Abstract
The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85–97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103–107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected.Key points• Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk• High specificity and sensitivity via hydrolysis probes against aprX and rpoB• Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential
Highlights
Several studies revealed a high variability of milk-associated Pseudomonas species and strains regarding their proteolytic potential, which has been proposed to be due to different gene expression and regulation mechanisms (Dufour et al 2008; Marchand et al 2009b; Bagliniere et al 2012; von Neubeck et al 2015; Caldera et al 2016)
AprX was chosen for species-specific detection, and the conserved rpoB gene for the quantification of total Pseudomonas counts
With respect to the seven chosen target species, the aprX tree exhibited a distribution of the 18 representative strains in four Pseudomonas subgroups, namely P. fluorescens, P. gessardii, P. fragi, and P. chlororaphis
Summary
Premature spoilage of ultra-high temperature (UHT) milk and milk products due to microbial extracellular enzymesAppl Microbiol Biotechnol (2021) 105:1693–1708 and Stepaniak 1997; Matéos et al 2015; Stoeckel et al 2016a; Marchand et al 2017).The alkaline zinc-metallopeptidase AprX, belonging to the serralysin protease family, has a molecular weight of 45–50 kDa and is encoded by the polycistronic aprX-lipA2 operon (Schokker and van Boekel 1997; Woods et al 2001; Marchand et al 2009b). Several studies revealed a high variability of milk-associated Pseudomonas species and strains regarding their proteolytic potential, which has been proposed to be due to different gene expression and regulation mechanisms (Dufour et al 2008; Marchand et al 2009b; Bagliniere et al 2012; von Neubeck et al 2015; Caldera et al 2016). While strains of P. proteolytica, P. lactis, P. protegens, P. gessardii, and P. fluorescens exhibited mainly high proteolytic activity, isolates of P. lundensis or P. fragi had middle or low proteolytic potential (Marchand et al 2009a; De Jonghe et al 2011; Baur et al 2015; von Neubeck et al 2015; Caldera et al 2016; Glück et al 2016; Maier et al 2020)
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