Reducing Foodborne Bacterial Biofilms on Produce and Packaging with an Engineered Enzyme

Biofilm formation is a major cause of post-harvest microbial persistence on fresh produce and packaging surfaces, as the biofilm matrix can provide pathogens and other bacteria with protection from common disinfection approaches. The potential of an enzyme-based food processing aide has been investigated as a green, targeted supplement to common disinfection practices for preventing bacterial adhesion and removing mature biofilms. Crystal violet staining of biofilms formed in multi-well polystyrene plates has been used to demonstrate the efficacy of enzymatic biofilm prevention and removal on E. coli O157:H7, E. coli 25922, Salmonella Typhimurium, and Listeria monocytogenes. In the presence of 0.1 mg/mL enzyme, biofilm development was significantly inhibited for all bacteria, with a maximum of 41 ± 7% reduction for E. coli O157:H7. Effectiveness of mature biofilm removal varied by bacteria species type, with a maximum of 35 ± 12% reduction for E. coli O157:H7. A microfluidic flow cell was used to directly observe and quantify the impact of enzyme rinses on E. coli O157:H7 cells adhered to spinach leaf surfaces. In the flow cell, enzyme rinses resulted in significantly greater cell removal than water, representing a reversal of initial phases of biofilm formation.

Initial biochemical characterization of the enzyme indicates a key role in surfacial extracellular polysaccharide degradation, suggesting multiple potential mechanisms of biofilm disruption. Transmission electron microscopy (TEM) of treated and untreated cells reveal major modification to surface polysaccharide structures, consistent with the predicted role as a polysaccharide-degrading enzyme. Additionally, changes in cell surface hydrophobicity were measured using the microbial adhesion to hydrocarbons (MATH) test and provide further evidence of changes to the cell surface. These results present a strong case for further development and optimization of enzyme activity for applications as a novel food processing and packaging aid to minimize food safety risks from bacterial pathogens.