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Ready-to-Eat Produce is potentially vulnerable to contamination in the field during growing and also during picking and post harvest. Conventional hypochlorite washing gives some protection but only typically only removes 90-99% of bacteria.
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Knowledge of the microbiology of crop surfaces is limited and it is not clear what the fate is of human pathogens that may be present. There is very little understanding of how these pathogens interact with the rest of the microbial flora prior to harvest and also following post harvest operations such as chopping, washing and storage. For example if the pathogens are normally incorporated into microbial biofilms they may not be killed so readily by chlorine washing as if they were present as solitary cells.
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This project has two parts with the joint purpose of minimising the chances of harmful bacteria being present at the time of consumption. On the one hand it will gain detailed information about the natural microbial flora and how that flora interacts with any pathogens and secondly it will examine the effects of novel washing approaches aimed at biofilm disruption to make washing more efficient. The principle behind this novel washing will be to use polysaccharidases to disrupt biofilm in conjunction with oxidants. The analytical techniques used will allow interpretation of the success of such protocols and also their refinement.
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Information from the first part of the project may be useful in itself (some components of the microbial flora may be antagonistic to pathogens for example). It will also provide information to the other part of the project running in tandem that may improve its chances of success.
The first approach was to identify the natural contamination of fresh produce at harvest and during factory processing using a molecular technique T-RFLP (terminal restriction fragment length polymorphism). More than twenty representative bacterial and fungal species were found including E. coli, Pseudomonas spp., Pantoea agglomerans, and Penicillium spp. Batches of watercress, lettuce, parsley and spinach from different sources that had been conventionally or organically produced were examined for changes in the microflora throughout washing, packing and storage. Data showed that the decontamination process reduced the sizes and number of peaks produced on a T-RFLP trace, reflecting the reduction in microbial load and diversity achieved by the washing process. During storage of salads, there was re-growth of the bacteria, predominantly from the Pseudomonad group, and later, re-growth of yeasts also occurred.
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This technique was also used to show that Salmonella and Listeria could survive on the surface of the plants containing natural microflora. T-RFLP analysis showed that S. typhimurium cells could be detected for at least one week following artificial inoculation at high levels and Listeria monocytogenes survived on lettuce for up to 12 days.
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The second phase of the project was to determine if novel approaches could increase the level of microbial reduction achieved during washing. In pilot scale studies, lettuce and spinach were inoculated with Salmonella or Pseudomonas and half the samples were treated with an enzyme (alginate lyase) before washing in order to attempt to disturb the biofilm present. The produce was then washed for 2 minutes with water, hypochlorite (20ppm free chlorine), chlorine dioxide (2-3 ppm) and ozone (2ppm). The presence of alginate lyase did not improve the level of microbial reduction seen which was in line with expectations based on previous studies i.e. a log reduction of between 0.75 and 2.5 dependent on produce type and treatment. Chlorine dioxide appeared to have a slighter greater effect than ozone, however, hypochlorite consistently achieved the greatest level of reduction of Salmonella and Pseudomonas.
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Decontamination treatments are often not as effective in an industrial situation as in pilot scale facilities as the volumes of produce being treated are much higher. Successful transfer of novel decontamination treatments from pilot scale to industrial scale was of particular importance to the project. Three different factory sites were used in these trials. Treatments tested were hypochlorite, ozone, chlorine dioxide, chlorine dioxide followed by hypochlorite, hypochlorite and ultrasound. The produce treated were salad mixes, spring onions, lettuce and carrots.
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Hypochlorite gave a higher level of microbial reduction compared to other treatments for naturally occurring organisms, i.e. TVC, Enterobacteriaceae and Coliforms. For other treatments, the level of microbial reduction achieved was generally lower under the factory conditions than previously found in pilot scale conditions. This is believed to be because the systems were more easily optimised for maximum benefit in the pilot scale facility. Where the factory scale systems were able to be optimised an improved level of microbial reduction was obtained.
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This work has demonstrated that alternatives to chlorine may need to be optimised in different factory environments to obtain the appropriate level of decontamination. In many cases the efficacy of chlorine dioxide, ozone and ultrasound was sufficient to achieve level of microbial reduction of up to 1.5 to 2 logs in factory tests which is an acceptable target for washing treatments. However, there did not appear to be any alternative treatment which gave consistently better results than hypochlorite.