Validation of chlorine level in sanitization system to avoid cross-contamination

Scientifically validated data are needed by regulators and commercial fresh-cut processors to determine the minimum chlorine concentration required to prevent pathogen cross-contamination in produce wash waters. This project will address this issue by development of a microfluidic mixer that simulates cross-contamination and pathogen survival scenarios in chlorinated produce wash water. The microfluidic mixer has the unique capability of manipulating solutions at a miniaturized scale within instantaneous response times. Thus, the mixer will provide a technological solution to the limitations encountered in macroscale testing. This project will investigate the relationship between contact time (0.1 second – 5 minutes) and chlorine level (0.125 to 50 ppm) to prevent cross-contamination in wash water. Minimal contact times for given chlorine levels will be determined for planktonic and biofilms of bacterial cells. Results will be further validated using industrial produce wash water to investigate the effects of operational variables (pH, temperature, organic load) on the contact time requirement at different chlorine levels. We anticipate the proposed study will provide insight on the relationship between minimal contact time and chlorine level, and therefore predict the processing requirement at given variables to effectively prevent cross-contamination in wash water.

It is important to determine the minimum chlorine concentration that results in the inactivation of pathogens quickly enough to avoid cross-contamination. This proposal addresses several needs by industry, specifically: 1) Development of a novel contact time x chlorine dose-sensitive method and response curves to determine the conditions needed to prevent cross-contamination by planktonic and biofilm E. coli O157:H7 and Salmonella in the presence of organic matter loads during fresh-cut wash water operations; 2) Identify salient operational variables for measuring the performance of hypochlorous acid wash water sanitizer for fresh-cut operations. Key features of this proposal include a microfluidic mixing device to simulate possible cross-contamination and pathogen survival scenarios in hypochlorous acid wash water; determination of contact time (0.1 second to 5 minutes) and chlorine dose (0.25 to 50 ppm) to prevent cross-contamination of planktonic cells and biofilm. The results will assist fresh produce industry and governmental agencies in the development of effective sanitization practices to prevent cross-contamination in wash water, and advance the safety and quality of fresh leafy green produce post-harvest sanitization

1. Develop a novel contact time x chlorine dose-sensitive method and response curves to determine the conditions needed to prevent cross-contamination by planktonic and biofilm E. coli O157:H7 and Salmonella in the presence of organic matter loads during fresh-cut wash water operations. 

2. Identify salient operational variables for measuring the performance of hypochlorous acid wash water sanitizer for fresh-cut operations.

Determination of the minimum free chlorine (FC) concentration needed to prevent pathogen survival and/or cross-contamination during produce washing is essential for the development of science-based food safety regulations and practices. A microfluidic mixer useful for assessing pathogen inactivation kinetics at less than 1.00 s was designed, fabricated, and tested. This device also was used to determine the time and dose-dependent response of pathogen inactivation via FC. Test results indicate that (i) E. coli O157:H7 inactivation is significantly affected by FC concentration (P < 0.0001), contact time (P < 0.0001), and their interactions (P < 0.0001); and (ii) a 5-log10 reduction of E. coli O157:H7 requires exposing E. coli O157:H7 cells to a solution containing 1.0 mg/L FC for at least 1.00 s, or a solution containing 10.0 mg/L FC for 0.25 s. These findings provide critical information regarding the determination of the minimum FC concentration required to prevent pathogen survival and crosscontamination during fresh produce wash operations. This study also provides an innovative tool for developing better processes for the produce industry. Future evaluations that build on results from this current study may need to incorporate the disinfection of process water containing varying organic loads, sanitizers, and pathogen strains that may have become adapted to chlorine. Further work should also include both chlorine-adapted and generic strains, and the validation should account for the difference between resistance and non-resistance strains. Chlorine is commonly used for preventing water-mediated cross-contamination during fresh produce washing. The sanitizing efficacy is constantly compromised by the organic load, which has been attributed to rapid chlorine depletion and unsuccessful maintenance of residual FC. However, little is known about whether chlorine performs equally effectively with or without organic load at the same, well-maintained residual level. In this project, the efficacy of E. coli O157:H7 inactivation by chlorine was evaluated at various FC levels, COD levels, and pH, under an experimental setting relevant to fresh produce washing procedures. A chlorine depletion technique was employed for the first time to maintain the above mentioned parameters over a sufficiently long period, allowing the accurate evaluation of sanitization efficacy under stable conditions. The chlorine depletion and chlorination byproduct formation proceeded at higher rates at either COD levels or lower pH. A significant decrease in the sanitization efficacy was observed in the presence of diluted lettuce extract exhibiting 450 and 900 mg/L COD, compared to that obtained without organic load. Such deterioration of performance was more noticeable at shorter residence time, higher COD levels, or elevated pH. In an extreme case, the sanitization efficacy of 1.25 mg/L FC (pH 6.5, 5 s residence time) was a 5.76- and 0.20-log reduction at 0 and 900 mg/L COD, respectively. Whereas 0.5 mg/L FC was sufficient to reduce the bacterial population by 5 log units, a minimum of 7.5 and 5 mg/L of FC was required to achieve a 5-log reduction at pH 6.5 within 5 and 20 s of residence time. The overestimation of the FC level in the presence of chloramines may partly have accounted for the decreased sanitization efficacy, but other factors associated with the formation and interaction between chlorination byproducts should be explored extensively. This study underscored the potential risk of insufficient bacterial inactivation in the presence of organic load at FC levels previously demonstrated effective for drinking water disinfection procedures (e.g., 0.5 mg/L), and it necessitates the proper validation of sanitization procedures based on specific produce type, processing facilities, and operational parameters. Accurate measurement of residual FC is critical for mitigating pathogen cross-contamination. Our study shows the significant interference by organic chlorination byproducts to the conventional DPD method, which could be minimized by using the indophenol method. In practice, the indophenol method cannot be used for rapid measurement of the residual FC during fresh-cut produce washing because the method reaction time takes 5 min. Rather, the indophenol method could be applied as a measurement to determine the bias of DPD colorimetric measurements from the true residual FC. However, for measuring very low residual FC, it should be noted that the DPD colorimetric method gives a considerable overestimation of residual FC in the wash water. When the DPD colorimetric method is applied, it is also important to measure the absorbance of the water sample at 515 nm (as a blank) before adding the DPD powder/solution in case significant color or turbidity interferes with the measurement.