Abstract:The efficacy of "gaseous" ozone in reducing numbers and re-growth of food-borne pathogens, (Escherichia coli and Listeria spp.), on leafy salads was investigated using spinach. A preliminary in vivo study showed 1-log reduction in six strains of E. coli and two species of Listeria spp. on spinach exposed to 1 ppm ozone for 10 min. A range of ozone treatments were explored to deliver optimal bacterial inactivation while maintaining the visual appearance (color) of produce. Exposure to a higher ozone concentration for a shorter duration (10 ppm for 2 min) significantly reduced E. coli and Listeria spp. viable counts by 1-log and the pathogens did not re-grow following treatment (over a nine-day storage period). Impacts of 1 and 10 ppm ozone treatments were not significantly different. Approximately 10% of the pathogen population was resistant to ozone treatment. We hypothesized that cell age may be one of several factors responsible for variation in ozone resistance. E. coli cells from older colonies demonstrated higher ozone resistance in subsequent experiments. Overall, we speculate that gaseous ozone treatment constitutes the basis for an alternative customer-friendly method to reduce food pathogen contamination of leafy produce and is worth exploring on a pilot-scale in an industrial setting.

Fresh leafy produce, such as lettuce and coriander, are subject to post-harvest 14 microbial contamination and decay. Because of increasing pesticide resistance and consumer 15 pressures, alternative residue-free treatments, such as ozone, are being actively explored and 16 encouraged to reduce microbial loads and curb spoilage of crops in storage/transit. However, 17 several researchers have reported that a component of the bacterial population on leaf surfaces 18 is resistant to ozone treatment. To investigate the potential reasons for this bacterial survival, 19confocal microscopy was used to visualise microbes on leaf surfaces before and after ozone 20 treatment. Direct observation (live/dead cell staining) of cells after ozone exposure showed that 21 some cells were still alive; this included cells in small colonies as well as individual cells. We 22 hypothesised that cell (colony) age and prior stress (cold) contributes to, or is responsible for, 23 the ozone resistance observed. Interestingly, cells derived from older agar-grown colonies (7-24 12-day-old) and cold stressed cells of a Pseudomonas sp. (isolated from coriander) showed 25 higher ozone resistance than that of control cells (4-day-old colonies). These findings suggest 26 that a range of factors are responsible for ozone resistance and further work to improve our 27 understanding of the mechanisms of ozone resistance may lead to improved methods to reduce 28 microbial spoilage of fresh produce. 29