Abstract:
Thermophilic bacilli and their spores are a concern in the dairy industry due to their resistance towards extreme temperature, pressure and chemicals, ability to form biofilm, and producing off-flavors in the product. It is of prime importance to control these contaminants in food products in order to meet customer’s requirements. Ultrasound treatment emerges as an effective alternative to traditional thermal treatment for food preservation through the mechanism of acoustic cavitation. In the present study, four isolates of thermophilic bacilli, identified as Geobacillus spp. and A.flavithermus using 16s rRNA sequencing, were considered. The vegetative cells and spores of these two species were subjected to 20 kHz low frequency ultrasound with power ~8 and 850 kHz high frequency ultrasound. Low frequency ultrasound had considerable effect on vegetative cells (5-log reduction in Geobacillus spp. and 1.6-log reduction in A.flavithermus), however, spores showed high resistance towards low frequency ultrasound treatment alone. TEM imaging of the ultrasonicated cells and spores showed an extensive damage both internally and externally in the vegetative cells. In case of spores, exosporium and outer coat layer was disrupted severely. These structural changes likely reduced their resistance towards sonication. Combined effects of low frequency ultrasound with different concentration of NaOH and H2O2 on the spores of Geobacillus spp. and A.flavithermus were also investigated. Combination of 0.12 M NaOH and 10 min ultrasonication completely inactivated 6 log spores of Geobacillus spp; and 7 log spores reduction for A.flavithermus was achieved by combining 0.17 M NaOH with 10 min ultrasound. These ultrasound treatment combinations are synergistic as they showed a higher spores reduction when compared to NaOH combined with high temperature (85°C). Ultrasound combined with 1% H2O2 had inactivated ~7 log Geobacillus spp. spores in 6 min and ~7 log A.flavithermus spores in 3 min. Combination of low frequency ultrasound with NaOH and H2O2 were found to be advantageous against these spores as low concentrations of chemicals and short treatment time were required. The preliminary studies on the effect of low-frequency ultrasound on biofilm was inconclusive as zero log reduction was obtained. This study clearly demonstrate that the combination of low frequency ultrasound with low concentration of NaOH or H2O2 could be an efficient method for the inactivation of Geobacillus spp. and A.flavithermus spores.