With the increasing demand for liquid milk, it is particularly important to ensure its quality and safety. The presence of heat-resistant microorganisms in liquid milk is a non-negligible cause of its quality defects. Therefore, this study employed high-throughput sequencing to explore the microbial diversity of raw milk. Heat-resistant microorganisms were isolated in pure culture and identified by 16S rDNA sequencing and their biological diversity and morphological characteristics were analyzed. Subsequently, the identified bacteria were inoculated into normal sterilized milk and skim milk to simulate the spoilage process of products contaminated by them, and their tolerance was examined to provide ideas to screen and control abnormal milk products. The results showed that the heat-resistant microorganisms in raw milk are mainly of the genus Bacillus, and three colony forms were found by Gram staining. Particle precipitation and delamination after inoculation of different types of Bacillus into sterilized milk; yellow color and bitter taste after inoculation into skim milk. All three Bacillus species had strong temperature tolerance but poor tolerance to peracetic acid. Milk enterprises can combine peracetic acid-based disinfectants with Clean-in-Place cleaning to effectively kill residual heat-resistant microorganisms on the production line.

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.