RAPD-based screening for spore-forming bacterial populations in Uruguayan commercial powdered milk

The occurrence of spore-forming bacteria in powdered milk is of concern to the dairy industry due to potential deleterious effects including those resulting from proteolytic and lipolytic activities. Twenty-two powdered milk samples representative of spring and summer production obtained from Uruguayan retail stores were analyzed for type and number of thermophilic and spore-forming bacterial species. Bacillus licheniformis isolates were found to be the most prominent milk powder contaminant followed by Anoxybacillus flavithermus representing 71.5 to 84% of the total microflora. Geobacillus stearothermophilus, however, was not found. B. licheniformis strains F and G were both found in this study but strain F was the prevalent isolate representing 98.9% of the total isolates of this species. A. flavithermus isolates corresponded to strain C in accordance with 16S rRNA gene sequence analysis, however, in contrast with other reports, the RAPD profiles showed three characteristic bands at approximately 650, 1000 and 1650 bp, but lacking a band at 1250 bp. A third group of isolates was identified corresponding to members of a Bacillus subtilis group and Bacillus megaterium. Isolates designated B. licheniformis, A. flavithermus, B. megaterium and the B. subtilis group represented 89.1 to 93.6% of those analyzed, and depended on previous heat treatment and incubation temperatures of the plates. The remaining isolates were Bacillus pumilus and unidentified spore-formers.