However, NCTC8239 spores incubated for 60 min in BHI broth plus AK showed a 1.6 DR, suggesting that the AK-induced germinated spores were inactivated at 55 C (Fig. 2). To test if a second heat treatment would increase spore inactivation, a heat inactivation treatment after various germination times was evaluated. Surprisingly, the addition of a second heat treatment (80 C, 10–20 min) after various incubation times increased spore inactivation from 2.8 to 3.6 DR (Fig. 3).