Quantification of psychrotrophic bacteria and molecular identification of Pseudomonas fluorescens in refrigerated raw milk

ABSTRACT In this study, we investigated the contamination of refrigerated raw milk produced in the western region of Paraná, southern Brazil, with psychrotrophic microorganisms, aiming to assay the proteolytic activity of the isolates and to identify Pseudomonas fluorescens, the main proteolytic species associated with the spoilage of milk products. Raw milk samples from 50 dairy farms were submitted to the counting of psychrotrophic microorganisms, being the microbiota characterized by its mesophilic behavior and proteolytic capacity, besides molecular identification of P. fluorescens. Of the samples evaluated, 94% had psychrotrophic counts ranging from 3 to 7.1 log CFU mL-1, and 48.5% of these showed mesophilic behavior. Of the isolates, 48.0% had proteolytic activity in at least one evaluated temperature (21 and 30°C), and 39.3% had proteolytic activity in both temperatures. Among the 61 isolates submitted to molecular identification by polymerase chain reaction (PCR), 86.8% contained the expression of the 16S gene characteristic for P. fluorescens. In this study, we demonstrated that P. fluorescens is the most prevalent psychrotrophic bacteria species in raw refrigerated milk and their proteolytic ability poses high risks to the dairy industry.摘要：本研究针对巴西南部巴拉那州西部产区的冷藏生鲜乳开展嗜冷微生物（psychrotrophic microorganisms）污染调查，旨在测定分离株的蛋白水解活性，并鉴定与乳品腐败密切相关的主要蛋白水解菌种——荧光假单胞菌（Pseudomonas fluorescens）。研究对50家奶牛场的生鲜乳样本进行嗜冷微生物计数，同时表征菌群的嗜温特性与蛋白水解能力，并开展荧光假单胞菌的分子鉴定。在所检测的样本中，94%的样本嗜冷微生物计数介于3至7.1 log CFU·mL⁻¹之间，其中48.5%的样本表现出嗜温特性。在分离得到的菌株中，48.0%的分离株在至少一个检测温度（21℃与30℃）下表现出蛋白水解活性，39.3%的分离株在两个温度下均具有蛋白水解活性。在通过聚合酶链式反应（polymerase chain reaction, PCR）进行分子鉴定的61株分离株中，86.8%的菌株携带荧光假单胞菌特征性16S基因的表达序列。本研究证实，荧光假单胞菌是冷藏生鲜乳中最为流行的嗜冷细菌菌种，其蛋白水解能力对乳品行业构成较高风险。