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Concentration and Detection of Pathogens in Bean Sprout Spent Irrigation Water using Microfiltration Coupled with Electrochemical Immuno-sensors


Despite the implementation of microbiological safety guidelines in the latter part of the 1990's there continues to be sporadic outbreaks of foodborne illness linked to contaminated sprouted seed. Part of the guidelines recommended screening spent irrigation water for Salmonella and Escherichia coli O157:H7 48h into sprout production, thereby preventing any contaminated product being released to market. However, research by the applicant has established that contamination within sprouting mung bean beds is heterogeneously distributed. Hence, relying on a single spent irrigation water sample, as recommended, does not provide a reliable index for assessing the microbiological status of the sprouting seed bed. In commercial terms, testing multiple spent irrigation water samples from a single seed bed would be economically unfeasible. A more practical approach would be to test a single composite sample of spent irrigation water collected from different points from under the sprouting seed bed. However, by compositing large sample volumes it is possible that contamination present would be diluted and unlikely to be detected using conventional microbiological methods.
The aim of the this work is to develop an integrated, on-site system that permits concentration of target pathogens from large sample volumes (10 liters) and detection via electrochemical based immuno-sensors.
Initial concentration of pathogens (Salmonella and E. coli O157:H7) from spent irrigation water (10 liters) will be achieved by initial pre-filtration using a 10 µm pore size membrane filter followed by circulation through a tangential cross-flow membrane filter (0.2ºm pore size). This will result in a 100 fold concentration of bacteria within the spent irrigation water. The retentate from the tangential filtration step (100 ml) will be introduced into a stream of carrier solution and pathogens captured by antibodies immobilized on the surface of a modified electrode. Detection of bound cells will be achieved through addition of a secondary enzyme labeled antibody or via detecting the metabolic activity of the bound cells (oxygen consumption, free radical formation, alkaline phosphatase, nitrite reductase, urease activity). Electrodes will be overlaid with perm-selective membranes to minimize non-specific binding events and facilitate selective amperometric detection of the electroactive species. Under optimized conditions the recovery of target cells will be close to 100% with the lower detection limit for the electro-immuno sensor being 100 cfu/ml.
The project brings together research scientists from the U.S. FDA and Guelph University with extensive experience in the microbiological safety of sprouted seeds. Major sprout producers within Ontario and the U.S. will also participate in the project.

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Expected Impact of Project Outcomes on Food Safety in Ontario:
The sprouted seed market within Ontario is valued at >$4m with more then 10% of Ontarians consuming sprouts on a regular basis. The popularity of sprouts can be attributed to the unique sensory properties and high nutritional content (high anti-oxidant, anti-carcinogens, anti-cholesterol constituents). However, health authorities cannot promote the consumption of raw sprouted seeds due to the inherent food safety risks associated with such products. This was underlined, by the major outbreak of salmonellosis within Ontario in 2005 that was subsequently traced to contaminated mung bean sprouts. In total, there have been 8 outbreaks (725 cases) and several product recalls of suspected contaminated sprouted seed in North America over the last 2 years. Therefore, more effective methods are required to remove (e.g. seed decontamination) or detect contamination in the course of sprout production. The key benefits of the proposed project will be to provide a technology that can both concentrate and detect contamination (if present) within large volumes (10 liters) of composite spent irrigation water in a semi- or fully automated rapid (4 h) process. This will facilitate on-site testing enabling the sprout producer to screen spent irrigation water later into the sprouting process when pathogen levels (if present) will be high. The initial cost of the unit is estimated at $4, 000. The cost per sample (taking into account regeneration of membrane and sensor interface) will be $10-40. Although the current application is focused on sprouted seeds the technology will find utility in other sectors were detecting low levels of pathogens in large sample volumes is required (e.g. water, fresh produce testing).
<P> For more information, please visit the <a href="; target="_blank">Ontario Ministry of Agriculture, Food & Rural Affairs (OMAFRA) Food Safety Research Program</a>.

Warriner, Keith
University of Guelph
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