Nisin-based antimircobial combination with cold plasma treatment inactivate Listeria monocytogenes on Granny Smith apples

Highlights

• Nisin-based antimicrobial and cold plasma treatments combination reduced microbial population on Granny Smith apples.

• Individual treatments alone did not cause significant reduction.

• The combination treatments for 3 min led to significant reduction listeria monocytogenes.

• SEM observation did not show changes cell morphology.

Abstract

A nisin-based antimicrobial and cold plasma combination treatments in reducing Listeria monocytogenes inoculated on apple surfaces purchased from a New Jersey farm and a supermarket in Philadelphia area was investigated. All apples were spot inoculated or by submersion in 10⁷ CFU/ml L. monocytogenes inoculum. Populations of L. monocytogenes recovered on farm and supermarket apples after spot inoculation averaged 5.8 ± 0.24 log CFU/g and 4.6 ± 0.12 log CFU/g, respectively and 4.1 ± 0.22 log CFU/g and 3.6 ± 0.12 log CFU/g, respectively on submerged apples. All apples were treated with antimicrobial solution for 30 s, 40 s, 3 min (180s) and 1 h (3600 s), cold plasma treatments for 30 and 40s, and a combination of antimicrobial and cold plasma treatments and the surface structure of apples were examined using scanning electron microscopy (SEM). Cold plasma treatment at 40s, followed immediately with antimicrobial treatments at 180s and 3600 s led to 2.5 and 4.6 log CFU/g inactivation of L. monocytogenes, respectively. SEM observation showed changes on apple surface structures but not on bacterial cell structure. This result suggests that this combination treatments is effective in killing L. monocytogenes on apple surfaces.

Introduction

Microbial populations of fruits and vegetables varies due to the fact they are frequently in contact with soil, insects, animals, or humans during growing or harvesting and in the processing plant (Ukuku, Olanya, Geveke, & Sommers, 2012). Fresh fruit and vegetable produce are ranked the fourth food category responsible for foodborne illnesses in the United States, implicated in 1.2 million illness, 7100 hospitalizations, 134 human deaths, and $1.4 billion in associated illness costs each year (Batz et al., 2004). Presence of human bacterial pathogens on fresh produce and outbreaks of diseases has led to costly recalls (CDC, 2011; FDA, 2008). In all outbreaks noted so far, reports mentioned produce from the farm, processing line or which had been precut and held at unknown temperatures for some period of time at retail prior to being purchased and consumed (Ukuku, Geveke, Chau, & Niemira, 2016). Washing with chlorinated water at 200 ppm is one of the very first processing operations to reduce dirt and microbial populations before fresh-cut processing; yet, produce surfaces are not free from natural contaminants. There is variation in total number of native microflora of apples due to location, weather and variety. For example, microbial populations of ground harvested apples ranged from 3.4 log CFU/g to 5.6 log CFU/g while those picked from the tree averaged 4.8 log CFU/g (Keller et al., 2004). Listeria monocytogenes is a food safety concern because it is widespread in the environment (Ukuku et al., 2012). L. monocytogenes has been implicated as a causative agent of several foodborne outbreaks, which resulted in both human illness and death (CDC, 2011; FDA, 2008). This pathogen has been isolated from soil, sewage sludge, vegetation, and water therefore, has the potential to contaminate apple surfaces (FDA, 2008). The level of sanitation at the farm, during processing, shipping and at retail outlets as well as the initial microbiological load are of primary importance to the quality, shelf stability and safety of fresh and fresh-cut produce (Ukuku, Sapers, & Fett, 2004, 2017). Presence of L. monocytogenes has been documented in fresh apples, sliced apples, and stone fruits including whole peaches, nectarines, plums, and pluots leading to several recalls. On January 6, 2015, Bidart Bros. (Bakersfield, California) voluntarily recalled Granny Smith and Gala apples as a result of environmental contamination with Listeria monocytogenes at the firm's apple-packing facility. Similarly, Happy Apples (California Snack Foods), and Merb's Candies each voluntarily recalled commercial produced and prepackaged caramel apples as result of Listeriosis outbreak associated with 34 people being hospitalized of which at least seven deaths were reported.

Physical and chemical treatments are used in food processing to eliminate or at least reduce the presence of pathogenic and spoilage microorganisms in foods (Ukuku, Latiful, Kassama, Mukhopadhyay, & Olanya, 2015). Thermal or minimal thermal treatments are still best option for microbial reduction in foods precluding fresh-cut vegetables and fruits. Produce including other fresh and fresh-cut vegetables and fruits are heat sensitive therefore any heat treatment can lead to changes in quality attributes of the fruits and vegetables. For now, processing of fruits and vegetables requires chemical washes using antimicrobial compounds such as ozone, chlorine, hydrogen peroxide, 1-MCP and control atmospheric storage to reduce bacterial populations including use of edible coating as a post-harvest treatment (Mahajan, Caleb, Singh, Watkins, & Geyer, 2014). There is much interest in developing non-thermal processing intervention technology that can kill bacteria on apple surface without damaging and or contribute to changes in the apples.

Cold plasma (also referred to as cool plasma or non-thermal plasma) is a non-thermal process. Its application have long been extended to surface treatment of materials such as electronics, textiles, polymers, and print surfaces (Niemira & Gutsol, 2010). It is relatively new and the food industry is beginning to recognize its antimicrobial activity (Niemira, 2012a and b; Surowsky, Frohling, Gottschalk, Schluter, & Knorr, 2014). This technology has shown promise as a direct treatment for fresh and fresh-cut fruits and vegetables, as well as for nuts and other foods (Hertrich, Boyd, Sites, & Niemira, 2017; Pankaj & Keener, 2017). There is limited information in the literature on the combination of cold plasma with antimicrobial solution for inactivation of bacterial pathogens on produce surfaces. There is a need to understand cold plasma interaction with produce surface structure and how it leads to bacterial inactivation. Such understanding including the mechanisms of bacterial inactivation on produce surfaces will be a welcome addition to the fresh produce and fresh-cut industry and the regulatory agencies. The objective of this study was to investigate the use of antimicrobials and cold plasma combination treatments to reduce native microflora and inoculated L. monocytogenes bacteria on apples, with particular attention directed to the bacterial populations at the apple calyx/stem region. Finally, the efficacy of the treatment combination in maintaining the microbial safety of Listeriae contaminated apples were investigated.