A novel micro fluidic mixer-based approach for determining inactivation kinetics of Escherichia coli O157:H7 in free chlorine solutions

Authors: ZHANG, BOCE - University Of Maryland; Luo, Yaguang - Sunny; ZHOU, BIN - University Of Maryland; Millner, Patricia

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2015
Publication Date: 2/11/2015
Publication URL: http://handle.nal.usda.gov/10113/61164
Citation: Zhang, B., Luo, Y., Zhou, B., Millner, P.D. 2015. A novel micro fluidic mixer-based approach for determining inactivation kinetics of Escherichia coli O157:H7 in free chlorine solutions. Food Microbiology. 49: 152-160.

Interpretive Summary: Sanitizers (chlorine) are routinely used to prevent the survival and dissemination of harmful bacteria during produce washing. Concentrations need to be high enough to inactivate bacteria, but low enough to not adversely affect produce quality. Therefore, information on the minimum concentration of sanitizer and optimal contact required to inactivate bacteria is very desirable; this information is also needed to support the implementation of the Food Safety Modernization Act. In this study, a novel automated micro fluidic device is described that can determine bacterial inactivation rates in as little as 0.1 second. The device was used to determine minimum contact times at different sanitizer concentrations. For example, at a concentration of 1 mg/L (current industry practice) a 1.2 seconds exposure achieved the FDA required 5-log reduction in E. coli O157:H7 population; whereas at 10 mg/L a 5-log reduction was acheived 0.2 seconds. Information obtained from this study will provide critical insight into the kinetics of bacterial inactivation for a broad range of sanitizers and produce wash operational conditions, thus facilitating the development and implementation of science-based food safety regulations and practices for improving food safety. This information will be useful to other scientists, the produce industry and to regulatory agencies.

Technical Abstract: Determination of the minimum free chlorine concentration needed to prevent pathogen survival/cross-contamination during produce washing is a critical need to develop science-based food safety regulations and practices. Although the trend of chlorine concentration-contact time on pathogen inactivation is generally understood, specific information on chlorine and the kinetics of pathogen inactivation at less than 1 second is urgently needed by the produce processing industry. However, conventional approaches to obtain this critical data have been unable to adequately determine very short time-span responses. This paper reports our development, fabrication, and test of a novel micro fluidic device, and its application to obtain the necessary data on pathogen inactivation by free chlorine in produce wash solution in as short as 0.1 second. A novel micro fluidic mixer was designed with three inlets for bacterial, chlorine and dechlorinating solutions, and one outlet for effluent collection. The master mold was fabricated on a silicon wafer with micro channels via photo polymerization. Polydimethylsiloxane replicas with patterned micro channels were prototyped via soft lithography. The replicas were further assembled into the micro mixer on glass via O2 plasma treatment, and the inlets were connected to a syringe pump for solution delivery. To determine the kinetics of free chlorine on pathogen inactivation, chlorine solutions of varying concentrations were first pumped into the micro mixer, together with the addition of bacterial suspension of E. coli O157:H7 through a separate inlet. This was followed by injection of dechlorinating solution to stop the chlorine-pathogen reaction. The effluent was collected and the surviving bacteria cells were enumerated using a modified ‘Most Probable Number’ method. Free chlorine concentration was determined using a standard colorimetric method, and the contact time was precisely controlled by adjusting the solution flow rate and quantitatively determined by computational fluid dynamics modeling. Results showed that 1) pathogen inactivation was significantly affected by free chlorine concentration (P < 0.001) and reaction time (P <0.001) and their interactions (P < 0.001); and 2) the current industry practices of 1 mg/L will require more than 1.2 seconds exposure to achieve a 5-log reduction in an E. coli O157:H7 population, whereas a 10 mg/L free chlorine solution will achieve 5-log reduction in as short as 0.2 seconds. Information obtained from this study will provide critical insight on kinetics of bacterial inactivation for a broad range of sanitizers and produce wash operational conditions, thus facilitating the development and implementation of science-based food safety regulations and practices for improving food safety.