Analysis of Neutral Electrolyzed Water anti-bacterial activity on contaminated eggshells with Salmonella enterica or Escherichia coli

Highlights

• Neutral electrolyzed solution had a neutral pH (7.12) and ORP value by 907.27 mV.

• NEW reduced in vitro bacterial titers by 99%.

• NEW reduced 99.16% and 99.99% of the Salmonella and E. coli load/egg.

• NEW caused external damage on Salmonella enterica and E. coli.

• CAS eliminates most of the cuticle and NEW left it morphologically intact.

Abstract

Neutral Electrolyzed Water (NEW) was tested in vitro and on artificially contaminated eggs against Salmonella enterica subsp. enterica or Escherichia coli. The antibacterial effect was measured 30 s after treatment. NEW microbicide activity results were compared against 2% citric acid and 0.9% saline solutions. NEW caused an in vitro decrease in Salmonella titers by ˃5.56 Log₁₀ CFU mL⁻¹ and in artificially contaminated eggs by ˃1.45 Log₁₀ CFU/egg. When it was tested against E. coli, it decreased in vitro bacterial titers by ˃3.28 Log₁₀ CFU mL⁻¹ and on artificially contaminated eggs by ˃6.39 Log₁₀ CFU/egg. The 2% citric acid solution caused an in vitro decrease of 0.4 Log₁₀ CFU mL⁻¹ of Salmonella and E. coli and on eggs artificially contaminated with E. coli or Salmonella there was a decrease of 0.06 and 0.62 Log₁₀ CFU/egg respectively. We evaluated egg cuticle integrity by scanning electron microscopy after treatments with evaluated solutions; the 2% citric acid solution caused damage to the cuticle and exposed eggshell pores and no interaction of NEW or NaCl with the cuticle was observed. NEW treatment showed a fast-bactericidal effect in vitro and table eggs.

Introduction

While the food industry tries to maintain food safe and with high quality, foodborne outbreaks still exist. Salmonella and Escherichia coli have been reported to be the principal pathogens related to foodborne disease outbreaks (CDC, 2010). Salmonella spp has an incidence of 20.9 and 17.2 cases per 100,000 and E. coli 1.82 and 2.2 per 100,000 in the European Union (EU) and US respectively (Food and Authority, 2017). Eggs and poultry are important vehicles for the transmission of Salmonella which has been classified as a food pathogen related to outbreak-associated illnesses (Report, 2011). Among Salmonella serotypes, S. Enteritidis and S. Typhimurium are frequently reported in outbreaks and are present in avian digestive tracts. Eggshells can be contaminated when they come into contact with feces, infested nests, or surrounding environments (Holck et al., 2018; Huang et al., 2008; Humphrey, 1994; Paramithiotis et al., 2017).

The washing process of eggshells of table eggs has not been implemented worldwide. This practice has been adopted in countries like Japan, the US, and Australia (Hutchison et al., 2004; Jones and Musgrove, 2005; Northcutt et al., 2005). The major advantage to egg washing is the decrease in bacterial load on eggshells. However, one of the major concerns about the egg washing process is the possible damage to protective structures like the cuticle (European Food Safety Authority (EFSA), 2005). Non-corrosive effects, low public health impacts, and being environmentally friendly are some important characteristics that disinfectants should have. Several sanitizers have been used or described in egg washing processes like organic acids (National Service for Agroalimentary Public Health [SENASICA], 2011) and chlorine based sanitizers (Northcutt et al., 2005; Soljour et al., 2004; Wang and Slavik, 1998).

The use of Electrolyzed Water (EW) is a promising strategy in the egg washing process. EW is generated by electrolysis of NaCl in water (Cheng et al., 2012) and its use has previously been reported in cutting boards (Venkitanarayanan et al., 1999) spinach (Guentzel et al., 2008), lettuce (Abadias et al., 2008; Koseki et al., 2004), meat (Fabrizio and Cutter, 2004), and strawberries (Udompijitkul et al., 2007) for the control of foodborne diseases. Its bactericidal effects are explained by three characteristics: Oxidation Reduction Potential (ORP), presence of reactive chlorine and oxygen species like hypochlorous acid (HOCl), and pH. High ORP causes modifications in metabolic flux and ATP production. It inhibits glucose oxidation, disrupts protein synthesis, inhibits oxygen uptake and oxidative phosphorylation which is coupled with the leakage of some macromolecules (Huang et al., 2008) and causes damage to cell membranes (Liao et al., 2007). Hypochlorous acid is the main component of EW and it produces a hydroxyl radical that acts on different pathogens (Len et al., 2002, Len et al., 2000; Len et al., 2002). pH is an important factor because bacteria generally grow in a pH range of 4 to 9 (Huang et al., 2008) and changing the pH cause proteins to denature. Based on pH, EW has been historically classified into two groups: acid electrolyzed water (AEW) and basic electrolyzed water (BEW). AEW (pH ̴ 2.0) has been reported in chicken meat (Fabrizio and Cutter, 2004), cutting boards (Venkitanarayanan et al., 1999), and eggs (Ni et al., 2014). Alkaline EW has been used against E. coli (Liao et al., 2007) and on eggs (Ni et al., 2014). There is also a slightly acidic solution (near neutral, pH 6.0–6.5) that has been tested in tomatoes (Deza et al., 2003), lettuce (Guentzel et al., 2008), grapes, peaches (Guentzel et al., 2010), chicken (Rahman et al., 2012), beef (Ding et al., 2010), and eggshells (Cao et al., 2009) as well as against the main foodborne pathogens like Salmonella, E. coli, Listeria monocytogenes and Enterococcus fecalis.

The goal of the present work is to evaluate a Neutral Electrolyzed Water (NEW) with a pH ̴ 7 against Salmonella enterica and Escherichia coli in vitro as well as on artificially contaminated eggshells. Additionally, cuticle and bacterial integrity were evaluated by transmission and scanning electronic microscopy to identify possible damage. To our knowledge, this type of EW has never been previously reported on eggs.