Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China.

The quality of raw milk is a key factor influencing the whole dairy processing chain. The richness and diversity of bacteria in raw milk affect its quality and safety. However, traditional microbial detection methods mainly depend on the known microbe culture and are often time consuming. Thus, the development of efficient ways for supervising any possible microbiological contamination is desiderated. In the current work, single-molecule real-time (SMRT) sequencing, developed by Pacific Biosciences (PacBio), was applied to acquire long reads and applied for discrimination of bacteria at species level. Forty samples of raw milk obtained from Beijing, Hebei, Inner Mongolia, Shanghai, and Guangdong in China during summer, autumn, and winter were investigated. Among 35 bacteria species identified in these samples, Acinetobacter albensis, Pseudomonas gessardii, Pseudomonas weihenstephanensis, and Rahnella inusitata were the bacteria with the highest relative abundance in the overall sample, whereas the bacteria with the highest relative abundance in raw milk samples of different origins and seasons are different. Significant differences in bacterial richness and bacterial community diversity in raw milk grouped according to different production areas and different sampling seasons were confirmed by Welch's t-test. Interestingly, the transport distance and transport time positively correlated with the relative abundance of Pseudomonas weihenstephanensis, suggesting that the content of this bacteria was expected to be a standard for evaluating the freshness of raw milk. Pathogens Bacillus cereus and Klebsiella pneumoniae were detected in most samples, indicating that the raw milk was at risk of contamination by pathogenic bacteria. Moreover, the findings of this study provide important evidence for quality and safety monitoring and biological control of raw milk.