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The overall objective of this research is directed towards enhancing disinfection of leafy vegetables and contributing new processes for further minimizing the recurring problem of foodborne illness related to retail and foodservice pre-packaged salad. In part, surface inaccessibility and possible internalization of human pathogens in lettuce and leafy vegetables lies at the heart of the problem of insufficient pathogen reduction by conventional sanitizing aqueous treatments. Therefore, application of a penetrating gaseous anti-microbial agent to would appear to represent a potential advantage to maximize consumer safety. Gas-phase ClO2 has the potential of achieving substantially increased pathogen reduction levels with minimal tissue injury or reduction in organoleptic quality. Current treatment technologies will be evaluated and modified to assess impedance to efficacious delivery by barriers to diffusion such as presented by the presence of films of air or water over microbial attachment sites on leafy vegetables such as the stomata or cut edges. The goals of the project are to establish the degree of efficacy of chlorine dioxide gas applications in disinfecting fresh-cut lettuce and other leafy vegetables, to establish the point of dose-dependent injury, and to further improve, extend and transfer treatment application methods to end-users so that adoption of a commercially feasible process becomes possible. Our anticipated outcome is a single-step intervention that contributes a pathogen reduction level in excess of 3 logs (99.9%) to diverse handling systems. <P>
Specific objectives include: <OL> <LI> Determine a preliminary injury threshold of ClO2 gas treatments for a range of lettuce types and other leafy green vegetables.<BR> 1.1 Identify factors limiting efficacy of ClO2 gas treatments in inactivating E. coli O157:H7 and other human pathogens on minimally-trimmed and fresh-cut leafy vegetables, or causing treatment-induced quality defects. <LI>Modify treatment conditions to improve efficacy and avoid injury to leafy vegetables.<BR> 2.1 Determine the efficacy of ClO2 gas treatments in inactivating naturally colonizing epiphytic coliforms and non-pathogenic E. coli as a comparative benchmark for performance expectations with environmentally acquired enteric bacterial pathogens.<BR> 2.2 Determine efficacy of ClO2 gas treatments in inactivating surrogates for E. coli O157:H7 and other human pathogens implicated in lettuce-associated outbreaks as a means of validating field tests and other trials where use of human pathogens would not be permitted. <LI>Design chambers for continuous application of above treatments that maximize exposure of leaf surfaces to the antimicrobial agents and provide for accurate control of treatment parameters and environmental conditions. <BR> 3.1. Verify absence of treatment-induced defects in product quality attributes and shelf- life, and if necessary, modify treatments to minimize any adverse effects. <LI> Evaluate pilot-scale treatment systems to demonstrate technical and economic feasibility.<BR> 4.1 Widely extend the outcomes of this research within and beyond the project period
NON-TECHNICAL SUMMARY: The risk of foodborne illness associated with packaged salads and leafy greens, consumed fresh, is widely recognized as a significant public health issue and threat to erode confidence in this consumer-benefit directed food category. This project seeks to identify commercially viable options for achieving disinfection goals in inactivating "E. coli" O157:H7 and other human pathogens that are economical for diverse applications within the broad industry sector.
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APPROACH: Our approach is to build on prior experience with ClO 2 disinfection of fresh produce and postharvest quality extension of lettuce. Evaluations would be conducted during the first year of this project and span the normal geographic and seasonal production and distribution of the crop but focus primarily on the Salinas Valley region. At least eight lettuce varieties a diversity of leafy greens (at least 15, including baby flat-leaf spinach and common "spring-mix" varieties) will be evaluated. For these preliminary assessments, Overall Visual Quality (OVQ) will be scored on a hedonic scale (1-severely damaged 5-limit of marketability 9-not affected relative to control). Within this goal we will conduct a feasibility assessment of the potential for monitoring PAL (phenylalanine ammonia-lyase) induction as an early indicator of treatment injury and reduced shelf-life following fresh-cut processing. In conjunction with this effort that should expedite the prediction of dose-dependent negative impacts, we will evaluate the potential for mild heat treatments to minimize browning disorders associated with fresh-cut processing in general and induced specifically by this gaseous phase disinfectant treatment. To support these evaluations towards economic feasibility assessments and commercial adoption, improved methods of pathogen detection and recovery will be incorporated into the study objectives. Based on our prior experience, for evaluations where a low, but potentially infectious survival rate of E.coli O157:H7 and high presence of competing background coliforms is anticipated, a monoclonal colony-lift detection procedure to specifically identify pathogenic colonies in a complex background following enrichment is needed. In combination with refinements in qRTPCR screening, this technical capability will be needed to provide the most compelling evidence for the degree of log reductions achieved by optimized ClO2 treatment. Finally, with collaborators, pilot-scale chambers for application of ClO2 gas to leafy vegetables in a continuous process would be designed to assure control of all key treatment parameters and environmental conditions and provide uniformity of sample treatment. The initial determination of economic feasibility would be achieved within this pilot-scale chamber and entail measurement of processing costs including ClO2 generation costs and ClO2 consumption rate, carrier gas consumption (if not air); power costs for operation of ventilation fans, conveyors, air compressors, and vacuum systems; laboratory costs for validation and verification of system efficacy; and amortized capital costs for treatment chamber or tunnel design and fabrication, ClO2 generator and monitoring instrumentation, and other process controls.
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PROGRESS: 2006/09 TO 2007/08 <BR>
Updates and description of this work in progress have been orally presented at several commodity group and leafy green grower association meetings as well as several fruit and produce food safety meetings and workshops. As the interest level in the potential and efficacy of gaseous chlorine dioxide remains high, despite challenges to non-injurious treatment of lettuce and leafy greens, the topic has been included in extension and outreach presentations to both industry groups and public health regulators. Written summary have not been made available as yet until current results are summarized.
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IMPACT: 2006/09 TO 2007/08<BR>
Lettuce leaves were inoculated with E. coli O157:H7 and treated with two levels of chlorine dioxide gas released from a granular formulation. After 48h, leaves were inspected for damage and then E. coli O157:H7 was recovered from the treated leaves. The leaves that were treated with a lower dose of chlorine dioxide gas maintained leaf quality but leaves treated with the upper limit, determined from earlier experiments, developed slight bleaching along the leaf edges. Leaves that were treated with chlorine dioxide gas, regardless of dose level, had a 3.2-3.3 log reduction of E. coli O157:H7. Although there was a visible difference in the leafy quality when the leaves were treated with the higher dose of chlorine dioxide gas there was no further reduction of E. coli O157:H7 on the leaves treated with a lower dose. Studies were conducted to assess the feasibility of monitoring induction of the enzyme phenylalanine ammonium lyase (PAL) as an early indicator of treatment injury, acute or sub-symptomatic, due to gaseous chlorine dioxide exposure and subsequent reduced shelf-life following fresh-cut processing: Extensive preliminary experimentation was done to determine the most efficient method of RNA extraction from the lettuce leaves, to determine the appropriate benchmarking genes to be used for the expression analysis of the PAL gene. During this period effort was directed to design primers for the PAL gene and to optimize the real-time PCR parameters for the RNA expression analysis. We found that the apparent degree of damage on the leaves from chlorine dioxide exposure corresponds to actual PAL gene expression increment but not visible injury. It would appear that chlorine dioxide gas does damage lettuce leaves and increases PAL gene expression which may be manifested in reduce shelf-life with storage. This preliminary data indicates therefore that monitoring PAL induction could be used as an early indicator of treatment injury and reduced shelf-life following fresh-cut processing and needs to be studied further.