Effects of cold plasma treatments on spot-inoculated Escherichia coli O157:H7 and quality of baby kale (Brassica oleracea) leaves

Highlights

• Disinfection and quality of spot-inoculated kale leaves by cold plasma was evaluated.

• Plasma treatment resulted in inactivation (0 CFU/mL) of E. coli O157:H7 on kale leaves.

• No significant color change was observed in leaves treated with plasma for 300 s.

• Refrigerated storage of treated leaves showed lower Browning Index values than untreated leaves.

Abstract

The efficacy of non-equilibrium atmospheric pressure-pulsed dielectric barrier discharge (cold plasma)-treated mist in disinfecting baby kale leaves, and its effect on color values and cuticle composition were evaluated. UV-treated baby kale leaves were spot-inoculated (0.04-mL of 10⁶ CFU/mL) with rifampicin-resistant strain E. coli O157:H7 ATCC 700728 incubated (0–4 °C) overnight and treated with plasma mist at 60, 120, 180, 240 or 300 s. Treated and untreated leaves were analyzed for change in color values (L*, a*, b*, chroma, hue, and browning index (BI)) using a Chroma meter. Functional groups (alcohols, esters, aldehydes, and ketones) were determined in the cuticle using FTIR. Color stability of treated leaves was also evaluated after refrigerated storage (4 °C) for 12 days. Levels of E. coli O157:H7 on the kale leaves were reduced below the detection limit of 5.5 × 10³ CFU/mL after plasma treatment for 300 s with no significant change in color values. Visible change in color (browning or leaf damage) was observed after 600 s of plasma treatment. Color stability of plasma-treated leaves was enhanced during refrigerated storage (4 °C), indicated by a lower BI value of 34.4 ± 5.4 at 120-s plasma treatment compared to untreated leaves after 12 days of storage. Fourier transform infrared (FTIR) spectroscopy analysis concluded that plasma treatment of kale did not negatively affect functional groups in the cuticle. This study demonstrated that cold plasma mist has the potential to reduce E. coli on the surface of baby kale leaves with no significant change in the color values.

Industrial relevance

Kale leaves are consumed worldwide owing to their health-promoting and nutritional benefits. However, in the past two decades there has been an increase in foodborne diseases outbreaks caused by microorganisms associated with leafy vegetables. The food industry faces challenges in improving food safety while maintaining quality of the food. Chemicals in wash water or heat treatment (70–121 °C), commonly used methods for killing microorganisms, can have negative effect on the aesthetic (color) and nutritional quality of the product. Cold plasma treatment of fresh produce, an emerging food preservation technology, is an efficient, quick, and environmentally-friendly method of disinfecting the product at low temperature (30–40 °C). This study demonstrated that treatment of baby kale with cold plasma mist not only inactivated E. coli, but also enhanced its color stability during refrigerated storage. Hence, cold plasma mist has a potential application as an efficient disinfection technology, thus enhancing shelf-life of fresh leafy greens.

Introduction

Fresh produce and leafy greens in particular have been implicated in foodborne disease outbreaks on a global level (Gajraj, Pooransingh, Hawker, & Olowokure, 2012). The number of leafy vegetable-associated outbreaks reported to Centers for Disease Control and Prevention (CDC) between 1973 and 2012 was higher than that of outbreaks caused by other food types (Herman, Hall, & Gould, 2015). Currently, antimicrobial interventions on fresh produce consist primarily of washes with a few approved chemical agents such as sodium hypochlorite and peroxyacetic acid/hydrogen peroxide. With these agents, it is necessary to find concentrations that are effective in inactivating undesirable microorganisms, but do not have negative effects on the quality of treated produce.

Cold plasma treatment is an emerging green process technology for decontamination of fresh fruit and vegetables (Pankaj et al., 2014). Plasma is a partially ionized gas consisting of ions, electrons, uncharged atoms and molecules, as well as radicals (Hoffmann, Berganza, & Zhang, 2013). Non-thermal plasma discharges can be generated under ambient temperatures. Therefore, cold plasma technology has the potential of reducing costs on an industrial scale compared to various non-thermal disinfection treatments such as ionizing radiation, high hydrostatic pressure, pulsed electric field, oscillating magnetic field and high power ultrasound (Kim et al., 2011).

Various studies have been carried out to evaluate the ability of cold plasma treatment in inactivating foodborne pathogens including gram-negative, gram-positive, and biofilm-forming bacteria. Liu et al. (2010) reported complete inactivation of Staphylococcus aureus suspended in aqueous solution after 16 min of treatment with a cold atmospheric pressure air plasma microjet. Their study also indicated that acidity of the liquid solution and interaction of plasma-activated species (radicals) resulted in peroxidation of fatty acids in the cell membrane that was responsible for inactivation of S. aureus in aqueous suspension. Decrease in microbial load was observed for apples inoculated with E. coli O157:H7, cantaloupes inoculated with Salmonella, and lettuce inoculated with L. monocytogenes when treated with a uniform glow discharge plasma (Critzer, Kelly-Wintenberg, South, & Golden, 2007). Biofilms produced by Bacillus subtilis spores on polycarbonate membranes supported by a layer of agar in a petri dish were inactivated by dielectric barrier discharge (DBD) with a helium plasma plume (Deng, Shi, & Kong, 2006). Their results showed that while reactive oxygen species (ROS) generated by plasma caused leakage of cytoplasmic components and complete rupture of the bacterial spore membrane, UV photons and the electric field made little or no contribution towards spore inactivation. In the present study, we evaluated the efficacy of cold plasma (non-equilibrium atmospheric pressure-pulsed dielectric barrier discharge)-treated mist in disinfecting baby kale leaves inoculated with E. coli O157:H7.

The aim of a disinfection technology is not only the inactivation of pathogens, but also to prolong shelf-life while maintaining nutritional and organoleptic food quality (Misra, Oliver, & PJ, 2016). Previous studies have reported both positive and negative effects of cold plasma on the quality of fresh produce. No change in quality was observed when fresh lettuce (color, antioxidant activity, and ascorbic acid) and strawberry (texture, firmness, color and pH) were treated with atmospheric plasma jets and DBD in-package plasma, respectively (Smeu et al., 2012; Misra et al., 2014). While carrots and tomatoes treated with a cold argon plasma needle showed insignificant change in color (0.5 to 10 min treatment), lettuce exhibited a change in color under the same treatment conditions beyond 7 min (Bermúdez-Aguirre, Wemlinger, Pedrow, Barbosa-Cánovas, & Garcia-Perez, 2013). Song et al. (2015) reported an increase in yellowness of lettuce after refrigerated storage of microwave-powered nitrogen cold plasma treatment. Another study reported loss of freshness, color, odor, texture, and overall acceptability of DBD plasma-treated radicchio leaves (Pasquali et al., 2016). Changes in food quality following cold plasma treatment can occur because reactive species catalyze oxidation and peroxidation processes inside the cell membrane not only of adhering microorganisms, but also the cells of produce being treated. Plants generally have cuticles covering the epidermal surfaces of leaves as a protective barrier (Chaumat & Chamel, 1991; Heredia-Guerrero et al., 2014). The wax components present in the cuticle are responsible for maximum resistance to moisture loss, insects, and pests (Lamikanra, Imam, & Ukuku, 2005; Znidarcic, Valič, & Trdan, 2008). The cuticle and its components (fatty acids, alcohols, aldehydes, esters, and alkanes) are critical in maintaining freshness and aesthetic quality of produce (Domínguez, Heredia-Guerrero, & Heredia, 2010). No study till date has investigated the effect of cold plasma treatment on cuticles of fresh leafy vegetables.

For the present study, a non-equilibrium atmospheric pressure-pulsed DBD with a single-dielectric layer on the inner electrode was designed. The effect of micrometer-sized water droplets (mist) passing through this system was investigated with respect to inactivation of E. coli O157:H7, color values and cuticle composition of baby kale leaves. Furthermore, the effect of DBD cold plasma mist treatment on color stability of baby kale leaves during storage at 4 °C for 12 days was determined.