Formulation-specific effects of commercial proteases combined with cleaning-relevant chemicals on Escherichia coli K-12 biofilms

Aims This study evaluated whether commercial proteases with distinct acidic and neutral-to-alkaline compatibility profiles could modify the effects of cleaning- and sanitation-relevant chemicals on Escherichia coli K-12 biofilms. Methods and Results Pepsin, acid-stable Aspergillus niger protease, native Bacillus subtilis protease, and an alkaline protease for detergent applications (APD) were characterized for apparent pH and temperature optima and stability, then tested alone and with sodium hypochlorite, sodium hydroxide, phosphoric acid, nitric acid, acetic acid, lactic acid, and EDTA. Planktonic minimum inhibitory concentration, crystal violet biomass, and TTC-reduction assays were used. Proteases alone did not completely inhibit planktonic growth at 0.1%, and protease addition produced only limited MIC decreases. Selected cotreatments improved inhibition of biofilm formation, particularly at submaximal chemical concentrations, but generally did not decrease residual CV-stainable biomass in preformed biofilms. Selected cotreatments also reduced TTC-reduction signals without proportional decreases in CV-stainable biomass. Conclusions Protease-assisted effects were formulation-, biofilm-stage-, and assay-dependent. These findings support formulation-specific screening of protease–chemical combinations based on the intended biofilm-control stage, compatibility with the cleaning chemistry, and performance relative to the corresponding chemical treatment.