Techniques to Detect and Quantify the Bacterial Metalloprotease AprX in Bovine Milk: A Review.

The heat-stable metalloprotease AprX, secreted by psychrotrophic Pseudomonas spp., is a major cause of quality deterioration in dairy products, particularly ultrahigh temperature (UHT) milk. This review synthesizes the evolution and current state of detection and quantification techniques for AprX in bovine milk, covering traditional immunological assays, enzymatic activity measurements, and zymography, alongside modern molecular approaches such as PCR-based methods, mass spectrometry, and advanced biosensors. Highly sensitive systems, including indirect ELISA (LOD 21.0 ng/mL), multiplex qPCR, and biosensor platforms, have enhanced the ability to detect AprX activity at low concentrations. Comparative analysis evaluates these methods in terms of sensitivity, specificity, turnaround time, cost, ease of use, and industrial applicability. Emerging directions such as multiomics integration, biosensor miniaturization, and artificial intelligence-driven data interpretation are also discussed. By critically assessing available and emerging tools, this review supports dairy scientists and industry stakeholders in selecting optimal strategies to detect, monitor, and mitigate AprX-associated spoilage in milk and dairy products.