Diversity and proteolytic activity of Pseudomonas species isolated from raw cow milk samples across China

Pseudomonas spp. are common microorganisms from cold-storage raw milk, and protease secreted by Pseudomonas spp. can cause the deterioration of stored milk. However, analyses of Pseudomonas spp. diversity and proteolytic activity in raw milk from different regions of China have not been extensively examined. With this aim, the diversity and proteolytic activity of Pseudomonas isolated from 25 raw cow milk samples from Inner Mongolia, Heilongjiang, Gansu, Henan, Anhui, Jiangsu, Chongqing and Hunan of China in different seasons were evaluated by PCR targeting 16S rDNA and rpoD, as well as TNBS method, respectively. A total of 116 Pseudomonas isolates from 25 raw cow milk samples were identified at the species level, including P. fluorescens, P. veronii, P. psychrophila, P. lundensis, P. lactis, P. azotoformans, P. granadensis, P. lurida, P. rhizosphaerae, P. rhodesiae and P. extremorientalis. P. fluorescens accounted for 75.8% of the total. Of all 116 Pseudomonas isolates, 68.9% of them displayed proteolytic activity at 4 °C, 81.9% at 10 °C and 85.3% at 25 °C, respectively. The aprX gene encoded a secreted and heat-resistant metalloprotease that was present in 60.3% of the Pseudomonas isolates tested. The proteases showed residual activity ranged from 73 ± 4% to 84 ± 7% residual activity after the heat treatment at 72 °C for 15 s and 62 ± 3% to 74 ± 2% after the heat treatment at 132 °C for 4 s. This is the first report to compare Pseudomonas spp. diversity and proteolytic activity at species levels in raw milk from different regions of China. The results of this study provide valuable data about the diversity and spoilage potential of Pseudomonas species in raw milk and the thermal resistance of the proteases. Therefore, these findings provide a reference for the importance to prevent Pseudomonas spp. contamination of raw cow milk to ensure the quality and safety of milk and dairy products.