Benzalkonium bromide is effective in removing Bacillus cereus biofilm on stainless steel when combined with cleaning-in-place

Abstract

Bacillus cereus is a common foodborne pathogen in dairy environment, which is hard to remove for its strong biofilm forming ability. In our study, Clean-in-place (CIP) regime and benzalkonium bromide (BK) were used in combination to enhance biofilm removal effectiveness. The CIP regime (1.5% NaOH at 65 °C for 10 min-water rinse-1% HNO3 at 65 °C for 10 min-water rinse) often used in dairy industry only achieved a 4.69 log reduction/cm2. A step of 5000 μg/mL BK following this CIP regime could effectively remove the biofilm cells on stainless steel, and biofilm cells were removed to below detection limit. The efficacy of BK in inhibiting Bacillus cereus was investigated. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of BK against Bacillus cereus were 6.25 μg/mL and 25 μg/mL. The antimicrobial activity of BK took effect by inducing intracellular proteins and electrolytes leakage. Moreover, the microstructure of extracellular matrix was broken under confocal microscopy (CLSM) and the amounts of proteins, polysaccharides and DNA in extracellular matrix reduced by 44.8%, 69.2% and 60.9% respectively after treatment of 5000 μg/mL BK. By detecting the zeta potential of biofilm extracellular matrix and contact angle assay, we found BK could bind to extracellular matrix with electrostatic interaction and increasing the hydrophilicity of biofilm, making biofilm removal easier. So, BK combined with CIP regime could be used as an effective strategy to remove Bacillus cereus biofilm.