| Summary: | The proteolytic traits of the psychrotrophic strains Pseudomonas poae LP5, Pseudomonas fluorescens LPF3, Chryseobacterium joostei LPR1, Pseudomonas fulva PS1, Citrobacter freundii PS37, Hafnia alvei PS46, and Serratia marcescens PS92 were initially investigated by phenotypic and genotypic approaches. Six strains elicited extracellular proteolytic activity, and five expressed the thermostable AprX or (likely) Ser1 enzymes. Then, the strains were inoculated (10<sup>4</sup> CFU/mL) in microfiltered pasteurized milk and kept at 4 °C for five days. All of the strains reached 10<sup>8</sup> CFU/mL at the end of storage and five produced thermostable extracellular proteolytic enzymes. The freshly inoculated samples and the corresponding samples at 10<sup>8</sup> CFU/mL were batch-sterilized (131 °C, 30 s) and kept at 45 °C up to 100 days. The former samples did not gel until the end of incubation, whereas the latter, containing <i>P. poae</i>, <i>P. fluorescens</i>, <i>C. joostei</i>, <i>C. freundii</i>, and <i>S. marcescens,</i> gelled within a few days of incubation. The thermostable proteolytic activity of strains affected the peptidomic profile, and specific proteolyzed zones of β-CN were recognized in the gelled samples. Overall, the results confirm some proteolytic traits of psychrotrophic Pseudomonas spp. strains and provide additional insights on the proteolytic activity of psychrotrophic bacteria potentially responsible for sterilized milk destabilization.
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