Bacillus and Paenibacillus spp. associated with extended shelf life milk

Abstract

Extended shelf life (ESL) milk combines longer shelf life and better organoleptic characteristics; a combination that is absent in both pasteurised and ultra-heat treated (UHT) milk. Bacterial spoilage remains the main cause of food loss worldwide, which also includes milk and dairy products, despite advances in food preservation technology. The objectives of this study were to identify and characterise the spore-forming population associated with ESL milk during processing and chilled storage as well as characterising Bacillus cereus isolates obtained from ESL milk processing and during storage. Characterisation of spore-formers associated with ESL milk was done by analysing bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI-TOF-MS. B. cereus strains obtained from ESL milk and filler nozzles were characterised using (GTG)5 Rep PCR fingerprinting; the presence of virulence genes; cytotoxin K (cytK), nonhemolytic enterotoxin A (nheA), emetic toxin cereulide (cer) and enterotoxin hemolysin BL (hblA). The isolates were further discriminated as psychrotrophic and mesophilic strains using 16S rDNA. Furthermore, B. cereus isolates were selected for 16S partial sequencing. Some of the B. cereus strains obtained from ESL milk and filler nozzles were further characterised using rpoB partial sequencing and multilocus sequence typing (MLST). Milk had spore counts < 2 log10 cfu/ml and 4 log10 cfu/ml during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. In addition B. subtilis, B. cereus, B. sonorensis, B. licheniformis and Paenibacillus spp. were among the main spore-formers identified in the study. Bacterial species were inoculated in sterile milk for a shelf life study and population change observed over 42 days at 7 oC. Despite high prevalence of cer, hblA and nheA; cytK was not widely distributed. There was 100% and 8% prevalence of mesophilic and psychrotrophic signatures, respectively in B. cereus isolates. Although ESL milk process was effective in the reduction of bacterial counts and species diversity, the presence of B. cereus shows a potential safety problem in ESL milk. Despite the large diversity of the B. cereus strains in this study, there is evidence that biofilms associated with filler nozzles and raw milk are a source of contamination of B. cereus in ESL milk. Furthermore, the study has also shown that rpoB partial sequencing and MLST can be used as a tool for source tracking in ESL milk processing.