Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana.
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana.
Biotechnol Prog. 2019 Nov;35(6):e2874. doi: 10.1002/btpr.2874. Epub 2019 Jul 8.
To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.
使用富集培养基在菠菜悬浮液中达到沙门氏菌检测阈值至少需要 24 小时。通过微滤和微沉淀分离和浓缩选定的微生物可以减少样品制备的时间，特别是在处理大量蔬菜悬浮液时。这有助于加速检测菠菜中的沙门氏菌，并有助于在沙门氏菌到达消费者之前对其进行有效监测。我们报告了一种基于微滤的加速样品制备方案，用于浓缩和回收≤1 个菌落形成单位 (CFU) 的无菌菠菜中的沙门氏菌。测试了市售的菠菜样品和在生产过程中经历两种环境条件（温度和光照暴露）的菠菜植株。整个过程包括用缓冲液提取、短时间的富集步骤、使用尼龙过滤器进行预过滤、错流中空纤维微滤以及将微生物细胞浓缩至检测水平的保留物离心。基于 1 CFU/g 冷冻菠菜中的沙门氏菌，以及 99%概率的泊松分布统计分析，我们计算出 3 小时的孵育时间，随后进行微滤，足以在 7 小时内达到沙门氏菌检测所需的 2 对数浓度。对于浓度低于 1 CFU/g 的即食菠菜，需要更长的富集时间（5 小时或更长时间）。使用聚合酶链反应 (PCR) 和平板计数法鉴定和确认回收的微生物细胞为沙门氏菌。在生产过程中测试的不同环境条件并未影响沙门氏菌的活力；这证明了沙门氏菌的广泛适应性，并强调需要能够有效监测生产过程中是否存在该病原体的方法。
