Abstract: Eight Bacillus cereus strains were isolated from 10 batches of raw milk and identified through 16S rDNA sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Out of these strains, one was selected on the basis of higher protease production and identified as B. cereus LY-6. We inoculated this strain back into skim milk and observed the resulting spoilage process. It was found that the milk turned to yellow, and had a bitter taste and an unpleasant odor. Moreover, the pH decreased and large amounts of proteases were found, indicating that the spoilage process is closely related to microbial protease production. Strain LY-6 was intolerant to ultra-high temperature sterilization. A distribution map of B. cereus in the dairy was drawn to control its spread. It was found that this bacterium mainly existed in places where milk stains are difficult to clean such as pasture bedding, towels, milk trucks, workshop floors, personnel shoe soles, and equipment surfaces. Therefore, special attention should be paid to the cleaning effect of the backend pipeline after the sterilization process to prevent potential food safety hazards caused by secondary contamination of B. cereus.

Key words: Bacillus cereus; raw milk; protease; bitterness; 16S rDNA; matrix-assisted laser desorption ionization time-of-flight mass spectrometry

摘要： 从10 批生牛乳中分离出8 株蜡样芽孢杆菌，同时采用16S rDNA测序和基质辅助激光解吸电离飞行时间质谱对该菌进行鉴定；通过产蛋白酶活力测试筛选到1 株高产蛋白酶的菌株蜡样芽孢杆菌LY-6，通过将其人工污染至正常脱脂牛乳中，观察其变质过程，发现色泽由乳白色变成黄色，口感出现苦味、臭味，pH值降低；且通过蛋白酶实验发现人工污染样品中含有大量蛋白酶，说明其变质过程与微生物产生的蛋白酶有很大关系。同时对该菌的耐热性进行分析，发现该菌不耐受超高温杀菌工艺。为了更好管控该微生物，建立蜡样芽孢杆菌在牛乳工厂的分布地图，发现蜡样芽孢杆菌主要存在于牧场垫床、毛巾、奶车残留清洗水、车间的地面、人员鞋底及设备表面等奶垢难清洗处。应重点关注杀菌工艺后端管路的清洗效果，防止蜡样芽孢杆菌的二次污染带来的食品安全隐患。

关键词: 蜡样芽孢杆菌；生牛乳；蛋白酶；苦味；16S?rDNA；基质辅助激光解吸电离飞行时间质谱