After electrophoretic separation, semidry blotting was performed. The proof of concept to detect heat-stable Pseudomonas AprX in milk by ELISA was established. The lowest AprX activity quantifiable in the spiked milk samples was 500 pkat Na-caseinate/OPA mL −1. The recovery of the ELISA was 92.3 ± 1.6 to 105 ± 4.7%. Milk samples were spiked with different AprX activity levels and evaluated by ELISA. 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 proteins or milk endogenous peptidases were not detected by the antibodies. 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. ![]() lactis and several other Pseudomonas spp. Western blot experiments showed that the binding affinity of these antibodies depended on the sequence homology of the AprX from P. Specific antibodies for purified AprX from Pseudomonas lactis were produced to establish the ELISA. The recovery of AprX in spiked milk samples after the 2-step treatment was 43 ± 0.1%. First, casein micelles were destabilized by the detraction of Ca 2+ using trisodium citrate then, AprX was concentrated 10-fold using hydrophobic interaction chromatography. We developed a 2-step sample treatment for milk contaminated with AprX to avoid the interference of milk proteins with the detection system. Therefore, we established an indirect ELISA for the detection of Pseudomonas AprX in milk. ![]() They can withstand UHT treatment and may cause quality defects over the shelf life of milk products. Heat-stable endopeptidases in raw milk, especially the alkaline metallopeptidase AprX secreted by Pseudomonas spp., are a well-known challenge for the dairy industry.
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