Composition and fate of heat-resistant anaerobic spore-formers in the milk powder production line

Anaerobic spore-forming bacteria are a continuous threat to the dairy industry due to their ability to withstand processing conditions, such as those during heat treatment. These ubiquitous microorganisms have ample opportunity for multiple entry points into the milk chain, creating food quality and safety issues. Certain spore-formers, namely bacilli and clostridia, are more problematic due to their ability to spoil dairy products and pathogenicity. In this study, we investigated how milk treatment and milk powder production influenced the composition and survival of anaerobic spore-formers. Samples were obtained on three different days (replicate blocks) during the production of dairy powders and examined in a culture-dependent manner using the most probable number method coupled with 16S rRNA amplicon sequencing and metagenomic analysis of the enriched samples. Results revealed that the milk separation greatly affected the spore-former presence and composition which were detected along the entire production line from raw material to milk powders. Throughout the various points of the production line, the occurrence of species belonging to the Bacillus cereus sensu lato was higher than that of clostridia. Sequence variants (SVs) belonging to the anaerobic spore-forming genus Clostridium were taxonomically assigned to two SVs groups and were detected in all three replicate blocks. A total of 19 metagenome-assembled genomes were recovered from nine enrichments. Four near-complete and two medium-quality genomes were found in raw milk/milk powder samples and further assigned as Clostridium tyrobutyricum and Clostridium diolis, which may constitute a problem in the finished dairy product. In conclusion, our findings highlight spore-formers' importance on dairy quality and may aid in their intervention and control in the dairy production line.