Ultrasound pretreatment enhances the inhibitory effects of nisin/carvacrol against germination, outgrowth and vegetative growth of spores of Bacillus subtilis ATCC6633 in laboratory medium and milk: Population and single-cell analysis.

This study evaluated the synergetic inhibitory effects of ultrasound and nisin/carvacrol on spore germination, outgrowth, and subsequent growth of vegetative cell of Bacillus subtilis in laboratory medium and milk. Ultrasound pretreatment (3.33 W/mL, 15 min) and nisin/carvacrol (0.01%, 0.02%) synergistically inhibited spore germination, outgrowth, and vegetative growth of spores in laboratory medium. Whereas no such inhibitory effect was observed in milk even with a 10-fold increase in the concentration (1%) of nisin. Flow cytometry analysis showed that the germination capacities of ultrasound pretreated spores combined with nisin/carvacrol (67.3% and 30.5%, respectively) was lower than that of the untreated spores (95.1%). These results quantitatively revealed the inhibitory effect of the combined treatments which were confirmed by phase-bright spore observations at single cell level. In general, the current work identified the combined ultrasound-carvacrol treatment as an effective strategy to control spores and vegetative cells of B. subtilis in the laboratory medium and milk during abusive storage.