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
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Date
2019
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Massey University
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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.
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Keywords
Enterobacter sakazakii, Listeria monocytogenes, Biofilms, Prevention, Enzymes, Industrial applications