Extended shelf life milk processing : effect of cleaning-in-place (CIP) on the survival of bacillus subtilis

Abstract

Bacillus subtilis vegetative cells have been found to remain in dairy processing equipment such as filler nozzles and heat exchangers even after cleaning processes such as Cleaning in Place (CIP). The cells form biofilms on the stainless steel and continue to grow and spread, detach, and move

to other processing areas and equipment further down the processing line, leading to cross- contamination and ultimately accelerated spoilage of Extended Shelf Life (ESL) milk during

storage post-processing at refrigeration temperatures. The objective of this study was to determine the effect of simulated CIP on the physiological state of B. subtilis cells, their attachment and subsequent growth in understanding the effectiveness of CIP and subsequent survival of B. subtilis vegetative cells. Three B. subtilis strains previously isolated from packaged ESL milk and ESL milk stored at 4 and 7 °C were subjected to a simulated CIP procedure with cells then subjected to flow cytometry and scanning electron microscopy. Enzymatic analysis was performed to

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determine the capability of the vegetative cells to produce proteolytic and lipolytic enzymes and the subsequent effect of these enzymes on the quality and shelf life of ESL milk. Flow cytometry results showed that approximately 98% of B. subtilis cells were physiologically dead after simulated CIP treatment, with 0.1% remaining viable. SEM revealed that the cells could reattach to stainless steel after simulated CIP treatment, and some cell multiplication was evidenced. The enzyme assays showed that all the B. subtilis strain cells continued to produce proteolytic enzymes after treatment, and only one strain could produce lipolytic enzymes. Over the 28 days of storage at 7 and 10 °C, the cells could grow in the milk. The results showed that simulated CIP treatment did not influence cell reattachment with bacterial growth evident 28 days at 7 and 10 °C after the

treatment, accelerating the deterioration of the ESL milk. The industry must identify easier non- evasive methodologies of identifying biofilm formation and develop new food processing

equipment coated surfaces that discourage the attachment of bacterial cells and spores.