Effective cleaning and disinfection remain challenging in the food industry due to the complex and highly variable nature of biofilms. Consequently, there is a growing need for standardised, reproducible, and safe methods to aid in cleaning processes.

This work presents a combined research approach aimed at establishing the scientific basis for biofilm imitations for cleaning validation applications. First, a systematic review identified the properties most relevant to cleaning behaviour. Building on this, differences in biofilm formation under static and dynamic cultivation conditions and on surfaces with varying roughness were investigated. Biofilms formed by food-relevant microorganisms, primarily Microbacterium lacticum, were characterised using microscopy, safranin staining, biomass quantification, CFU determination, and EPS analysis. To translate these findings into practical cleaning validation tools, food-safe hydrogel systems based on agar, κ-carrageenan, and bovine and porcine gelatine were evaluated as biofilm imitation materials. Adhesion behaviour, rheological properties, and thermal stability were systematically characterised for both base formulations and additive-enhanced systems.

Adhesion, viscoelasticity, cohesion, and thermal stability were identified as key determinants influencing biofilm removal in the review. Biofilm characterisation revealed growth-condition-dependent differences in biofilm properties. The investigated hydrogels covered adhesion energies from 0.039 ± 0.008 J/m² to 0.633 ± 0.256 J/m² and elastic moduli ranging from 60 Pa to 15 kPa, reproducing characteristic ranges reported for microbial biofilms.

Overall, the findings demonstrate the potential of tailored hydrogel systems as reproducible biofilm imitations for standardised cleaning validation and optimisation in food processing environments. Cleaning tests are still outstanding to test the applicability of imitation systems.