Phylogenetic affiliations of Bacillus amyloliquefaciens isolates produced by a bacteriocin-like substance in goat milk

Eight isolates identified as belonging to the genus Bacillus were obtained from Aloqt (a crusty dried product made from goat milk). The cell-free culture supernatant (CFCS) from the eight isolates possessed an inhibitory spectrum against Staphylococcus aureus and Escherichia coli. The eight unknown bacterial isolates were identified by PCR amplification of their 16S ribosomal RNA gene and sequencing of the resulting PCR products. All of the isolates were classified as members of Bacillus amyloliquefaciens, as their 16S rDNA similarities to the respective species were greater than 99%. The phylogenetic analysis grouped two isolates with strain BCRC 11601 and the remaining six isolates with strains MPA 1034 and BCRC 11601. The inhibitory activity of the CFCS was either highly reduced or fully inactivated when treated by proteolytic enzymes, suggesting the possible involvement of a protein/polypeptide bacteriocin-like inhibitory substance (BLIS) in their antagonism. The optimum growth conditions for maximum inhibitory activity were achieved at an initial pH of 7, incubation temperature of 37 °C, and NaCl concentration of (1%). A considerable decrease and/or complete loss of activity occurred at values above and below the optimum. The maximum inhibitory activity occurred after 24 h of incubation; as the incubation time increased, a decrease in the activity was observed until a complete loss of activity occurred after 72 h of incubation.