2.2. Combined treatments
A schematic for the experimental design is shown in Fig. 1. Experiments were conducted to evaluate the effect of PL pre-treatment (0.5 or 1 J/cm2) on the resistance of B. subtilis spores to subsequent heating (90 °C, 10 min) or PL treatment (0.5 J/cm2), as well as the resistance to PL treatments after sublethal heat processing (90 °C: 5, 10, 15 or 20 min). After the samples were pre-treated, spore suspensions were immediately subjected to a second treatment or suspended in distilled water or TSB and stored at 4 or 30 °C for different time intervals before further treatment. When spores were suspended in TSB, cells were washed twice by centrifugation at 10.000 ×g for 10 min at 4 °C and resuspended in distilled water prior to second treatment. Culturability of B. subtilis spores after combined treatments was compared with that of spores subjected to prior treatment without subsequent processing (pre-treatment control) and spores incubated and subjected to second treatment without pre-treatment (treatment control). Each condition treatment was repeated at least three times. Untreated inoculated samples were used as controls.