Disinfection efficacy and mechanism of slightly acidic electrolyzed water on Staphylococcus aureus in pure culture

Highlights

• Disinfection mechanism of slightly acidic electrolyzed water was investigated against Staphylococcus aureus.

• SAEW treatment had higher disinfection efficacy compared to NaClO and HCl.

• SAEW had significantly effects on the permeability of cell membranes.

• The ranking of partly agglutinated cellular inclusion formation was HCl > SAEW > NaClO.

Abstract

Slightly acidic electrolyzed water (SAEW), considered as a broad-spectrum and high-performance bactericide are increasingly applied in the food industry. However, its disinfection mechanism has not been completely elucidated. This study aims to examine the disinfection efficacy and mechanism of SAEW on Staphylococcus aureus, compared with that of sodium hypochlorite (NaClO) and hydrochloric acid (HCl). SAEW treatment significantly reduced S. aureus by 5.8 log CFU/mL in 1 min, while 3.26 and 2.73 log reductions were obtained with NaClO and HCl treatments, respectively. A series of biological changes including intracellular potassium leakage, TTC-dehydrogenase relative activity and bacterial ultrastructure destruction were studied following disinfection treatment of S. aureus. The results showed that SAEW decreased the relative activity of TTC-dehydrogenase by 65.84%. Comparing intracellular potassium leakage, the SAEW treatment caused a greater percent of protein leakage (108.34%) than the NaClO (18.75%) or HCl (0.84%) treatments. These results demonstrated the potent impact SAEW had on the permeability of cell membranes. In addition, the ranking of partly agglutinated cellular inclusion formation was HCl > SAEW > NaClO. It appeared that HCl, along with its low pH value, are responsible for most of the cytoplasmic disruptions. Overall, this study demonstrated that the disinfection mechanism of SAEW was disrupting the permeability of cell membrane and the cytoplasmic ultrastructures in S. aureus cells.

Introduction

Staphylococcus aureus, a gram-positive microorganism, is capable of secreting several different kinds of enterotoxins associated with specific staphylococcal infections (Ding et al., 2010). The risks associated with consuming foods contaminated with S. aureus have caught the attention of major public and governmental organizations in recent years. To date, there are at least 30 countries on six continents that have reported S. aureus infection in humans. In Japan, about 32.5% of the contaminated food showed the presence of S. aureus and particularly for raw milk, a 76.3% higher rate of contamination was found (Tian, Ji, Yang, Li, & Liu, 2007). The percentage of bacterial food poisoning cases caused by S. aureus was estimated to be 33% and 45% in United States and Canada, respectively (Shao, Dong, Zhao, Kong, & Liu, 2013). Similar poisoning cases have also occurred in China where more than 100 children were infected when they drank contaminated calcium milk produced by the Weiwei Daheng Dairy Co., Ltd. in 2008 (Xia et al., 2012).

In order to produce high-quality, microbiologically safe food for human consumption, numerous disinfection methods have been studied and/or used in the food industry. A few of these methods are the use of chemical disinfectants (hypochlorite, chlorine dioxide, hydrogen peroxide, hydrochloric acid, ozone etc.), physical treatments (heat and irradiation etc.), and their combinations (Koide et al., 2011, Zhang et al., 2011). The mechanisms concerning the traditional disinfection methods have been sufficiently studied by many researchers (Juneja and Sofos, 2002, Komanapalli and Lau, 1996, Setlow et al., 2002, Wang et al., 2015). The physical heat treatment method, where the bacteria are subjected to inhospitable temperatures, is regarded as the primary factor affecting microbial inactivation, as it can inhibit the activity of intracellular proteins and enzymes, and damage the cellular membranes and nucleic acids (Juneja & Sofos, 2002). Setlow et al. 2002 reported that the disinfection mechanism of strong acid or alkali treatment on Bacillus subtilis spores was due to the inactivation of the intracellular enzymes and the disruption of the permeability barrier. To date only a few researchers have investigated the disinfection mechanism of electrolyzed oxidizing water (EOW) (Zeng et al., 2010, Zeng et al., 2011). These researchers reported that EOW caused damage to the cell walls, nucleus and outer membranes, which lead to the rapid leakage of intracellular K⁺, DNA and proteins, and a decrease in the dehydrogenase activities of S. aureus. It is generally accepted that the high oxidation reduction potential (ORP) of EOW could significantly influence EOW's disinfection power by penetrating the outer and inner membranes (Liao, Chen, & Xiao, 2007), and the low pH was also a main factor in EOW's bactericidal efficacy (Waters & Hung, 2014). On the other hand, slightly acidic electrolyzed water (SAEW) has a relatively low ORP value (less than 1000 mV), a pH near neutrality pH 5.0–6.5 and HClO as the main chlorine compound (Cao et al., 2009, Koide et al., 2009, Koide et al., 2011). To date, there are very few studies investigating the disinfection mechanism of SAEW.

Therefore, the aim of this study was to investigate the disinfection efficacy and mechanism of SAEW against S. aureus in comparison with NaClO and HCl where the main forms of the chlorine compounds are ClO⁻ and Cl⁻. The inactivation of S. aureus by SAEW, NaClO, and HCl was determined by plate count while the disinfection mechanism was investigated by measuring changes to intracellular potassium leakage, TTC-dehydrogenase relative activity and cellular ultrastructure as indicators.