Metagenomics approach to explore the changes of bacterial community in raw milk during refrigerated storage

Abstract: Refrigeration storage is one of the important methods to inhibit the reproduction of microorganisms in raw milk. But the psychrophilic bacteria still grow during this time, particularly on synthesizing phospholipids at the relatively low storage temperature. Meanwhile, these psychrophilic bacteria hydrolyze protein and fat can cause discoloration, odor, viscosity increase, gelation, rancidity, and other defects phenomena in the raw milk. So far, there are few reports on the changes of the flora in the raw milk during cold storage. In this study, an IlluminaHiseq sequencing platform was established to conduct metagenomic analysis of bacteria in the raw milk, in order to investigate the bacterial community succession and functional characteristics of raw milk with the extension of refrigeration time. During 72 hours of storage, the number of culturable microorganisms observably increased in the raw milk, including the number of colonies, psychrophilic bacteria, lactic acid bacteria, and coliform bacteria. Taxonomy annotation indicated that there was a significant effect for the microbial succession of raw milk during cold storage. At the genus level, the dominant flora gradually evolved from Acinetobacter, Streptococcus, Anaplasmaand and Clostridium to Flavobacterium, Pseudomonasand Lactococcus. At the species level, it evolved from Acinetobacter sp. TTH0-4, Anaplasmaphagocytophilum, Streptococcus pneumoniae, and Streptococcus mutans to Flavobacteriumfrigidarium, Pseudomonas fluorescensand Lactococcuspiscium. There were few reports on Acinetobacter sp. TTH0-4, Flavobacteriumfrigidarium, and Lactococcuspiscium. The results of COG functional annotation showed that the L (replication, recombination and repair), J (translation, ribosomal structure and biogenesis), M (cell wall/membrane/envelope biogenesis) had relatively higher abundance in the early stage of refrigeration (24 h), suggesting that the raw milk microbes were more resistant to the variable milk environment during this period. The relative abundance of E (Amino acid transport and metabolism) and G (Carbohydrate transport and metabolism) were higher in the late stage of refrigeration (72 h), indicating that the utilization of carbon and nitrogen sources by microbes increased at this stage. The results of correlation analysis between dominant microorganisms and main functions show that Acinetobacter was significantly related to the lipid metabolism. Combining with its characteristics of accumulating fatty acids, it infers that Acinetobacter had a greater impact on the composition of raw milk lipids. However, Pseudomonas was significantly related to the carbohydrate metabolism and amino acid metabolism, where to produce protease and lipase, and thereby pose a great influence on the decomposition of raw milk protein and the formation of flavor. In addition, the characteristics of Lactococcus, decomposing lactose to produce acid and inhibit bacteria, were closely related to the microbial control of raw milk and chess processing. The finding can provide new ideas for the research of raw milk microorganisms in the preservation of raw milk, sterilization control of liquid milk, and processing of dairy products. Further in-depth study of this subject can be conducted in the correlation analysis between metabolomics and metagenomics, and thereby to discover the mechanism of quality changes in the raw milk.