Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; State Key Laboratory of Grassland Agro-Ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China; Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Sci Total Environ. 2022 Sep 10;838(Pt 3):156382. doi: 10.1016/j.scitotenv.2022.156382. Epub 2022 May 31.
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.
假单胞菌是冷藏生牛乳中常见的微生物，假单胞菌分泌的蛋白酶会导致贮存牛奶的变质。然而，中国不同地区生牛乳中假单胞菌的多样性和蛋白水解活性的分析尚未广泛研究。为此，本研究分别采用 PCR 靶向 16S rDNA 和 rpoD 以及 TNBS 法，评估了中国内蒙古、黑龙江、甘肃、河南、安徽、江苏、重庆和湖南不同季节 25 份生牛乳样本中分离的假单胞菌的多样性和蛋白水解活性。从 25 份生牛乳样本中获得的 116 株假单胞菌经鉴定达到种的水平，包括荧光假单胞菌、 Verona 假单胞菌、嗜冷假单胞菌、Lundensis 假单胞菌、乳杆菌、固氮假单胞菌、Granadensis 假单胞菌、Lurida 假单胞菌、根际假单胞菌、Rhodesiae 假单胞菌和 extremorientalis 假单胞菌。荧光假单胞菌占总数的 75.8%。在所有 116 株假单胞菌中，68.9%在 4°C 时具有蛋白水解活性，81.9%在 10°C 时具有蛋白水解活性，85.3%在 25°C 时具有蛋白水解活性。aprX 基因编码一种分泌的耐热金属蛋白酶，在 60.3%的测试假单胞菌中存在。蛋白酶经 72°C、15s 热处理后的残留活性为 73±4%至 84±7%，经 132°C、4s 热处理后的残留活性为 62±3%至 74±2%。这是首次比较中国不同地区生牛乳中假单胞菌属的多样性和蛋白水解活性。本研究结果为生牛乳中假单胞菌属的多样性和腐败潜力以及蛋白酶的耐热性提供了有价值的数据。因此，这些发现为防止生牛乳中假单胞菌属污染以确保牛奶和乳制品的质量和安全提供了参考。
