Synergistic inactivation and mechanism of thermal and ultrasound treatments against Bacillus subtilis spores

Abstract

Some Bacillus species are causative agents of food spoilage and a wide array of diseases. Due to their ability to form highly heat-resistant spores, it is of great interest to develop more effective inactivation strategies whereby these spores could be inactivated. Therefore, this work assessed inactivation of thermal and ultrasound treatments against Bacillus subtilis spores. The study further investigated the thermosonication (thermal and ultrasound, TS) -induced inactivation to the spores through a combination of morphology observation and internal factor analyses. The results of TS indicated that the TS combination synergistically inactivated spores by the maximum log reduction of 2.43 ± 0.08 at 80 °C and 20 W/ml and caused severe cell damage. The visual images revealed that the destructive mode of action of TS had multitarget sites, including coat, cortex, and inner membrane. Three distinct sub-populations were detected by Flow cytometry (FCM), and an unknown step with some physical compromise of the spore's inner membrane and partially hydrolyzed cortex involving the three steps model of inactivation was suggested. The combination of DPA (pyridine-2,6 dicarboxylic acid) content and the relative viabilities of the fractions suggested that during the TS treatment DPA release took place largely after spore death. The dead spores that retained DPA germinated relatively normally, but outgrow poorly, indicating that some key enzymes of intermediary metabolism has been damaged by TS treatment. Such understanding of the lethal action of TS may lead to the development of novel strategies involving spore destruction.