Division of Food Science, Toyo Institute of Food Technology, 23-2, 4-chome, Minami-hanayashiki, Kawanishi, Hyogo 666-0026, Japan.
Int J Food Microbiol. 2020 Jun 16;323:108573. doi: 10.1016/j.ijfoodmicro.2020.108573. Epub 2020 Mar 14.
Spoilage microorganisms can occur at many points throughout food production systems. Bacillus spp. and Paenibacillus spp. are important aerobic spoilage bacteria in various sectors of the food industry. In this study, we developed a rapid detection and quantification technique for Bacillus group-specific and the genus Paenibacillus by using multiplex quantitative PCR (mqPCR). The 1st was the Bacillus cereus group containing B. cereus and B. weihenstephanensis; the 2nd was the B. subtilis group containing B. subtilis, B. licheniformis, B. safensis, and B. pumilus; the 3rd was the B. simplex group containing B. megaterium and B. simplex; and the 4th was the genus Paenibacillus. Depending on the assays, the detection limit was 10 copy numbers. In addition, mqPCR assays were validated by spiking potato salad and milk samples with four strains; B. weihenstephanensis, B. licheniformis, B. megaterium, and P. lautus. The detection dynamic range for potato salad was 10 CFU/mL-10 CFU/mL with B. weihenstephanensis and B. licheniformis, and 10 CFU/mL-10 CFU/mL with B. megaterium and P. lautus, while, for milk, all strains were 10 CFU/mL-10 CFU/mL. We also stored these food matrices spiked with four bacterial suspensions (approximately 10 CFU/mL) at various temperatures. Results showed that B. weihenstephanensis and B. licheniformis were able to grow in potato salad, whereas, the populations of B. weihenstephanensis, B. licheniformis, and P. lautus increased in milk. Consequently, the mqPCR assays developed here in facilitated the differentiation, quantification, and confirmation of the presence of the psychrophilic and psychrotolerant Bacillus group and Paenibacillus spp.
腐败微生物可以在食品生产系统的许多环节中出现。芽孢杆菌属和类芽孢杆菌属是食品工业各领域中重要的需氧腐败细菌。在本研究中，我们使用多重定量 PCR（mqPCR）开发了一种快速检测和定量芽孢杆菌属特异性和类芽孢杆菌属的技术。第 1 组是包含蜡样芽孢杆菌和魏氏芽孢杆菌的蜡样芽孢杆菌组；第 2 组是包含枯草芽孢杆菌、地衣芽孢杆菌、沙福芽孢杆菌和短小芽孢杆菌的枯草芽孢杆菌组；第 3 组是包含巨大芽孢杆菌和简单芽孢杆菌的简单芽孢杆菌组；第 4 组是类芽孢杆菌属。根据检测，检测限为 10 个拷贝数。此外，通过向土豆沙拉和牛奶样品中添加 4 株菌（魏氏芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌和类劳特氏菌）对 mqPCR 检测进行了验证。土豆沙拉的检测动态范围为 10 CFU/mL-10 CFU/mL，含魏氏芽孢杆菌和地衣芽孢杆菌，10 CFU/mL-10 CFU/mL，含巨大芽孢杆菌和类劳特氏菌，而牛奶中所有菌株均为 10 CFU/mL-10 CFU/mL。我们还将这些含有 4 种细菌悬液（约 10 CFU/mL）的食物基质在不同温度下储存。结果表明，魏氏芽孢杆菌和地衣芽孢杆菌能够在土豆沙拉中生长，而魏氏芽孢杆菌、地衣芽孢杆菌和类劳特氏菌的种群在牛奶中增加。因此，本研究中开发的 mqPCR 检测方法有助于区分、定量和确认低温和耐冷的芽孢杆菌组和类芽孢杆菌属的存在。
