Introduction: Bifidobacteria are typical commensals in the gut of various hosts. Raw milk and its products are also sources of bifidobacteria, which is attributable to secondary contamination. The aims of our work were to quantify and identify bifidobacteria in raw milk from various farms and ruminant hosts to detect their species variability, and based on their typical occurrence and taxonomy, to evaluate the possible origin.

Methods: Bifidobacteria were quantified and isolated using bifidobacterial mupirocin media under anaerobic/microaerobic conditions, at 37 °C/room temperature. Colony identification was performed using MALDI-TOF MS and 16S rRNA gene sequencing. TBX medium was used to detect Escherichia coli and other coliforms (aerobically, at 37 °C), which was intended to serve as a control for fecal contamination.

Results: Bifidobacterium crudilactis and B. animalis subsp. lactis were repeatedly detected in raw milk using anaerobic cultivation at 37 °C. Other cultivation conditions reduced bifidobacterial capture and allowed the growth of a larger number of other microorganisms. Also, other bifidobacterial species typical of ruminants were detected in some samples. The detection of β-glucuronidase-positive E. coli on TBX agar was quite frequent; however, it did not fully correspond with the occurrence of bifidobacteria.

Conclusions: B. animalis subsp. lactis is a well-known probiotic species used in dairy production, as well as a multi-host species typical of the gut microbiota. By contrast, the occurrence of B. crudilactis has not yet been confirmed in the gut microbiota, which, together with our results, indicates its environmental characteristic. However, future strain physiological characterization is needed.

Acknowledgements: This work was supported by the METROFOOD-CZ research infrastructure project (MEYS Grant No: LM2023064) including access to its facilities.