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

Summary

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.

Abstract

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 °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.

Introduction

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 strawberries^3,4, 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 blueberries^10,11. 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 settings^19,20,21.

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 spoilage^22,23. 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 storage^25,26. 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.

Protocol

1. Measurement of Gaseous ClO₂ in the Headspace of a Closed Chamber

1. 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.
2. Attach the ClO₂ pouch to the lid using double-sided tape.
3. Close the chamber by sealing the lid with petroleum jelly.
4. Connect the inlet and outlet of a ClO₂ gas detector to the chamber.
   NOTE: This is a gas circulation system, and no gas loss occurred when taking measurements.
5. Switch on the inlet and outlet gas flow and measure the ClO₂ concentration in the chamber after incubating for 0, 1, 2, 3, 4, 24, 26, 28, and 48 h.
6. Monitor the temperature and relative humidity (RH) in the chamber with temperature and RH data loggers.

2. Fruit Preparation and Storage

1. Obtain 15 kg of fresh grape tomatoes (Solanum lycopersicum var. cerasiforme) from a local retailer. Ensure that the fruits are healthy and have no visual flaws.
2. Preparation of inoculum
   1. Use strains of E. coli (wild type) and A. alternata from citrus fruit surfaces²⁷ for inoculation.
   2. Culture E. coli on E. coli agar (ECA) at 35 °C for 1 day²⁷ and then re-culture the organisms on a new plate for 1 day. Confirm the organisms by sampling the ECA plates with a bac-loop, streaking the bacteria on Levine eosin methylene blue (EMB) agar, and incubating for 24 h at 35 °C; cultures that turn reflective, metallic green are positive for E. coli.
   3. Culture A. alternata on potato dextrose agar (PDA) at 25 °C until spores appear.
   4. Scrape the E. coli cells from the agar plate into 50 mL of sterile distilled water until the estimated concentration reaches 9 log CFU/mL using a comparison with McFarland equivalence turbidity standards. Add 1,950 mL of sterile water containing 0.1% Tween-20 to make 2 L total of the final inoculum.
   5. Verify the cell concentration by dilution plating on EC agar plates. Scrape the A. alternata spores from the culture medium and suspend them into 2 L of sterile distilled water containing 0.1% Tween-20.
      NOTE: The final E. coli population was 7.5 log CFU/g, and the A. alternata population was 5.5 log CFU/g.
3. Place 7 kg of the tomatoes into a 10 L stainless steel pan that is completely covered by an autoclavable bag. Place the bag and pan in a safety hood. Apply the inoculum solution (2 L) to the fruits using a trigger sprayer applied from the top while gently stirring the fruits with a gloved hand.
   1. After 5 min, place the tomatoes in a single layer on sterilized sheets and allow them to air dry for 2 h. Put about 200 g of fruit each into twent-four 1 lb (~1.14 L) perforated clamshells.
4. Carefully fold the contaminated foils and place them into the steel pan. Remove the gloves and put them into the pan. Wrap the autoclavable bag and autoclave all contaminated supplies at 121 °C for 25 min.
5. Attach ClO₂ pouches to the lids of 12 clamshells. Use the other 12 clamshells as controls. Weigh each whole clamshell. Store the fruit at 20 °C for 14 days.
6. Take samples on days 3, 7, 10, and 14. Sample three clamshells, representing 3 replicates, per treatment per day.

3. Monitoring of ClO₂ Concentration in the Clamshells

1. Insert the inlet and outlet tubing of the ClO₂ gas detector into the center of the clamshells, with a 2 cm distance between the two ends, and take the ClO₂ measurement on days 3, 7, 10, and 14.

4. Determination of Microbial Population and Fruit Quality Attributes

1. Agitate 5 fruits (about 60 g) from each replicate at 100 rpm for 1 h in a sterilized sampling bag along with 99 mL of sterile potassium phosphate buffer (0.01 M, pH 7.2) on an orbital shaker.
   1. Plate serial dilutions (1-, 10-, and 100-fold) of the buffer wash, 50 µL each, on ECA (for E. coli) and PDA (for A. alternata) using a spiral plater.
   2. Incubate the ECA plates at 35 °C for 24 h and the PDA plates at 25 °C for 3 days. Read the microbial colony count using an optical plate reader. Sanitize all equipment which contacted the contaminated fruit after use.
2. Measure fruit firmness with a fruit firmness tester using the manufacturer's protocol. Calibrate the tester before each use. Measure 20 fruit for each replicate and express the results as the pressure force, Newton (N), required to compress the fruit by 1 mm (converted to N·m⁻¹).
3. Weigh the whole clamshell with the fruit at the beginning of and during storage and calculate the weight loss in comparison to the initial weight.

5. Statistical Analysis

1. Replicate all experiments in triplicate. Analyze the data using analysis of variance (ANOVA). Determine the mean separation by Duncan's multiple range test; the significance is defined at p <0.05.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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