Seasonality and geography have a greater influence than the use of chlorine-based cleaning agents on the microbiota of bulk tank raw milk.

Cleaning of the production environment is vital to ensure the safety and quality of dairy products. Although cleaning with chlorine-based agents is widely adopted, it has been associated with detrimental effects on milk quality and safety, which has garnered increasing interest in chlorine-free cleaning. However, the influence of these methods on the milk microbiota is not well documented. This study investigated the factors that influence the raw milk microbiota, with a focus on the differences when chlorine-based and chlorine-free cleaning of milking equipment are used. Bulk tank raw milk was sampled at three sampling months (Apr, Aug and Nov), from farms across Ireland selected to capture the use of different cleaning methods, i.e., exclusively chlorine-based (n = 51) and chlorine-free cleaning (n = 92), and farms that used chlorine-free agents for the bulk tank and chlorine-based cleaning agents for the rest of the equipment (n = 28). Shotgun metagenomic analysis revealed the significant influence of seasonal and geographic factors on the bulk tank milk microbiota, indicated by differences in diversity, taxonomic composition, and functional characteristics. Taxonomic and functional profiles of samples collected in November clustered separately from other months. In contrast, cleaning methods only accounted for 1% of the variation in the bulk tank milk bacterial community, and samples collected from farms using chlorine relative to chlorine-free cleaning did not differ significantly, suggesting that chlorine-free approaches used did not negatively impact microbiological quality. This study shows the value of shotgun metagenomics in advancing our knowledge of the raw milk microbiota.