluorometric FITC-casein method are not sensitive enough for fast detection of low AprX levels (reviewed by Zhang et al 2019). The same probably applies for total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) which was recently investigated as an alternative rapid technique to assess proteolysis in UHT milk stored for up to 4 months (Ranvir et al 2020).…”

“…As reviewed by Zhang et al . (2019), there is a consensus in literature that AprX of pseudomonads preferentially hydrolyses κ‐casein, but nevertheless a relatively low number of peptides from this casein type have been previously identified by LC‐MS in comparison to β‐casein. It is proposed that this is due to the specific region in which κ‐casein is cleaved and the difficulty in detecting the resulting glycosylated peptides using mass spectrometry.…”

Effect of Temperature-Dependent Bacterial Growth during Milk Protein Fractionation by Means of 0.1 µM Microfiltration on the Length of Possible Production Cycle Times

“…The activity of proteolytic enzymes was also considered as a possible factor for the rapid pH decrease at 55 • C since enzymatic protein hydrolysis leads to a reduced pH. However, Zhang et al [34] showed an autolytic temperature at 55 • C for the AprX protease produced by Pseudomonas. Furthermore, Kroll and Klostermeyer [35] also reported an increased inactivation of proteinase produced by Pseudomonas fluorescens at 55 • C. Therefore, an increased protease activity did not seem to be the main effect with regard to the sudden drop in pH observed at 55 • C.…”