Removal of Pseudomonas putida Biofilm and Associated Extracellular Polymeric Substances from Stainless Steel by Alkali Cleaning

Abstract

Alkali (NaOH)-based compounds are commonly used in the food industry to clean food contact surfaces. However, little information is available on the ability of alkali and alkali-based cleaning compounds to remove extracellular polymeric substances (EPS) produced by biofilm bacteria. The objectives of this study were to determine the temperature and NaOH concentration necessary to remove biofilm EPS from stainless steel under turbulent flow conditions (clean-in-place simulation) and to determine the ability of a commercial alkaline cleaner to remove biofilm EPS from stainless steel when applied under static conditions without heat. Biofilms were produced by growing Pseudomonas putida on stainless steel for 72 h at 25°C in a 1:10 dilution of Trypticase soy broth. The biofilms were treated using NaOH at concentrations of 1.28 to 6.0% and temperatures ranging from 66 to 70°C. Other biofilms were treated with commercial alkaline cleaner at 25 or 4°C for 1 to 30 min. Removal of EPS was determined by direct microscopic observation of samples stained with fluorescent-labeled peanut agglutinin lectin. Treatment with 1.2% NaOH at 66°C for 3 min was insufficient to remove biofilm EPS. A minimum of 2.5% NaOH at 66°C and 2.0% NaOH at 68°C for 3 min were both effective for EPS removal. Commercial alkaline cleaner removed over 99% of biofilm EPS within 1 min at 4 and 25°C under static conditions. Selection of appropriated cleaning agent formulation and use at recommended concentrations and temperatures is critical for removal of biofilm EPS from stainless steel.