Survival of spoilage bacteria subjected to sequential eugenol and temperature treatments

• Efficacy of sublethal treatments depended on time and order of application.
• At 42 °C, a temperature first–eugenol later treatment was least effective.
• Initial broad stress adaptations may increase resistance against subsequent stresses.
• Differences were observed between simultaneous and sequential treatments.

Effects of a sequential application of eugenol and temperature on the survival of two model spoilage organisms, Staphylococcus carnosus LTH1502 and Escherichia coli K12 C600, were studied. To assess effects of a “temperature first–antimicrobial later” treatment, cultures were treated with eugenol at 20, 37 and 42 °C at the beginning of the incubation period, and after 3 h and 8 h. To assess effects of an “antimicrobial first–temperature later” treatment, eugenol was added at the beginning of the incubation period at 37 °C and temperature was changed to 20 or 42 °C after 3 or 8 h. Cell numbers were determined in regular intervals during the incubation period using plate counts. Partitioning of eugenol was measured by HPLC, and cell morphology was assessed by electron microscopy. Combined treatments were more effective against the Gram negative E. coli than against S. carnosus. Order of application influenced the effectiveness of treatments, especially at 42 °C. There, the temperature first–eugenol later treatment was less effective than other treatments, likely due to temperature-induced adaptation processes occurring in cellular membranes making them more resistant against a later eugenol treatment. Results are of significance in situations where combinations of sublethal stresses are used to build a hurdle concept for food preservation.