Milk Quality Improvement Program, Department of Food Science, Cornell University, Ithaca, NY 14853.
Milk Quality Improvement Program, Department of Food Science, Cornell University, Ithaca, NY 14853.
J Dairy Sci. 2018 Sep;101(9):7746-7756. doi: 10.3168/jds.2017-14216. Epub 2018 Jun 13.
Microbial spoilage of pasteurized fluid milk is typically due to either (1) postpasteurization contamination (PPC) with psychrotolerant gram-negative bacteria (predominantly Pseudomonas) or (2) growth of psychrotolerant sporeformers (e.g., Paenibacillus) that have the ability to survive pasteurization when present as spores in raw milk, and to subsequently grow at refrigeration temperatures. While fluid milk quality has improved over the last several decades, continued reduction of PPC is hampered by the lack of rapid, sensitive, and specific methods that allow for detection of PPC in fluid milk, with fluid milk processors still often using time-consuming methods (e.g., Moseley keeping quality test). The goal of this project was to utilize a set of commercial fluid milk samples that are characterized by a mixture of samples with PPC due to psychrotolerant gram-negative bacteria and samples with presence and growth of psychrotolerant sporeforming bacteria to evaluate different approaches for rapid detection of PPC. Comprehensive microbiological shelf-life characterization of 105 pasteurized fluid milk samples obtained from 20 dairy processing plants showed that 60/105 samples reached bacterial counts >20,000 cfu/mL over the shelf-life due to PPC with gram-negative bacteria. Among these 60 samples with evidence of gram-negative PPC spoilage over the shelf-life, 100% (60/60) showed evidence of contamination with noncoliform, non-Enterobacteriaceae (EB) gram-negative bacteria (e.g., Pseudomonas), 20% (12/60) showed evidence of contamination with coliforms, and 7% (4/60) showed evidence of contamination with noncoliform EB. Among the remaining 45 samples, 28 showed levels of gram-positive bacteria above 20,000 cfu/mL and the remaining 17 samples did not exceed 20,000 cfu/mL over the shelf-life. Evaluation of the same set of 105 samples using 6 different approaches {all possible combinations of 2 different enrichment protocols (13°C or 21°C for 18 h) and 3 different plating media [crystal violet tetrazolium agar, EB Petrifilm (3M, St. Paul, MN), and Coliform Petrifilm]} showed that enrichment at 21°C for 18 h, followed by plating on crystal violet tetrazolium agar provided for the most sensitive, accelerated detection of samples that reached >20,000 cfu/mL due to PPC with psychrotolerant gram-negatives (70% sensitivity). These results show that tests still required and traditionally used in the dairy industry (e.g., coliform testing) are not suitable for monitoring for PPC. Rather, approaches that allow for detection of all gram-negative bacteria are essential for improved detection of PPC in fluid milk.
（1） 巴氏杀菌后污染（PPC），存在耐冷革兰氏阴性菌（主要是假单胞菌）；（2） 耐冷孢子形成菌（例如，芽孢杆菌）的生长，这些菌在生乳中以孢子形式存在时能够耐受巴氏杀菌，随后在冷藏温度下生长。尽管过去几十年液态奶质量有所提高，但由于缺乏快速、敏感和特异性的方法来检测液态奶中的 PPC，PPC 的持续减少仍受到阻碍，液态奶加工商仍经常使用耗时的方法（例如，Moseley 保持质量测试）。本项目的目标是利用一组商业液态奶样本，这些样本的特点是既有因耐冷革兰氏阴性菌导致的 PPC 样本，也有存在耐冷孢子形成菌并生长的样本，以评估快速检测 PPC 的不同方法。对从 20 家乳制品加工厂获得的 105 个巴氏杀菌液态奶样本进行全面微生物货架期特征描述，结果表明，由于耐冷革兰氏阴性菌的 PPC，60/105 个样本在货架期内的细菌计数超过 20,000 cfu/mL。在这些在货架期内出现革兰氏阴性 PPC 腐败的 60 个样本中，100%（60/60）显示出受到非大肠菌群、非肠杆菌科（EB）革兰氏阴性菌（如假单胞菌）污染的证据，20%（12/60）显示出受到大肠菌群污染的证据，7%（4/60）显示出受到非大肠菌群 EB 污染的证据。在其余 45 个样本中，28 个样本的革兰氏阳性菌水平超过 20,000 cfu/mL，其余 17 个样本在货架期内未超过 20,000 cfu/mL。使用 6 种不同方法（13°C 或 21°C 下 18 小时的 2 种不同富集方案和 3 种不同的平板培养基[结晶紫四唑琼脂、EB Petrifilm（3M，圣保罗，明尼苏达州）和大肠菌群 Petrifilm]的组合）对同一组 105 个样本进行评估，结果表明，在 21°C 下 18 小时的富集，随后在结晶紫四唑琼脂上平板培养，可提供最敏感、加速检测因耐冷革兰氏阴性菌导致的>20,000 cfu/mL 的样本（70%的灵敏度）。这些结果表明，乳品行业仍需要且传统使用的检测方法（例如大肠菌群检测）不适合监测 PPC。相反，能够检测所有革兰氏阴性菌的方法对于提高液态奶中 PPC 的检测至关重要。
