Removal of Cronobacter sakazakii and Listeria monocytogenes biofilms using enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand

Abstract

C. sakazakii and L. monocytogenes are pathogens that may occur in dairy manufacturing plants with the potential cause serious diseases in neonates if dairy products containing these bacteria are consumed. Control of these pathogens is through cleaning with chemicals such as caustics and acids. Enzymes have been considered as alternative cleaning agent as they are more environmentally friendly compared with traditional chemical-based cleaning. However, the use of enzymes to remove biofilms of these pathogens has not been well studied. The objectives of this study were (1) to explore the biofilm formation of C. sakazakii and L. monocytogenes on hydrophilic and hydrophobic surfaces; (2) test the efficacy of α-amylase, proteases and endoglucanase on removal of these biofilms. The first part of this study focused on screening for the robust strains that can form strong biofilm in reconstituted infant milk formula using a microtiter plate assay and stainless steel coupons. The second part evaluated the efficacy of enzymes on the removal of biofilms by impedance detection and epifluorescence microscopy. The results showed that 12 of 14 strains of C. sakazakii strains and half of the L. monocytogenes strains formed strong biofilm. The ability to form biofilm varied with the origin of the isolates with clinical isolates of C. sakazakii and food isolates of L. monocytogenes forming strong biofilm on microtiter plates and stainless steel surfaces. For the evaluation of enzyme efficacy, the biofilms were treated with α-amylase, proteases and endoglucanase at 85°C, pH 3 for 15 min. The results from the microtiter plate assay based on the absorbance at 550 nm showed that the enzymes especially proteases and endoglucanase were effective in removing biofilms. The effectiveness of cleaning by enzymes demonstrated by plate counting and impedance detection was supported by epifluorescence microscopy results. In conclusion, these results demonstrated the efficacy of enzymes on removal of biofilms of C. sakazakii and L. monocytogenes. Compared with traditional method of cleaning, enzymes are more effective in removing extracellular polymeric substance of biofilms.