Influence of application sequence and timing of eugenol and lauric arginate (LAE) on survival of spoilage organisms.

The effectiveness of sequential applications of the antimicrobials eugenol and lauric arginate (LAE) was investigated against Staphylococcus carnosus, Listeria innocua, Escherichia coli K12, and Pseudomonas fluorescens. The antimicrobials were applied simultaneously at half of their minimum lethal concentrations (MLC) or sequentially at t = 0 h and t = 3, 4, 6 or 8 h. Bacterial survival was determined by direct plate counts. Survivals kinetic were fitted to a growth and mortality model to obtain characteristic parameters that described time-dependent changes from growth to mortality or vice versa. The most effective was a simultaneous exposure of both antimicrobials to the spoilage organisms at the beginning of the incubation period. Efficiency decreases depending on order and timing of the two antimicrobials were observed upon sequential treatments. These were most effective when antimicrobials where applied within a short time period (3-4 h) and when eugenol was first applied against S. carnosus and P. fluorescens. No sequence effects were observed for L. innocua, and sequential treatments proved to be ineffective against E. coli K12. These results were attributed to cells adapting to the first applied antimicrobial. In some cases, this provided protection against the second antimicrobial rendering the overall treatment less effective.

Survival of spoilage bacteria subjected to sequential eugenol and temperature 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 3h and 8h. 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 8h. 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.