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

Abstract

Identification of the most proteolytic Pseudomonas strains that can produce heat-resistant proteases and contribute to the Ultra High Temperature (UHT) milk destabilization is of great interest. In the present study, among the 146 Pseudomonas isolates that encoded the aprX gene, five isolates with the highest proteolytic activity were selected and identified based on 16S rRNA, rpoD and gyrB gene sequences data. The identification results were confirmed by phylogenetic analysis based on multilocus sequence analysis and identified the representative isolates as P. jessenii (two isolates) and P. gessardii (three isolates). Casein zymography demonstrated the ability of these species to produce heat-resistant enzymes, AprX, with molecular mass of about 48 kDa during storage at 7° C for 72 h. In sterilized milk samples, the residual activity of AprX caused a considerable enhancement in the degree of protein hydrolysis, non-protein nitrogen and non-casein nitrogen contents of the samples during a two-month storage. This enhancement was slightly higher in samples containing enzyme produced by P. jessenii compared to P. gessardii ones, resulting in earlier onset of sterilized milk destabilization. Hence, this study revealed that P. jessenii and P. gessardii can play a considerable role in deterioration of Iranian commercial long-life milk.

The effect of extracellular protease secreted by Pseudomonas fluorescens W3 on the quality of UHT milk

“…P. fluorescens W3 could secrete a heat‐resistant protease with a molecular weight of approximately 45 kDa (Figure 1a). The molecular weight of protease AprX was usually between 40 and 50 kDa (Zarei et al, 2020). In our previous study, the protease AprX was identified from the extracellular secretions of P. fluorescens W3 by proteomics (Wang et al, 2021), and this also proved that the heat‐resistant protease secreted by P. fluorescens W3 was AprX.…”

Identification, proteolytic activity quantification and biofilm-forming characterization of Gram-positive, proteolytic, psychrotrophic bacteria isolated from cold raw milk

“…One tube was frozen at -20˚C (blank) and the others were incubated at 7˚C and 25˚C for 2 weeks [10,30,31]. Hydrolysis of proteins was measured by the O-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC) fluorometric assay to determine free amino groups of milk, as explained previously [24,32]. The fraction of free amino groups (F/F0) was the relative fluorescence unit (RFU) of milk sample after treatment divided by their initial RFU (blank).…”

“…In light of the major economic impact of proteolytic psychrotrophic bacteria on the dairy industry, these bacteria have been, and continue to be, extensively studied with the main purpose of establishing effective control measures and appropriate regulations to ensure that milk and dairy products are of a high standard. To date, the majority of studies have been performed to identify Gram-negative, proteolytic, psychrotrophic bacteria in raw milk, and to evaluate their capacity to produce proteolytic enzymes and biofilms [3,[19][20][21][22][23][24][25]. Although, various genera of Gram-positive bacteria, e.g., Bacillus, Lactococcus, Staphylococcus, Microbacterium and Enterococcus with the ability to produce biofilm and/or proteolytic enzymes have been identified in raw milk from different geographical regions [20,[26][27][28], still little is known about the biofilm formation and proteolytic activity of Gram-positive psychrotrophic bacteria, which constitute approximately 10% of the microbial population in cold raw milk that has been stored.…”