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.
