Use of gaseous ozone to prevent post-harvest microbial spoilage of leafy produce

PhD ThesisFresh leafy salads suffer from post-harvest microbial contamination and decay. Due to increasing pesticide resistance and consumer pressures, residue free alternatives, such as ozone, are being actively explored and encouraged to reduce microbial loads of crops in storage/transit. Previous work has demonstrated that long-term exposure to low concentrations of ozone can be effective in retarding the degradation of some fruit and vegetable. Much less is known about the potential of ozone-exposure to protect leafy produce. The first goal of this project was to determine ozone exposure levels that did not damage produce, but reduced microbial loads significantly. Different produce types exhibited varying abilities to resist ozone damage, e.g. coriander and rocket were relatively resistant to ozone (10 ppm for 10 min); while spinach, watercress and lettuce were more sensitive (1 ppm for 10 min). These ozone exposure levels reduced bacterial loads by at least 1-log. Confocal microscopy confirmed that some bacterial cells (1−10%) survived ozone treatment. These visual observations demonstrated heterogeneity in the resistance of the leaf surface microflora to ozone treatment. It was tested if colony age and/or stress (e.g. cold) may be responsible for the variation in ozone resistance observed. Stressed cells of Pseudomonas sp. isolated from coriander exhibited greater resistance to ozone than control cells. Subsequent gene expression analysis using RNA-Seq technology of stressed cells showed significant changes in the expression of genes related to stress resistance compared to controls. In particular, it was observed that in aged colonies, about 90% of the changes in gene expression mapped to one gene, a non-coding RNA that is part of RNase P. Many of the genes showing differential expression were involved in energy production and transport, motility or cell wall/membrane integrity. This improved mechanistic understanding of ozone resistance may lead to novel anti-microbial treatments. As there are growing concerns about the contamination of leafy products with pathogens resulting in food poisoning the final part of this work focused on the potential of ozone to inactivate food pathogens on leafy produce. Results showed that this treatment significantly reduced E. coli and Listeria spp. on spinach, and the pathogens did not re-grow after treatment over a 9-day storage period. v It was concluded that gaseous ozone treatment is worthy of further exploration as a potential commercial tool to improve the safety of fresh leafy salads and herbs, and reduce microbial spoilage.HDC (Horticultural Development Company) for funding my PhD Studentship in conjunction with Vitacress Salads Ltd, Intercrop Ltd, Geneius Laboratories Ltd and Biofresh Ltd

Investigation of potential reasons for bacterial survival on ‘ready-to-eat’ leafy produce during exposure to gaseous ozone

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

Effect of Ozone Treatment on Inactivation of Escherichia coli and Listeria sp. on Spinach

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

Effect of Ozone Treatment on Inactivation of Escherichia coli and Listeria sp. on Spinach

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

Abstracts of Scientifica 2022

This book contains the abstracts of the papers presented at Scientifica 2022, Organized by the Sancheti Institute College of Physiotherapy, Pune, Maharashtra, India, held on 12–13 March 2022. This conference helps bring researchers together across the globe on one platform to help benefit the young researchers. There were six invited talks from different fields of Physiotherapy and seven panel discussions including over thirty speakers across the globe which made the conference interesting due to the diversity of topics covered during the conference. Conference Title:  Scientifica 2022Conference Date: 12–13 March 2022Conference Location: Sancheti Institute College of PhysiotherapyConference Organizer: Sancheti Institute College of Physiotherapy, Pune, Maharashtra, Indi