Extended shelf life milk processing: Effect of cleaning in place (CIP) on the germination and attachment of Bacillus cereus spores

Abstract

Bacillus cereus spores are resistant to heat and chemicals and can attach and form biofilms on stainless steel surfaces, indicating that the spores may contribute to the contamination of extended shelf life (ESL) milk. Filler nozzles account for the most contamination in ESL milk. Ultimately, spores are dispensed into the final ESL milk and could germinate and multiply at refrigeration temperatures. The objective of this study was to determine the effect of simulated cleaning in place (CIP) on the structure, viability, growth and attachment of B. cereus spores with the aim of improving the shelf life and safety of ESL milk. In this study, three B. cereus strains isolated from biofilms from the filler nozzles of an ESL milk processing plant and raw milk were subjected to simulated CIP processes. Flow cytometry (FCM), epifluorescence microscopy and Transmission Electron Microscopy (TEM) were used to analyse the effect of simulated CIP treatment on spore structures. Following simulated CIP treatment, a biofilm assay was conducted to analyse biofilm formation and the attachment of B. cereus spores to stainless steel strips was analysed by Scanning Electron Microscopy (SEM). In addition, the growth kinetics of spores from B. cereus as well as the viability of B. cereus spores in milk over 28 days at refrigerated temperatures (5 °C) were determined. FCM and TEM revealed that CIP structurally damaged 92% of spores from B. cereus strains. However, 0.1% of spores survived and remained intact. A biofilm assay and SEM indicated that B. cereus strains were capable of forming biofilms and attaching to stainless steel strips following simulated CIP treatment. Furthermore, over 28 days, spores were capable of germination and growth under refrigerated conditions. It was concluded that CIP has an effect on the structure of spores. However, CIP is not entirely adequate as some spores are able to survive CIP. CIP has no effect on the attachment of B. cereus spores to stainless steel or on the ability of spores to germinate and grow in milk over 28 days if stored at 5 °C. Variation among strains is evident as strains isolated from filler nozzles are more resistant to CIP. B. cereus strains that survive CIP are less susceptible to subsequent CIP. We have shown that these spores may attach to filler nozzles in dairy processing plants and germinate when favourable conditions arise and contaminate ESL milk. If ESL milk is stored for an extended period of time, B. cereus spores will germinate. This may lead to a reduced shelf life and potentially be a safety risk in ESL milk with a prolonged shelf life.