Identification of microbial hotspots and biofilm-forming bacteria in a high output industrial cheese plant: Comparative evaluation of enzymatic versus sodium hydroxide-based cleaning-in-place (CIP) for biofilm removal

Effective cleaning and sanitation are essential in cheese manufacturing to control microbial contamination and prevent biofilm formation. A multi-step approach was undertaken to assess microbial persistence and cleaning-in-place (CIP) strategies at an industrial Cheddar cheese facility with a production capacity of ∼40,000 tonnes. Pre- and post-CIP environmental swabs were analysed using plate counts and high-throughput (16S rRNA amplicon and shotgun) sequencing to identify microbial hotspots. Traditional CIP was broadly effective, however residual contamination persisted in critical zones such as the Alfomatic weir, curd distribution tank (CDT), and block formers. Industrial-scale trials comparing caustic and acid-based CIP with an enzymatic alternative revealed complete microbial elimination post traditional CIP, while enzymatic CIP left DNA signatures at one site, culturable cells were not detected. Sequencing identified Pseudomonas fluorescens as the dominant non-starter bacterium, suggesting possible biofilm formation. Parallel laboratory-scale studies of P. fluorescens biofilm removal using a CDC Biofilm Reactor® coupled with Scanning Electron Microscopy (SEM) compared caustic and enzymatic CIP. Caustic treatment completely removed the biofilm matrix but left adherent cells, while enzymatic treatments degraded the matrix but showed residual matrix fragments and adherent cells. Plate counts confirmed significant differences in residual biofilm populations, with average reductions of 3.75 log, 2.56 log, and 1.87 log after caustic, enzyme cocktail, and protease treatments, respectively (p < 0.0001), underscoring the incomplete nature of these approaches. Caustic CIP improves microbial reduction, while enzymes disrupt biofilm matrices. However, neither alone achieves complete removal, supporting combined strategies to enhance cleaning efficacy and sustainability in dairy processing.