Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

Abstract

Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

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

“…As described in section Hydrolysis Patterns of Milk Proteins by AprX, AprX cannot only cleave the peptide bond Phe 105 -Met 106 of κ-casein, like chymosin (Recio, García-risco, Ramos, & López-fandiño, 2000), but also nonspecifically cleave in the region of the soluble hydrophilic caseino-macropeptide (CMP) which is normally present as the "hairy layer" or "polyelectrolyte brush" on casein micelles (Gaucher et al, 2011). Such cleavage Andreani et al (2016) and Samaržija et al (2012).…”

“…Genetic methods, especially PCR, have been considered as the "gold standard" method for bacterial identification (Vithanage, 2017). Recently, many researchers have also tried to develop a screening test for detecting the aprX gene (Bach, Hartmann, Schloter, & Munch, 2001;Machado, Bazzolli, & Vanetti, 2013;Marchand et al, 2009b;Martins et al, 2005). The principle of this method is determining the presence of the aprX gene in bacterial cells as an indirect indicator of the spoilage potential using specific primers.…”

“…The principle of this method is determining the presence of the aprX gene in bacterial cells as an indirect indicator of the spoilage potential using specific primers. Bach et al (2001) first indicated that PCR primers targeting the genes apr, npr, and sub could be used for detection of a wide range of proteolytic bacteria, as these genes are all part of the same genomic region as explained in section aprX Gene, Amino Acid Sequence, and Protein Structure. Later on, Martins et al (2005) found that the apr gene could be directly amplified from pasteurized milk inoculated with 10 8 cfu·mL −1 of P. fluorescens.…”

“…Age gelation is an irreversible condition, described as coagulation, sweet‐curd formation, thixotropic thickening, or lumpiness, after the product has been stored for weeks to months. AprX has been reported to promote the occurrence of age gelation by mediating the hydrolysis of caseins (Andreani et al., ; Baglinière et al., ; Stoeckel et al., ).…”

Identification ofPseudomonas jesseniiandPseudomonas gessardiias the most proteolyticPseudomonasisolates in Iranian raw milk and their impact on stability of sterilized milk during storage

“…Producing heat-resistant extracellular proteases during growth is very common for Pseudomonas spp. The fact that current industrial heat treatment is not severe enough to deactivate them illustrates their importance in the dairy industry (Baur et al ., 2015; Andreani et al ., 2016; Glück et al ., 2016).…”