Contamination of beer arises in 50% of all events at the late stages of production, in the filling area. This is where biofilms, a consortia of microorganisms embedded in a matrix composed of extracellular polymeric substances, play a critical role. To date, most studies have focused on the presence of (biofilm-forming) microorganisms in the filling environment. Our aim was to characterize the microbial status as well as the presence of possible biofilms at a can filling line for beer by determining the presence of microorganisms and their associated matrix components (carbohydrates, proteins and extracellular DNA [eDNA]). For 23 sampling sites, targeted quantitative PCR confirmed the presence of microorganisms at 10 sites during operation and at 3 sites after cleaning. The evaluation of carbohydrates, eDNA, and proteins showed that 16 sites were positive for at least one component during operation and 4 after cleaning. We identified one potential biofilm hotspot, namely the struts below the filler, harboring high loads of bacteria and yeast, eDNA, carbohydrates, and proteins. The protein pattern was different from that of beer. This work deepens our understanding of biofilms and microorganisms found at the filling line of beer beverages at sites critical for production.

  • One biofilm hotspot was identified at a can filling line during operation.

  • No biofilm could be detected after the automated cleaning procedure.

  • Automated cleaning procedure was not effective to remove all microorganisms and carbohydrates.

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