Inactivation of Enterobacter aerogenes on the surfaces of fresh-cut purple lettuce, kale, and baby spinach leaves using plasma activated mist (PAM)

https://doi.org/10.1016/j.ifset.2021.102868Get rights and content

Highlights

  • Plasma activated mist (PAM) was more effective against E. aerogenes attached to smooth surfaces than uneven surfaces.

  • Twenty minutes exposure to PAM reduced the concentration of E. aerogenes on agar plate by ~5.5 log CFU/plate.

  • Twenty minutes exposure to PAM reduced the concentration of E. aerogenes on leafy greens by ~2.0 log CFU/leaf.

Abstract

Dielectric barrier discharge plasma-activated mist (PAM) is a surface treatment that has been shown to have antimicrobial effects on microorganisms attached to food contact surfaces. In this study, tryptic soy agar, purple lettuce, kale, and baby spinach leaves, were surface-inoculated with Enterobacter aerogenes inoculum (4 × 107 CFU/ml) and held for 30 min at room temperature (25 °C), then subsequently exposed to PAM in an enclosure (0.04 m3) from 5 to 20 min. Reductions ranging from 3.8 ± 0.1 log CFU/plate to 5.6 ± 0.3 log CFU/plate were observed on agar plates after exposure to PAM for 5 to 20 min. The leaves were either dip-inoculated or spot-inoculated. Extending PAM treatment time from 5 to 20 min increased microbial reduction on dip-inoculated leaves from 0.4 ± 0.2, 0.8 ± 0.1, and 0.9 ± 0.1 log CFU/g to 0.9 ± 0.1, 1.3 ± 0.1, and 2.0 ± 0.2 log CFU/g for purple lettuce, kale, and baby spinach leaves, respectively, and similar bacterial inactivations were observed on spot-inoculated leaves.

Introduction

Fresh-cut fruits and vegetables, namely leafy vegetables, are considered high risk in terms of food safety when consumed raw. According to the most recently released data from Center for Disease Control and Prevention (CDC, USA), there were 1339 illnesses associated with fresh-cut fruits and vegetables in 2017. Possible sources of microbial contamination of fresh-cut fruit and vegetables include soil, wash water, and food contact equipment, such as belt conveyors and flumes. To ensure the safety of fresh-cut fruits and vegetables, they have to be properly washed with a sanitizer and rinsed before reaching the consumer market.

Available sanitation/cleaning approaches for fresh-cut leafy vegetables include using chemical sanitizers, such as chlorinated water and peracetic acid solution, using highly turbulent wash water to detach bacteria and other contaminants from the vegetable surfaces, and the combination of the wash water and sanitizers (Gil, Selma, López-Gálvez, & Allende, 2009). Although some alternative technologies, such as high pressure (HPP) (Narwankar, Flimlin, Schaffner, Tepper, & Karwe, 2011; Tan & Kerr, 2015), high-intensity pulsed electric field (HIPEF) (Scott, Dickinson, & Shama, 2021), ultraviolet radiation (UV) (Kim et al., 2013), radio frequency (RF) (Usall, Ippolito, Sisquella, & Neri, 2016), ionizing radiation (Fan, Niemira, & Sokorai, 2003), and gas cold atmospheric pressure plasma (Silvetti et al., 2021) have been reported to be effective for sanitizing leafy vegetables and offering some benefits (e.g., non-destructive and non-thermal), using chemical sanitizers and water to clean or spray fresh-cut leafy vegetables remains the most frequently used sanitization approach for the fresh produce processors because it is cost-effective. Legislative concerns regarding the scaling up of the new technologies limit their use in the food industry (Artés-Hernández, Martínez-Hernández, Aguayo, Gómez, & Artés, 2017) at least as of now.

In the past decade, atmospheric cold plasma and plasma-based sanitizers, such as plasma-activated water (PAW) and plasma-activated mist (PAM), have received increased attention and have been explored as eco-friendly sanitizers for surface decontamination (Sysolyatina et al., 2020; Xu, Tian, Ma, Liu, & Zhang, 2016). Air plasma consists of ionized or partially ionized gases, photons, electrons, positive and negative ions, and free radicals (Hepbasli, Colak, Hancioglu, Icier, & Erbay, 2010; Misra, Tiwari, Raghavarao, & Cullen, 2011). Air plasma discharge has been explored as a surface decontamination approach to inactivate the bacteria or biofilm on the surface of vegetables (Mošovská et al., 2018; Patange et al., 2019), in planktonic systems (Ziuzina, Han, Cullen, & Bourke, 2015), cereal grains, and seeds (Billah et al., 2020; Los, Ziuzina, Boehm, Cullen, & Bourke, 2017), and in meats (Bhide, Salvi, Schaffner, & Karwe, 2017; Misra & Jo, 2017; Wang, Zhuang, Hinton, & Zhang, 2016).

Plasma-activated water (PAW) and plasma-activated mist (PAM) are obtained by exposing water or water mist, respectively, to air-based plasma discharge. Reactive oxygen and nitrogen species (RONS) such as H2O2 (hydrogen peroxide), NO3 (nitrate), NO2 (nitrite), and ONOO (peroxynitrite) generated in PAW and in PAM have been reported to have antimicrobial effects on several major food pathogens, including Staphylococcus aureus (Ma et al., 2015), Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella (Critzer et al., 2007; Jiang et al., 2017). The generation method for plasma, such as dielectric barrier discharge or gliding arc, affects the presence and concentrations of RONS.

Many previous studies have validated the antimicrobial effects of PAW and explored the applications of PAW as a sanitizer for foods such as bean sprouts, mushrooms, berries, grapes, and fresh-cut lettuces (Guo et al., 2017; Kučerová, Henselová, Slováková, Bačovčinová, & Hensel, 2021; Ma et al., 2016; Xu et al., 2016) and food contact surfaces (Joshi, Salvi, Schaffner, & Karwe, 2018a; Juzhong Tan & Karwe, 2021).

However, the antimicrobial effects and the potential applications of PAM have not been well-studied. In one previous study, they studied the effect of PAM on seed germination and found that PAM can increase the germination rate of selected seeds (El Shaer et al., 2020). Only a few studies have reported the antimicrobial effect of PAM on L. monocytogenes, Salmonella Typhimurium, and E. coli O157:H7 on agar surfaces (Sysolyatina et al., 2020). The antimicrobial effects of PAM on food pathogens attached to the surfaces of fresh produce, especially leafy vegetables, are still not well documented. Compared to using chemical sanitizer sprays, PAM offers several advantages, including low water usage and is free of chemical sanitizer residuals.

The objective of this study was to investigate the effects of PAM treatment time, inoculation methods, and inoculation locations on inactivating E. aerogenes inoculated on agar plates and three types of vegetable leaves (purple lettuce, kale, and baby spinach leaves).

Section snippets

Materials and methods

A PAM generator and an exposure chamber were custom built. Inoculated TSA agar plates and dip- or spot- inoculated fresh-cut vegetable leaves were exposed to PAM in the exposure chamber at room temperature and atmospheric pressure.

Characterizations of PAM condensate

The pH, ORP, EC, and the concentrations of hydrogen peroxide (H2O2), nitrites (NO2), and nitrates (NO3) in PAM condensate are shown in Table 1. The pH of the tap water that was used to generate the mist was 6.8, and the pH of PAM condensate was 4.1, which was similar to the pH of plasma-activated water (PAW) reported in previous studies (Joshi et al., 2018b; Li et al., 2017; Juzhong Tan & Karwe, 2021). The concentration of nitrates in the PAM concentrate was 240 ± 11 ppm, and the

Conclusions

PAM was found to be an effective antimicrobial agent against E. aerogenes attached to agar surfaces and the surfaces of baby spinach, kale, and lettuce leaves. PAM was found to cause more microbial inactivation when the surface was flat and smooth. Curled, folded, or cracked surfaces seem to reduce the antimicrobial effectiveness of PAM due to the inability of PAM to penetrate the deeper regions. The antimicrobial effect of PAM was slightly reduced on dip-inoculated vegetable leaves compared to

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Juzhong Tan: Conceptualization, Methodology, Validation, Investigation, Software, Writing – original draft, Formal analysis, Visualization. Mukund V. Karwe: Supervision, Resources, Project administration, Writing – review & editing.

Acknowledgments

This research was supported and funded by USDA NIFA under Federal Award No. 2018-67018-28146 and 2016-51181-25403. Authors are thankful to Dr. Donald W. Schaffner and his students the Department of Food Science at Rutgers University, for providing microbial culture, training, and advice.

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