Name: controlled-release of chlorine dioxide in a perforated packaging system to extend the storage life and improve the safety of grape tomatoes
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1. Measurement of Gaseous ClO2 in the Headspace of a Closed Chamber
<ol>
	<li>Obtain the materials: ClO2 pouch (0.5 g of ClO2 slurry (9.5% a.i.) in a polymer film selected for its release rate (total surface area of 6 cm2); the exact components are proprietary), a glass chamber (19.14 L), and a lid with switchable gas inlet and outlet.</li>
	<li>Attach the ClO2 pouch to the lid using double-sided tape.</li>
	<li>Close the chamber by sealing the lid with petroleum ...

<ol>
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J., Chung, K. S., Won, M., Bin Song, ., K, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aqueous+chlorine+dioxide+treatment+on+the+microbial+growth+and+qualities+of+strawberries+during+storage.">Effect of aqueous chlorine dioxide treatment on the microbial growth and qualities of strawberries during storage.</a> Food Sci Biotechnol. 16 (6), 1018-1022 (2007).</li><li>Sun, X. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+of+controlled-release+chlorine+dioxide+gas+on+fresh+blueberries.">Antimicrobial activity of controlled-release chlorine dioxide gas on fresh blueberries.</a> J Food Protect. 77 (7), 1127-1132 (2014).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+storability+of+fresh+strawberries+with+controlled+release+chlorine+dioxide+in+perforated+clamshell+packaging.">Improving storability of fresh strawberries with controlled release chlorine dioxide in perforated clamshell packaging.</a> Food Bioprocess Technol. 7 (12), 3516-3524 (2014).</li><li>Narciso, J. A., Ference, C. M., Ritenour, M. A., Widmer, W. 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Chlorine dioxide is an ideal biocide to prevent food decay. However, it is unstable at high concentrations and non-transportable, requiring costly generators or inefficient two-part powder mixing. This study examined the application of a stable, ready-to-use form of chlorine dioxide to reduce food spoilage and the incidence of foodborne illness. In contrast to other chlorine dioxide application technologies currently in use, the commercial ClO2 used here is cost effective, has a long shelf life, and does not r...

A diet rich in fresh fruits and vegetables may help to reduce the risk of many diseases, including coronary heart disease and specific types of cancers1. However, there are a number of foodborne microbial pathogens, such as Escherichia coli, Salmonella enterica, and Listeria monocytogenes, associated with the consumption of fresh fruits and vegetables that can cause illness or even death among consumers who eat contaminated produce2. For example, E. coli O157:H7 outbreaks have been associated with grapes, tomatoes, and strawberries3,4, and hepatitis A outbreaks have been associated with fresh blueberries5. In addition, microbial contamination can cause substantial product loss through postharvest decay6. Alternaria alternata is an important plant pathogenic fungus that is known to cause leaf spots and other diseases in over 380 host species of plants7. It has been shown to be the cause of an Alternaria black spot8, a stem canker disease and a leaf blight of tomatoes9. Therefore, a safe and effective postharvest decontamination treatment is needed to both control foodborne pathogens and to prevent postharvest decay in fresh produce.
Low- and non-residue technologies are new trends for alternative sanitizers. A variety of postharvest fungicides have been used to reduce spoilage organisms and to prevent foodborne illness. Ozone, a strong antimicrobial agent, has been shown to preserve the quality and freshness of strawberries and blueberries10,11. However, ozone may cause oxidation of fruit surface tissue and can result in discoloration and the deterioration of flavor quality12. Chlorine has been used to sanitize fresh produce, such as blueberries and apples13. While effective, chlorine can react with nitrogen-containing compounds or ammonia, resulting in carcinogenic byproducts14, especially when used for the sanitization of fresh fruit15.
Chlorine dioxide (ClO2), an alternative sanitizer, was approved by both China and the US for the postharvest treatment of fruits and vegetables16. ClO2 is a water-soluble oxidizing agent with an oxidation capacity 2.5 times greater than that of free chlorine17. ClO2 is highly effective at low concentrations and with a short contact time18. ClO2 has low toxicity and minimal corrosiveness at the concentrations used for disinfection, and it is recognized as one of the most effective bactericidal and fungicidal agents for use in a variety of settings19,20,21.
Numerous research results have shown that ClO2 can control foodborne pathogens and postharvest decay16. For example, ClO2 gas has been used to inactivate L. monocytogenes, Salmonella, and E. coli O157:H7 and to prevent blueberry and strawberry spoilage22,23. ClO2 gas reduces the risk of microbial contamination while maintaining the attributes of fresh fruit, and it was effective at controlling the postharvest decay of strawberries24. However, it is unstable at high concentrations and non-transportable, historically requiring costly generators on site or inefficient two-part powder mixing.
However, a new ClO2 product with a ready-made, controlled-release formulation (i.e., it does not require a generator or the premixing of ingredients) has been shown to be highly effective at controlling food spoilage organisms and pathogens in preliminary experiments25. It is a safe, cost-effective, non-corrosive, easily transportable, and controlled-release form of ClO2, with no adverse effects on the environment. Previous experiments have demonstrated that this slow-release ClO2 powder wrapped in filtration material and placed in clamshell packaging significantly reduced the decay of fresh blueberries and strawberries, decreased berry water loss, and maintained fruit firmness during postharvest storage25,26. Recently, a controlled-release ClO2 packet was developed by sealing a slurry form of ClO2 in a semipermeable polymer film. The objectives of this work were to: 1) monitor ClO2 gas release properties in both a closed container and in perforated clamshells, 2) investigate the effect of a controlled-release ClO2 pouch enclosed in a container on foodborne pathogens and the decay of grape tomatoes, and 3) evaluate the effects of the controlled-release ClO2 on the storage quality of grape tomatoes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Curoxin chlorine dioxide pouch</td>
			<td>Worrell Water Technologies</td>
			<td></td>
			<td>Slurry, a.i. 9.5% in sealed permeable polymer film</td>
		</tr>
		<tr>
			<td>Grape tomato</td>
			<td>Santa Sweets, Inc</td>
			<td></td>
			<td>Santa Sweets Authentic&#160;</td>
		</tr>
		<tr>
			<td>ClO2 gas detector</td>
			<td>Analytical Technology, Inc., Collegeville, PA</td>
			<td></td>
			<td>PortaSens II&#160;</td>
		</tr>
		<tr>
			<td>Perforated clamshell</td>
			<td>Packaging Plus LLC, Yakima, WA</td>
			<td></td>
			<td>OSU #1, 1 lb</td>
		</tr>
		<tr>
			<td>Escherichia coli&#160;</td>
			<td></td>
			<td></td>
			<td>Wild Type (WT) from fruit surface</td>
		</tr>
		<tr>
			<td>Alternaria alternata</td>
			<td></td>
			<td></td>
			<td>from fruit surface</td>
		</tr>
		<tr>
			<td>E. coli agar&#160;</td>
			<td>EC Broth, Oxoid, UK</td>
			<td></td>
			<td>EC Broth with 1.5% agar</td>
		</tr>
		<tr>
			<td>Potato dextrose agar&#160;</td>
			<td>BD Difco, Sparks, MD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Levine eosin methylene blue agar</td>
			<td>BD Difco, Sparks, MD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trigger spray bottle&#160;</td>
			<td>Impact Products, LLC., Toledo, OH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilized sampling bag&#160;</td>
			<td>Fisherbrand, Fisher Scientific, Pittsburgh, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbit shaker&#160;</td>
			<td>New Brunswick Scientific, New Brunswick, NJ</td>
			<td></td>
			<td>Innova 2100</td>
		</tr>
		<tr>
			<td>IUL Instruments Neutec Eddy jet spiral plater inoculation plating system</td>
			<td>Neutec Group Inc., Farmingdale, NY</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ micro optical plate reader&#160;</td>
			<td>Synoptics, Ltd., Cambridge, UK</td>
			<td></td>
			<td>ProtoCOL</td>
		</tr>
		<tr>
			<td>Fruit firmness tester&#160;</td>
			<td>Bioworks Inc, Wamego, KS</td>
			<td></td>
			<td>FirmTech 2&#160;</td>
		</tr>
		<tr>
			<td>Tinytag temperature and RH data logger</td>
			<td>Gemini Data Loggers, West Sussex, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McFarland equivalence turbidity standard</td>
			<td>Fisherbrand, Fisher Scientific, Pittsburgh, PA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled-release of chlorine dioxide in a perforated packaging system to extend the storage life and improve the safety of grape tomatoes

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties of the pouch were monitored in containers with or without fruit. The pouch was affixed to the inside of a perforated clamshell containing grape tomatoes, and the effect on microbial population, firmness, and weight loss was evaluated during a 14 day storage period at 20 &#176;C. Within 3 days, the ClO2 concentration in the clamshells reached 3.5 ppm and remained constant until day 10. Thereafter, it decreased to 2 ppm by day 14. The ClO2 pouch exhibited strong antimicrobial activity, reducing Escherichia coli populations by 3.08 log CFU/g and Alternaria alternata populations by 2.85 log CFU/g after 14 days of storage. The ClO2 treatment also reduced softening and weight loss and extended the overall shelf life of the tomatoes. Our results suggest that ClO2 treatment is useful for extending the shelf life and improving the microbial safety of tomatoes during storage without impairing their quality.

The release of ClO2 exhibited a linear pattern over the first few hours. The concentration increased about 2.38 ppm/h over the first 4 h. The release speed slowed after 24 h of incubation, and the ClO2 concentration reached 25.4 ppm. However, the concentration tended to be stable after 24 h of incubation (Figure 1).
The headspace ClO2 concentration in the clamshell with grape tom...

Watch this Scientific Journal Video about Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes at JoVE.com

Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata.

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties ...

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.

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