Characterization of Pseudomonas spp. and Associated Proteolytic Properties in Raw Milk Stored at Low Temperatures

Abstract

Milk spoilage is caused by the presence of proteolytic enzymes produced by <i>Pseudomonas</i> spp. during storage at low temperatures. The aim of this study was to identify <i>Pseudomonas</i> spp. in raw milk and investigate their associated proteolytic properties at low temperatures. Raw milk samples (<i>n</i> = 87) were collected from 87 bulk tanks in Shaanxi Province in China. <i>Pseudomonas</i> spp. were identified using <i>Pseudomonas</i> specific 16S, universal <i>16S rRNA</i> sequencing, and <i>rpo</i>B gene sequencing. The proteolytic properties of <i>Pseudomonas</i> spp. were examined using milk agar, quantitative trinitrobenzenesulfonic acid assay, and by the presence of alkaline metallopeptidase gene (<i>apr</i>X). A total 143 isolates from all 87 samples were confirmed as <i>Pseudomonas</i>, and were identified as belonging to 14 <i>Pseudomonas</i> species. Of these, 40 (28.0%) isolates revealed proteolysis on milk agar at 2°C, 74 (51.8%) at 4°C, 104 (72.7%) at 7°C, and 102 (71.3%) at 10°C. However, proteolytic activity of 45 (31.5%) isolates exceeded 2 μmol of glycine equivalents per mL at 7°C, followed by 43 (30.1%) at 10°C, 18 (12.6%) at 4°C, and 7 (4.9%) at 2°C. The results reveal proteolytic activity of <i>Pseudomonas</i> spp. present in milk and their spoilage potential at different temperatures.