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

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.     

An indirect ELISA system for the detection of heat-stable Pseudomonas endopeptidases (AprX) in milk

Heat-stable endopeptidases in raw milk, especially the alkaline metallopeptidase AprX secreted by Pseudomonas spp., are a well-known challenge for the dairy industry. They can withstand UHT treatment and may cause quality defects over the shelf life of milk products. Therefore, we established an indirect ELISA for the detection of Pseudomonas AprX in milk. We developed a 2-step sample treatment for milk contaminated with AprX to avoid the interference of milk proteins with the detection system. First, casein micelles were destabilized by the detraction of Ca²⁺ using trisodium citrate; then, AprX was concentrated 10-fold using hydrophobic interaction chromatography. The recovery of AprX in spiked milk samples after the 2-step treatment was 43 ± 0.1%. Specific antibodies for purified AprX from Pseudomonas lactis were produced to establish the ELISA. Western blot experiments showed that the binding affinity of these antibodies depended on the sequence homology of the AprX from P. lactis and several other Pseudomonas spp. The indirect ELISA, which was completed in 6 to 7 h, had a limit of detection of 21.0 ng mL^?1 and a limit of quantification of 25.7 ng mL^?1. Milk proteins or milk endogenous peptidases were not detected by the antibodies. The ELISA had high precision, with a CV between 0.2 and 0.8% measured on the same day (intraday) and 5.6 and 6.8% measured on 5 separate days (interday). Milk samples were spiked with different AprX activity levels [7.5–150 nkat Na-caseinate/o-phthalaldehyde (OPA) mL^?1] and evaluated by ELISA. The recovery of the ELISA was 92.3 ± 1.6 to 105 ± 4.7%. The lowest AprX activity quantifiable in the spiked milk samples was 500 pkat Na-caseinate/OPA mL^?1. The proof of concept to detect heat-stable Pseudomonas AprX in milk by ELISA was established.