Effect of microbial loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella enterica serovar Typhimurium

In recent years the application of cold atmospheric gas plasma (CAP) aimed at the removal of microbial contamination from fresh and minimally processed food has received increased attention. For CAP to be successfully adopted by the food production industry, factors which affect its potential for microbial inactivation must be evaluated. In this study, we examined the effects of initial microbial concentration, present on filter discs, on the inactivation of Salmonella enterica serovar Typhimurium (S. Typhimurium) with nitrogen CAP. It was found that the rate of inactivation of S. Typhimurium is inversely proportional to initial bacterial concentration, with the D-value observed at the highest cell concentration assayed (108 CFU/filter) being 14 fold higher than seen at the lowest starting concentration (105 CFU/filter). Addition of increasing concentrations of Pseudomonas fluorescens cells to a Salmonella population of 105 CFU/filter resulted in an exponential decrease in the rate of killing of the Salmonella cells. However, whilst the addition of heat-killed S. Typhimurium cells to 105 CFU/filter live S. Typhimurium cells resulted in a significant decrease in the killing rate, this effect was dose independent. This suggests that although biomass plays a role in the protection against CAP inactivation seen at high cell densities, dead cells and their components released during the heating period are not as effective as viable cells. Fluorescence microscopy showed that, unlike the single dispersed cells observed at low cell densities, at higher cell densities bacteria were present in a multilayered structure. This phenomenon could explain the reduced inactivation by the plasma, since the top layer may present a physical barrier that protects underlying cells. In conclusion, this work clearly shows a link between bacterial cell density and the efficacy of CAP inactivation, making an important contribution to the understanding of this alternative food processing technology, which should be taken into account in both further studies and in the practical application of this technique to the food industry.

Research Highlights
► Cold atmospheric plasma can be successfully applied to inactivate S. Typhimurium.
► The inactivation of S. Typhimurium is inversely proportional to microbial loading.
► The effect of biomass could compromise the CAP induced inactivation of S. Typhimurium.
► Multilayered bacterial structures may provide physical protection against plasma.