Abstract:Excessive psychrophilic bacteria in raw milk can produce high levels of heat-stable protease and lipase, both of which are important causes of quality problems when cow's milk enters the shelf life. In order to explore the microbial diversity of cows' living environment in pasture and different stages in the raw milk circulation process, the pollution source of psychrophilic bacteria in the pasture was found out. In this study, we collected five samples in a large-scale dairy farm in Hebei Province: TMR feed, cowshed pad soil, the first three milks, the normal milk after the first three milks, and the raw milk in the milk tank, and analyzed the bacterial community structure, psychrophilic species, abundance of psychrophilic bacteria in all samples by using metagenomic shotgun sequencing technology. The results showed that the mainly microbial were Proteobacteria, Firmicutes, and Actinobacteria at phylum level. At the genus level, the main genera were Anaplasma, Streptococcus, Enterococci, Spirospirita, Bacillus, and Pseudomonas. The soil of the barn contains 99.63% and 96.39% of the species of Pseudomonas in the normal milk and the total milk of the pasture storage tank, indicating that the soil of the barn mat is a potential source of pollution of various types of Pseudomonas.