Sensitivities of germinating spores and carvacrol-adapted vegetative cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment

Abstract

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.

FIG. 1

Inactivation of germinating spores (BHI broth, 10× diluted, 20°C) by PEF treatment (27 kV/cm, 60 2-μs pulses, single pass). Symbols: ▴, control; ●, control with heat treatment (10 min, 70°C); ⧫, PEF treatment; ■, PEF treatment with additional heat treatment (10 min, 70°C). Standard deviations are indicated by error bars.

FIG. 2

Inactivation of germinated spores (BHI broth, 10× diluted, 20°C) by nisin (1.25 μg/ml). Symbols: ▴, control (untreated); ●, control with heat treatment (10 min, 70°C); ⧫, nisin treatment; ■, nisin treatment with additional heat treatment (10 min, 70°C). Standard deviations are indicated by error bars.

FIG. 3

Inactivation of germinated spores (BHI broth, 10× diluted, 20°C) by a combination of nisin (0.6 μg/ml) and PEF treatment (27 kV/cm, 60 2-μs pulses, single pass). Symbols: □, control (untreated); ●, control with heat treatment (10 min, 70°C); ▴, nisin treatment; ⧫, nisin treatment with additional heat treatment (10 min, 70°C); ×, nisin treatment combined with PEF treatment; ■, PEF treatment with additional heat treatment (10 min, 70°C).

FIG. 4

Influence of precultivation in the presence of carvacrol on the sensitivity of B. cereus to nisin. (A) Effects of nisin (0.3 μg/ml) (■), carvacrol (0.3 mM) (▴), and a combination of the two compounds (●) on the viability of control cells of B. cereus. (B) Effect of nisin on the viability of B. cereus grown on carvacrol. Symbols: ■, 0.3 μg of nisin per ml (control cells); ⧫, 0.16 μg of nisin per ml and precultivation in the presence of 0.2 mM carvacrol; ●, 0.16 μg of nisin per ml and precultivation in the presence of 0.3 mM carvacrol; ▴, 0.3 μg of nisin per ml and precultivation in the presence of 0.3 mM carvacrol.

FIG. 5

Influence of precultivation in the presence of nisin on the sensitivity of B. cereus. Symbols: ⧫, control; ▴ and ▵, 0.3 mM carvacrol; ■ and □, 0.3 μg of nisin per ml; ● and ○, 0.3 μg of nisin per ml and 0.3 mM carvacrol; open symbols, control cells; solid symbols, cells adapted to 0.3 μg of nisin per ml.

FIG. 6

Influence of incubation in the presence of carvacrol on activity of nisin and/or PEF treatment with cells of B. cereus (30°C). (A) Effect of PEF treatment (20 kV/cm, 30 2-μs pulses, recirculation) on control cells (⧫) and adapted cells (●) and effect of a combination of PEF treatment and nisin (0.04 μg/ml) on control cells (■) and adapted cells (▴). (B) Effect of nisin (0.08 μg/ml) on control (■) and adapted (●) cells of B. cereus. Standard deviations are indicated by error bars.