Bacillus dominance and biofilm dynamics in liquid milk filling lines: high-throughput sequencing reveals critical control points

• HTS identifies filling processes as the main contamination source, with Bacillus dominant. • Bacillus forms denser biofilms on rough stainless steel via enhanced anchoring. • CLSM reveals 3D biofilm architectures strengthening sanitizer resistance. • Simulated CIP shows Bacillus biofilm persists with proliferative capacity in milk. • Product-specific pathway: UHT milk links to filling heads, pasteurized, to storage tanks. Due to the limitations of macro sequencing, high-throughput sequencing (HTS) cannot reflect the microbial community structure in sterilized food. This study combines HTS with cultivable cell counting to confirm the presence of bacterial contamination in both commercial factory-produced liquid milk, demonstrating that most contamination. We further traced the main source of bacterial contamination to the filling process. Bacillus dominates throughout the filling line, after enrichment, cultivable Bacillus counts in filling head samples reach 3.81 CFU/cm², highlighting their persistence. Liquid milk’s nutrient-rich environment supports Bacillus growth, while rough stainless steel surfaces in filling equipment promote biofilm formation. Confocal microscopy revealed multi-layered biofilms with extracellular matrices, making them resistant to Cleaning-in-Place (CIP) processes. After CIP, Bacillus counts remained at 4.69 CFU/mL, suggesting survival and potential quality risks. This study highlights Bacillus biofilm persistence in filling lines and offers insights for improving CIP protocols to maintain a sterile filling environment for liquid milk.