The overall goal of this experiment is to use a novel controlled release chlorine dioxide product to reduce the risk of food borne illness and food spoilage, and extend the shelf life of fresh fruit. This method can help answer key questions in the agricultural post harvest field about the preservation of fresh fruits and vegetables. The main advantage of this technique is that the release dosage and the chlorine dioxide treatment duration can be controlled throughout the entire storage and handling of the packaged produce.
Though this method can provide insight into the controlled release of antimicrobial agents for produce preservation, it can also be applied to other systems such as water sanitization and the deodorization. Before beginning the procedure, attach the chlorine dioxide pouch to the lid of the glass chamber with double sided tape, and seal the chamber with petroleum jelly. Connect the inlet and outlet of a chlorine dioxide gas detector to the chamber, and switch on the inlet and outlet gas flow.
Then, measure the chlorine dioxide concentration in the chamber at the indicated time points, and monitor the temperature and relative humidity with temperature and relative humidity data loggers. Next, revive the E.coli by thawing the frozen bacterial stock, and plate the suspension on E.coli agar for a 24 hour incubation at 35 degrees Celsius. The next day, reculture the organisms on a new plate for a 24 hour, 35 degree Celsius incubation.
Then, streak a colony onto a Levine Eosin-Methylene Blue Agar plate for another 24 hours at 35 degrees Celsius. Cultures that turn a reflective metallic green are positive for E.coli. Scrape the identified E.coli cells from their agar plate into 50 milliliters of sterile distilled water until the estimated concentration reaches nine log CFU per milliliter as compared to McFarland equivalence turbidity standards, and then pour the 50 milliliters of bacteria into 1950 milliliters of sterile water containing 0.1%Tween 20 to the bacteria to obtain a final inoculum volume of two liters.
For A.alternata, culture the fungi on potato dextrose agar at 25 degrees Celsius until spores appear, and prepare a two liters volume sample inoculum as just demonstrated. When the inoculum is ready, place seven kilograms of tomatoes into a 10 liter stainless steel pan that is completely enclosed within an autoclavable bag. Move the bag and pan into a safety hood, and use a trigger sprayer to apply the experimental inoculum solution of interest, while gently stirring the fruits with a gloved hand.
After five minutes, place the tomatoes in a single layer, on sterilized foils for two hours. When the fruits are dry, transfer 200 grams of tomatoes into 24 individual one pound perforated clam shells. Carefully fold the contaminated foils, and place them and the gloves into the pan for autoclave sterilization.
Now, attach chlorine dioxide pouches to the lids of half of the clam shells, and weigh each whole clam shell. Store all of the clam shells at 20 degrees Celsius for 14 days. Then at the appropriate experimental time points, insert the inlet and outlet tubing of the chlorine dioxide gas detector into the center of the clam shells with a two centimeters distance between the two ends, and obtain the chlorine dioxide measurement.
To calculate the weight loss in comparison to the initial weight, weigh the whole clam shell with the fruit. For measurement of the microbial population present on the fruit surface at each time point, agitate five fruits from each replicate with 99 milliliters of sterile phosphate buffered saline in a sterilized sampling bag at 100 revolutions per minute for one hour on an orbital shaker. At the end of the agitation, use a spiral plater to seed 50 microliters serial dilutions of the buffer wash on to the appropriate agar plate for the appropriate incubation period, and use an optical plate reader to measure the microbial colony count.
To measure the fruit firmness, use a fruit firmness tester according to the manufacturer's instructions to analyze 20 fruits from each replicate, remembering to sanitize the equipment after use, expressing the results as the pressure force required to compress the fruit by one millimeter. Then, analyze the data using analysis of variance and determine the mean separation by Duncan's multiple range test. Chlorine dioxide release initially exhibits a linear pattern with an increase in concentration of about 2.38 parts per million per hour over the first four hours.
The release speed slows over the next 20 hours to a chlorine dioxide concentration of 25.4 parts per million, at which point the gas concentration appears to stabilize. The head space chlorine dioxide concentration in a clam shell of grape tomatoes typically measures to be about four parts per million between days three and ten, at which point the gas concentration decreases until it reaches about two parts per million on day 14. The initial bacterial population concentrations on the fruit after inoculation, are 4.3 log colony forming units per gram for E.coli, and 3.4 log colony forming units per gram for A.alternata, levels which are reduced by 3.08 and 2.85 log colony forming units per gram respectively after 14 days of storage in chlorine dioxide treated clam shells.
Further, chlorine dioxide treatment not only maintains the firmness and weight of the fruit compared to control untreated tomatoes, but these effects also appear to be augmented over time. After watching this video, you should have a good understanding of how to utilize this new chlorine dioxide product for preserving and extending the shelf life of fresh produce.
