Performing Department
Nutrition and Food Science
Non Technical Summary
Sanitizer wash is crucial for mitigating pathogenic contamination and prevent cross-contamination for fresh produce. Chlorine is the predominant sanitizer for fresh produce industry, but its efficacy is compromised by its physical/chemical interactions with organic materials generated from produce and debris during washing. The knowledge gap on the nature and effect of those interactions impedes the development and validation of sanitizing procedures. Moreover, detection and characterization of inadequate bacterial removal at local areas (e.g., wounds, crevices, and cracks) remains challenging without suitable analytical tools. Herein, we propose a novel experimental platform consisting of two components: (1) a stable polymeric substrate with a separable design containing artificial cracks/crevices, and (2) a coating film having chlorine sensing capability and locally adjustable chlorine demand. A systematically designed washing process will be undertaken to observe the sanitizing efficacy and chlorine availability. We anticipate that this approach will provide an important research platform for understanding the basics in bacteria-produce-sanitizer interactions, as well as for the development of technology to enhanced inactivation of food-borne human pathogens and improve public health.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Goals / Objectives
Objective 1. Fabrication of chlorine sensing chip to mimic fresh-cut and cracks of produce. We will develop a polymeric sensor chip with separable design, rapid chlorine indicating capacity, and adjustable, localized chlorine demand.Objective 2. Investigation on the sanitization process using the sensing chip under simulated washing conditions. We will investigate bacterial attachment, chlorine availability, and sanitizing efficacy under various conditions mimicking the fresh produce washing process, using the novel experimental platform.
Project Methods
Objective 1. Fabrication of Chlorine Sensing Chip to Mimic Fresh-Cut and Cracks of Produce1.1 Fabrication of Polydimethylsiloxane (PDMS) Base of the Sensing Chip withScheme 1 illustrates the "plug" design of the sensor chip, which consist of multiple male and female plugs. When these parts are combined, the cross sections of the plugs create an artificial crack within the chip. After the simulated washing, these parts can be separated to expose the cracks for subsequent analyses. On the other hand, the outer smooth surface will be utilized to represents intact produce surface. Both the artificial cracks and outer surface will be functionalized with a layer of chlorine or chloramine sensing film for the locally sensing of chlorine and chloramines.Individual polypropylene mold for each "plug" part will be prepared by 3D printing. The walls of the mold that have direct contact with the "crack" will be designed to be attached by nuts and bolts. The walls having direct contact with the "surface" will be joint to the main compartment by dovetail and groove design, respectively, so that these walls are removable. When fully assembled, the mold can be used in the formation of PDMS base of the sensor chip. In this case, the mold will be filled with PDMS precursors (Sylgard 184, Dow Corning) and the curing agent (10:1, w/w) and heated at 120 oC for 8 min and then cooled to room temperature. The hardened PDMS chip will be kept in the mold for subsequent film casting procedures.1.1 Casting Chlorine/Chloramine-Sensing Films on PDMS base1.1.1 Preparation of Coating SolutionHydrophobic proteins including zein and gliadin will be chosen as the coating material due to their well-established film forming stability, encapsulating capacity, moderate wettability, and suitability for chemical modification. Commercial chlorine indicating reagents including DPD and indophenol powder will be dissolved in ethanol and incorporated into the coating solution. To prevent the oxidation of DPD reagent by gaseous oxygen during encapsulation, all liquid preparation and mixing will be conducted under a constant flow of nitrogen gas.1.1.2 Casting Chlorine/ Chloramine Sensing Film on PDMS baseThe PDMS base obtained from Objective 1.1 will be kept in the plastic mold, with only one face ("surface" or "crack") exposed for film casting each time. Different volumes of casting solution (e.g., 5, 10, and 20 mL) will be transferred into the mold to cover the exposed PDMS face. The mold will then be placed in a vacuum oven and dried overnight under reduced pressure. Film samples with different thicknesses (depending on the volume of coating solution) will be collected and stored in a sealed desiccator at room temperature until subsequent analyses.1.1.3 Spots of High Chlorine DemandCoating solution that incorporates chemical compounds with known chlorine demand (e.g., phenols such as gallic acid and antioxidants such as ascorbic acid) will be casted on top of selective location on the as-prepared DPD- or indophenol-polymeric matrix to mimic the wounds on the fresh produce. Optionally, an array of spots with different chlorine demand will be formed on the chip for high throughput assays. The chip will be dried under reduced pressure, and a second layer of film with chlorine demand will be formed.1.2 Characterization of the Sensing ChipsSEM and FTIR will be applied to demonstrate successful coating on the substrate 7,8. In addition, the formed film will be characterized with the focus on several key properties. In specific, the dissolvation of film in the wash water will be determined by fluorescent labelling with FITC. The reactivity of film with FC or chloramine will be measured by colorimetric observation upon the application of different chlorine doses. Alternatively, the film will be immersed into the abovementioned solutions for 30 s to 2 min and removed from the liquid. The correlation between the measured color values and chlorine level or contact time will be measured.Objective 2. Evaluation of chlorine availability and sanitization efficacy using the novel platformWe will employ the chlorine sensing chip fabricated in Objective 1 for the investigation of local chlorine availability and the affecting factors resulted from various simulated washing conditions. Importantly, the capability of our platform to gain localized information of the pathogen infection/migration pathways and their interruption by chlorine during washing will offer new opportunity to better quality controls in terms of microbial safety.a. Produce-to-produce or produce-to-water migration. The chips will be divided in two groups: donor (sterilized and pre-inoculated) and receiver (sterilized without inoculation) chips. Both groups will be submerged simultaneously in the model washing water under pre-determined conditions. After the test, both will be analyzed for the bacteria population and chlorine availability.b. Water-to-produce migration. Inoculum will be added to the wash water at pre-determined intervals during the washing process. Sterile receiver chips without prior inoculation will be immersed in this solution and then analyzed. After the test, the receiver chips will be analyzed for the bacteria population and chlorine availability.2.1 Bacterial CultureThe BFP-E.coli bearing pRSET/BFP plasmid will be inoculated into TSB from a frozen stock culture, and incubated for 24 hrs at 37°C. Cells will be harvested, rinsed, and diluted phosphate buffer to contain approximately 107 CFU/mL of bacterial cells.2.2 Chip pretreatmentThe PDMS chip without coating will be autoclaved and rinsed with aqueous ethanol (ethanol/water=70/30, v/v). The coating solution will be sterilized by filtering through a 0.22 um syringe filter before deposited onto the desired surface on the substrate.2.2.1 Sanitization testsTo mimic the washing procedures in the fresh produce industry 7,9,10, a glass jar containing 4 L of washing water solutions of hypochlorous acid varying in free chlorine content (from 0 to 100 mg/L) and pH (3, 5, 6.5, 7, and 7.5). The solution will be agitated using a magnetic stirring bar at various speeds ranging from 10 to 1,500 rpm. To investigate the effect of the organic load, freshly prepared lettuce extract will be diluted to the chlorinated water to give different chemical oxygen demand (COD) levels ranging from 200 to 2000 mg/L, which are representative of the COD levels in the real lettuce washing facilities according to our prior field study. Alternatively, solutions of model compounds including glucose (model sugar), soy protein isolate (model protein), gallic acid (model phenol), and ascorbic acid (model antioxidant) will also be used as testing solutions. The free chlorine and chloramine levels of the solution in the presence of organic load will be determined using the conventional DPD colorimetric method before mixing with washing water solutions.The chlorine sensor chips with different materials, surface properties 11,12, and pretreatments13 will be immersed in the chlorine solution to initiate the washing trial. In order to minimize the artifacts arising from sample-to-sample variation, all sensor chips will be prepared from a same negative stamp, so that they all possess identical hierarchic structure.The following working parameters will be investigated: produce/water ratio (0.5, 1.0, and 2.0 kg/L, FC content (0.5 to 50 mg/L), sanitizing time (30, 60, 90, and 120 s), source of organic matter (Romaine lettuce, iceberg lettuce, etc.), COD level (500, 1000, 1500, and 2000 mg/L), and agitation speed (0, 500, 1000, and 1500 rpm).After desired period of time, sodium thiosulfate (Na2S2O3) will be added at the concentration of 10 mg/mL to neutralize the remaining chlorine. The chips will be then subjected to subsequent observations. The water sample will also be collected to measure the free chlorine content and bacterial population.?