Characterization of B. cereus in Extended Shelf-life (ESL) Milk

Introduction: Despite the existence of Food Safety Management Systems, B. cereus spore contamination continues to be a global concern more so in mild heat treated products including dairy products. Unlike most ESL milk products that are based on ultra-pasteurization (94 - 100°C) a recently introduced milk product on the South African market is produced by pasteurization (72 - 73°C), bactofugation and aseptic cold filling.

Purpose: The objective of this research was to isolate B. cereus from the entire ESL milk processing chain, processing equipment (filler nozzles) and the final chill stored ESL milk product to determine the characteristics and similarities of B. cereus strains isolated.

Methods: Spore formers were isolated at selected sampling locations using standard plating methods. Isolates were identified using biochemical, morphological tests and MALDI-TOF-MS.  (GTG)₅-Rep-PCR fingerprinting was performed to determine similarities between the B. cereus isolates. A discriminatory PCR was carried out to identify psychrotrophic strains of B. cereus.

Results: MALDI TOF MS dendrogram showed that there were six groups of B. cereus. All B. cereus showed ability to digest blood agar and were positive on proteolytic activity. (GTG)₅-Rep-PCR revealed the presence of two major clusters with subgroups. Results of the discriminatory PCR showed the presence of cspA gene in all isolates therefore indicating that the isolates were psychrotrophs. Isolates were sequenced using the 16S RNA gene to confirm the identity of strains.

Significance: The close relationship between isolates from the ESL milk product and those from filler nozzles shows processing equipment is a source of product contamination with psychrotrophic B. cereus which is of both safety and spoilage concern. This research can be utilized for validation of Food Safety Management Systems and risk assessment of B. cereus in ESL milk.