Efficacy of Acidic and Alkaline Electrolyzed Water for Inactivating Escherichia coli O104:H4,...

Accepted Manuscript

Efficacy of Acidic and Alkaline Electrolyzed Water for Inactivating Escherichia coliO104:H4, Listeria monocytogenes, Campylobacter jejuni, Aeromonas hydrophila, andVibrio parahaemolyticus in Cell Suspensions

Mahmoudreza Ovissipour, Hamzah M. Al-Qadiri, Shyam S. Sablani, Byju N.Govindan, Nivin Al-Alami, Barbara Rasco

PII: S0956-7135(15)00019-5

DOI: 10.1016/j.foodcont.2015.01.006

Reference: JFCO 4237

To appear in: Food Control

Received Date: 10 November 2014

Revised Date: 8 January 2015

Accepted Date: 10 January 2015

Please cite this article as: Ovissipour M., Al-Qadiri H.M., Sablani S.S., Govindan B.N., Al-Alami N. &Rasco B., Efficacy of Acidic and Alkaline Electrolyzed Water for Inactivating Escherichia coli O104:H4,Listeria monocytogenes, Campylobacter jejuni, Aeromonas hydrophila, and Vibrio parahaemolyticus inCell Suspensions, Food Control (2015), doi: 10.1016/j.foodcont.2015.01.006.

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http://dx.doi.org/10.1016/j.foodcont.2015.01.006

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Efficacy of Acidic and Alkaline Electrolyzed Water for Inactivating 1

Escherichia coli O104:H4, Listeria monocytogenes, Campylobacter 2

jejuni, Aeromonas hydrophila, and Vibrio parahaemolyticus in Cell 3

Suspensions 4

Mahmoudreza Ovissipour1, Hamzah M. Al-Qadiri1,2, Shyam S. Sablani3*, Byju N. 5

Govindan3, Nivin Al-Alami4, Barbara Rasco1 6

1School of Food Science, Washington State University, Pullman, WA 99164, USA 7

2Department of Nutrition and Food Technology, Faculty of Agriculture, The 8

University of Jordan, Amman 11942 Jordan 9

3Department of Biological Systems Engineering, Washington State University, 10

Pullman, WA 99164, USA 11

4Water, Energy and Environment Center, The University of Jordan, Amman 11942 12

Jordan 13

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Running Title: Effect of electrolyzed water on pathogenic bacteria cell suspensions 17

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*Corresponding author. Tel: +1 509 335 7745; Fax: +1 509 335 2722. Email address: 19

[email protected] 20

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Abstract 21

This study investigated the effect of electrolyzed water on pathogenic bacteria cell suspensions. 22

Specifically, we evaluated the efficacy of strong and weak acidic electrolyzed waters (SACEW, 23

WACEW) and strong and weak alkaline electrolyzed waters (SALEW, WALEW) on Vibrio 24

parahaemolyticus, Listeria monocytogenes, Aeromonas hydrophila, Campylobacter jejuni, and 25

Escherichia coli O104:H4 in suspensions of (107 to 109 CFU/mL) in 1% NaCl. SACEW and 26

WACEW were applied at available chlorine concentrations (ACC) of 20 and 10 mg/mL, pH 3.1 27

and 3.55 and oxygen reduction potentials (ORP) of 1150 and 950 mV, respectively. Results 28

show that no viable cells were recovered for V. parahaemolyticus, L. monocytogenes, A. 29

hydrophila, C. jejuni within 2 min at 20oC. However, E. coli O104:H4 was significantly more 30

resistant to ALEW compared to ACEW. Results also show that the bactericidal activity of 31

SACEW (20 mg/mL ACC) was more effective than WACEW (10 mg/mL ACC) in terms of 32

inactivating E. coli O104:H4. Alkaline-electrolyzed waters were found to reduce cell numbers by 33

1 to 3 log (P < 0.05). However, alkaline electrolyzed water was less effective (P < 0.05) than 34

acidic electrolyzed treatment. 35

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Key words: Acidic electrolyzed water, alkaline electrolyzed water, pathogenic bacteria, cell 37

suspensions 38

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1. Introduction 42

Worldwide demand for whole and cut fresh produce items with high-quality and microbiological 43

safety is increasing. In May 2011, an outbreak caused by sprouts contaminated with Escherichia 44

coli O104:H4 occurred in northern Germany, leading to 3,222 infected patients (39 of whom 45

died within a month). Approximately 25% of the patients showed hemolytic-uremic syndrome 46

(HUS), which is higher than the rate reported for those infected with Shiga toxin-producing E. 47

coli (Qin et al., 2011). 48

Vibrio parahaemolyticus occurs naturally in marine environments, is one of the leading causes of 49

foodborne infections associated with molluscan shellfish consumption in the United States and 50

China (McLaughlin et al., 2005; Ren and Su, 2006; Rong, Lin, Wang, Khan, & Li, 2014; Su and 51

Liu, 2007). Campylobacter jejuni infection is the most common bacterial cause of human 52

gastroenteritis (Al-Qadiri, Lu, Al-Alami, & Rasco, 2011; Altekruse, Stern, Fields, & Swerdlow, 53

1999), and develops through consumption of contaminated meat, poultry, dairy products and 54

bottled water (Al-Qadiri et al., 2011; Eideh and Al-Qadiri, 2011; Park, Hung, & Brackett, 2002). 55

In fact, there are about 6 billion cases annually of gastrointestinal illness from drinking 56

contaminated water annually (Al-Qadiri et al., 2011). There is a relatively high frequency of C. 57

jejuni contamination in poultry, and the microbe lives on chicken skin and contact surfaces (Park 58

et al., 2002). There are no available data regarding C. jejuni in fish products, with the exception 59

of a foodborne outbreak of C. jejuni infection in tuna salad. However, this was likely due to egg 60

ingredients (Roels et al., 1998) or cross-contamination. Both V. parahaemolyticus and C. jejuni 61

can cause acute gastroenteritis, leading to diarrhea, headache and vomiting. 62

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Over the past twenty years, Aeromonas species have become a more common cause of 63

gastroenteritis, causing acute diarrhea in children and adults, and may be an important cause of 64

traveler’s diarrhea (Joseph, 1996). Aeromonas spp. have been reported in well water (Massa, 65

Altieri, & D'Angela, 2001) and drinking water production plants (Huys et al., 1995) as well as 66

fresh vegetables, fish, shellfish, meat and poultry (Isonhood and Drake, 2002; Villari, Crispino, 67

Montuori, & Stanzione, 2000). Aeromonas is a psychrotrophic bacteria that can grow well at 68

refrigeration temperatures from 4 to7°C, causing food spoilage and food-borne disease (Isonhood 69

and Drake, 2002). Aeromonas hydrophila causes disease in fish and human, and is also called 70

Motile Aeromonas Septicemia (MAS), Hemorrhagic Septicemia, Ulcer Disease, or Red-Sore 71

Disease. Aeromonas hydrophila is a major problem for the aquaculture industry, affecting 72

seafood quality and causing severe losses for production and marketing (Vivekanandhan, Hatha, 73

& Lakshmanaperumalsamy, 2005). Listeria monocytogenes also a psychrotroph, is widely 74

distributed in nature, and has caused several fatal outbreaks of foodborne illness. It has been 75

isolated at rates of up to 60% from ready-to-eat fish products (Al-Holy, Lin, & Rasco, 2005). 76

Therefore, it is critical to develop methods of eliminating L. monocytogenes from ready-to eat 77

food products. In fact, the U.S. Food and Drug Administration has set a zero tolerance level for 78

L. monocytogenes in ready-to-eat seafood products. 79

This study attempts to develop effective methods to reduce these pathogens in food and seafood-80

production environments. The use of electrolyzed water (EW) is a novel technology developed in 81

Japan, and involves electrolysis of deionized water containing a low concentration of sodium 82

chloride (0.1%) in an electrolysis chamber. Anode and cathode electrodes are separated by a 83

diaphragm, and a strong oxidant imparts powerful bactericidal and virucidal properties to the 84

water collected at the anode. At the anode, the acidic electrolyzed water (ACEW) has a pH of 2.7 85

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or lower, with an oxidation-reduction potential (ORP) greater than 1,100 mV, and an available 86

chlorine concentration (ACC) of 10 to 80 mg/mL. The alkaline electrolyzed water (ALEW) has a 87

pH of 11.6, and an ORP of -795 mV is generated at the cathode (Fabrizio & Cutter, 2003; 88

Venkitanarayanan, Ezeike, Hung & Doyle, 1999). 89

The properties of the ACEW and ALEW water depend on salt concentration, electrolysis time, 90

and water flow into the electrolysis chamber (Hsu, 2003). This topic has attracted considerable 91

attention from the food, medical, and agricultural industries (Katayose, Yoshida, Achiwa, & 92

Equchi, 2007). The antimicrobial effect of EW in fisheries has been reported (Kasai, Ishikawa, 93

Hori, Watanabe, & Yoshimizu, 2000; Kasai, Watanabe, & Yoshimizu, 2001a) and applied for 94

surface sanitization of seafood products. It has also been applied for sanitation at aquaculture 95

facilities (Huang et al., 2006; Jorquera, Valencia, Eguchi, Katayose, & Riquelme, 2002; Ren and 96

Su, 2006), including disinfection of waste seawater (Kasai, Watanabe, & Yoshimizu, 2001b; 97

Kasai, Yoshimizu, & Ezura, 2002). Although ACEW has strong antimicrobial properties, ALEW 98

may also decrease bacterial populations (Ayebah, Hung, & Frank, 2005; Fabrizio and Cutter, 99

2003; Koseki, Yoshida, Kamitani, Lsobe, & Itoh, 2004; Park, Alexander, Taylor, Costa, & Kang, 100

2008; Rahman, Jin, & Oh, 2011; Xie, Sun, Pan, & Zhao, 2012). 101

However, few studies have examined the effect of ALEW water on microbial inactivation. 102

Studies on microbial inactivation have investigated the effect of strong and weak ACEW with 103

high and low ACC on bacterial viability. For example, complete inactivation of 10 log10 104

CFU/mL E. coli O157:H7 and L. monocytogenes was achieved with ACEW (pH 2.6) at an ORP 105

of 1160 mV and an available chlorine concentration (ACC) of 56 mg/L for 30 s at 24°C (Kim, 106

Hung, & Brackett, 2000). Efficacy of electrolyzed water with higher available chlorine 107

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concentrations on bacteria inactivation has also been reported (Quan, Choi, Chung, & Shin, 108

2010). 109

Due to the need for more controlled studies on EW, we investigated the effect of strong and 110

weak acidic and alkaline electrolyzed waters on bacterial viability for E. coli O104:H4, L. 111

monocytogenes, A. hydrophila, V. parahaemolyticus, and C. jejuni in suspension. 112

2. Materials and methods 113

2.1. Bacterial strains and growth conditions 114

American Type Culture Collection (ATCC) strains were obtained from Microbiologics®, 115

Inc. (St. Cloud, MN). All strains were cultured to yield a cell count of approximately 108 to 109 116

CFU/mL. E. coli O104:H4 ATCC BAA-2326, L. monocytogenes ATCC 19111, and A. 117

hydrophila ATCC 35654 were cultured and activated by inoculating a Kwik-Stik swab into 50 118

mL of tryptic soy broth TSB (BactoTM) and incubated at 37°C for a maximum of 24 h. C. jejuni 119

ATCC 29428 strain was activated by inoculating a Kwik-Stik swab in 50 mL of Campylobacter 120

enrichment broth, consisting of Campylobacter nutrient broth no. 2 (CM0067, Oxoid Ltd.) and 121

supplemented with Campylobacter growth supplement (SR0232E, Oxoid Ltd.). C. jejuni broth 122

was then incubated in an anaerobic jar at 37°C for 48 h in a microoxic atmosphere (O2~6-7%) 123

with CampyGen sachets (CN0025, Oxoid Ltd.). V. parahaemolyticus ATCC 17802 was cultured 124

by inoculating a Kwik-Stik swab into 50 mL of tryptic soy broth (BactoTM) supplemented with 125

2% NaCl and then incubated at 37°C for a maximum of 24 h. 126

127

2.2. Cell suspension preparation 128

After appropriate incubation of bacterial cultures, 10 mL broth of each strain was 129

transferred under aseptic conditions to a sterile centrifuge tube, and then centrifuged for 15 min 130

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at 5,000 rpm (3,380 x g) to harvest bacterial cells (AccuSpinTM model 400 bench top centrifuge, 131

Fisher Thermo Scientific, Pittsburgh, PA). To eliminate any effect of broth components and 132

bacterial metabolites, the resultant pellets were resuspended in 10 mL of sterile saline solution 133

(0.85 % (w/v) NaCl). After the second centrifugation, the supernatant was decanted, and the 134

resulting washed pellets were then resuspended in sterile 10 mL aliquots as before. These were 135

then used as pure cell suspensions to study the effect of acidic and alkaline electrolyzed waters 136

on their activity. The approximate initial cell number for V. parahaemolyticus (7.08 log10 137

CFU/mL), L. monocytogenes (8.10 log10 CFU/mL), A. hydrophila (7.48 log10 CFU/mL), C. 138

jejuni (8.30 log10 CFU/mL), and E. coli O104:H4 (9.11 log10 CFU/mL) were used as “cell 139

suspension” in this study. Two replicate experiments were run, with the same experimental 140

conditions. 141

142

2.3. Electrolyzed water 143

Electrolyzed water was generated at 9-12 V direct current (dc) for 15 min using a two-144

compartment batch scale electrolysis apparatus (Super Oxseed Labo, Electrolyzed Water 145

Generator, Aoi Electronic Corp., Kannami, Shizuoka, Japan), with the anode and cathode sides 146

of the chamber divided by an ion exchange diaphragm. To optimize the chlorine concentration, 147

Oxidation-Reduction Potential (ORP), and pH in the acidic and alkaline electrolyzed water, a 148

series of preliminary experiments were conducted with different concentrations of sodium 149

chloride. Table 1 present the experimental design and properties of electrolyzed water. 150

The ORP and pH were measured with a pocket-sized redox meter (HI 98201, HANNA® 151

Instruments, Ann Arbor, Michigan, USA) and a pH meter (FE20, Mettler-Toledo, Columbus, 152

OH, USA), respectively. The free chlorine concentration of the EW was measured with a DPD 153

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assay (ColorimeterTM Analysis System, Hach Co., Loveland, CO, USA) according to 154

manufacturer instructions. 155

2.4. Electrolyzed water treatment of bacterial cell suspensions 156

To investigate the influence of ACEW and ALEW on bacterial suspension, 2 mL of cell 157

suspension was transferred to sterile 50 mL test tubes, and 38 mL of electrolyzed water was 158

added. Four different types of electrolyzed water were tested: strong acidic electrolyzed water 159

(SACEO), weak acidic electrolyzed water (WACEW), strong alkaline electrolyzed water 160

(SALEW), and weak alkaline electrolyzed water (WALEW) (Table 1) at 2, 4, and 6 min at room 161

temperature (20°C) (n=3). At each time interval, one mL of the treated cell suspension was 162

sampled and added to 9 mL of sterile 0.85% NaCl solution. To neutralize the available chlorine 163

(ACC), 1 mL of 3.0% sterile sodium thiosulfate was added to all solutions in advance. Treatment 164

with sterile 0.85% NaCl solution served as a control. 165

2.5. Recovery of bacteria and culture media 166

To recover the surviving bacteria, treated samples were examined in duplicate using 167

selective and nonselective culture media. One mL of the homogenized treated suspension was 168

serially diluted (dilution range: 100-10-5) in 9 mL sterile 0.1% peptone. Samples were then 169

examined in duplicate using the spread plate technique using 0.2 mL of the aliquots for E. coli 170

O104:H4, and cultured on m-Endo agar LES (DifcoTM). Plates were aerobically incubated at 171

37°C for 24 h, and the number of viable cells was determined as CFU/mL or E. coli O104:H4 172

(Al-Qadiri et al., 2008). For L. monocytogenes, A. hydrophila, and V. parahaemolyticus, samples 173

were spread plated onto PALCAM agar supplemented with antibiotics (CM0877, Oxoid Ltd.) 174

(Liu and Busse, 2010), selective Aeromonas medium (RYAN) (CM0833, Oxoid Ltd) 175

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(Warburton, McCormick, & Bowen, 1994), and selective TCBS Cholera medium (CM0333, 176

Oxoid Ltd.), respectively. Plates were incubated at 37°C for 24-48 h, and the number of viable 177

cells was determined as CFU/mL. TSA (BactoTM) was used as a nonselective culture medium 178

with the addition of 2% NaCl to culture V. parahaemolyticus. Nonselective Campylobacter 179

Blood-Free Agar (Modified CCDA-Preston, CM0739, Oxoid Ltd.) and selective agar prepared 180

with the addition of CCDA selective supplement (SR0155, Oxoid Ltd.) were used to recover and 181

enumerate surviving C. jejuni bacteria. Plates were incubated at 37°C for 48 h under 182

microaerophilic conditions. 183

184

2.6. Statistical analysis 185

We applied mixed-effect analysis to the data set using the PROC MIXED procedure in SAS 186

(Version 9.2, SAS, 2008) according to SAS software manual. To test whether the rate of 187

reduction in bacterial counts differed among and within the four non-selective treatment groups 188

over time, we examined the treatment x time interaction effect. Three replications for each of the 189

four treatments (3 x 4 = 12) were considered as random subjects. We ran the Tukey-Kramer 190

HSD test for pairwise comparison of bacterial counts between treatments for given time steps, 191

and within treatments at each time step against the initial count (time step 0). 192

193

3. Results and discussion 194

This study evaluated the effect of two acidic and two alkaline electrolyzed waters on bacterial 195

viability in selective and non-selective media. Weak ACEW (10 mg/mL chlorine), strong ACEW 196

(20 mg/mL chlorine), weak ALEW (ORP: -715 mV) and strong ALEW (ORP: -840 mV) were 197

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evaluated (Table 1). Results show that SACEW and WACEW can completely inactivate L. 198

monocytogenes, C. jejuni, A. hydrophila, and V. parahaemolyticus at concentrations of 107 199

CFU/mL or higher within 2 min in both selective and non-selective media (Table 2-5). However, 200

within 2 min, the population of E. coli O104:H4 was decreased only 3.90 and 3.77 log in 201

selective and non-selective media, respectively, with complete inactivation after 4 min only in 202

SACEW. However, the population of E. coli O104:H4 was significantly lower after 4 min 203

compared to 2 min in WACEW (P < 0.05). Results showed that E. coli O104:H4 was more 204

resistant to WACEW, and within 4 min, 5.65 and 6.88 log reductions were observed for non-205

selective and selective media, respectively. E.coli O104:H4 was undetectable after 6 min of 206

exposure to WACEW (Table 6). 207

These results differ from those of Kim et al. (2000), who tested EW with 10 mg/L chlorine and 208

the ORP of 1123 mV against L. monocytogenes and E. coli O157:H7 in 0.1% peptone water. 209

They found that L. monocytogenes was more resistant to ACEW due to differences in the cell 210

wall structure of Gram-negative and Gram-positive bacteria. Venkitanarayanan et al. (1999) 211

reported complete inactivation of E. coli O157:H7 and L. monocytogenes (cell concentrations) 212

after 10 min using 80 mg/L chlorine ACEW. Park, Hung, and Chung (2004) reported that E.coli 213

O157:H7 was more sensitive to ACEW compared to L. monocytogenes. Guentzel, Lam, Callan, 214

Emmons and Dunham (2008) also reported inactivation of E. coli O157:H7 and L. 215

monocytogenes after 10 min for acidic electrolyzed water with 20 mg/L chlorine. They found 216

that L. monocytogenes was more resistant than E. coli O157:H7. Most E. coli O157:H7 strains 217

are very sensitive to chlorine, with a reduction of more than 7 log10 CFU/mL at levels as low as 218

0.25 mg/L of free chlorine (Zhao, Doyle, Zhao, Blake, & Wu, 2001). 219

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In response to SACEW, 3.9 and 5.10 log reductions were observed in non-selective and 220

selective media in E. coli O104:H4 within 2 min. Complete inactivation was observed within 4 221

min treatments using SACEW. Little is known about the emerging food-borne pathogen E. coli 222

O104:H4 (Grad et al., 2013), especially how to control it. Therefore, these results are significant 223

for disinfection of water, produce and food surfaces. Previous treatments for good manufacturing 224

and hygiene practices, as well as those set forth by HACCP (Hazard Analysis Critical Control 225

Point) programs, are insufficient for inactivating this harmful pathogen. 226

A. hydrophila was completely inactivated by applying either SACEW or WACEW. 227

Uyttendaele, Neyts, Vanderswalmen, Notebaert, and Debevere (2004) reported that no 228

significant differences in survival of A. hydrophila and A. caviae between chlorinated (0.1-0.5 229

mg/L free chlorine) and chlorine-free water after 120 min. Other researchers have reported that 230

use of chlorine alone is ineffective to eliminate Aeromonas spp. from contaminated water 231

supplies. Chamorey, Forel, and Drancourt (1999) suggest a minimum concentration of chlorine 232

of 0.95 mg/L to reduce A. hydrophila. Brandi, Sisti, Giardini, Schiavano, and Albano (1999) 233

found that Aeromonas spp. can survive in chlorinated water containing 0.18 mg/L. Sisti, Albano 234

and Brandi (1998) reported that tap water with 0.2–0.25 mg/L chlorine cannot reduce Aeromonas 235

spp. The susceptibility of A. hydrophila strains to chlorine depends on the origin of the strains. 236

Those isolated from chlorinated water having greater resistance to chlorination than strains 237

isolated from untreated waters (Massa, Armuzzi, Tosques, Canganella, & Trovatelli, 1999). 238

SACEW and WACEW can completely inactivate V. parahaemolyticus (7 log10 CFU/mL) 239

within 2 min (Table 2) at lower concentrations than reported by others if the treatment time is 240

extended. Chiu, Duan, Liu, and Su (2006) applied EW with a high ACC (40 mg/L) for sanitizing 241

different contact surfaces inoculated by V. parahaemolyticus. They found that EW could 242

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inactivate V. parahaemolyticus within 1 min on plastic cutting board surfaces. According to 243

Quan et al. (2010), EW has never been applied against V. parahaemolyticus in pure culture. 244

Quan et al. (2010) used weak acidic electrolyzed water (ACC: 35 mg/L, pH: 5.9, ORP: 798 mV) 245

against V. parahaemolyticus. They reported that V. parahaemolyticus suspension could be 246

completely inactivated in 0.5 min. 247

For C. jejuni, complete inactivation (8 log10 CFU/mL) occurred within 2 min using SACEW and 248

WACEW with no recoverable injured cells. Park, Hung, and Brackett (2002) applied EW (25 249

and 50 mg/L ACC) and chlorinated water (25 and 50 mg/L ACC) to a C. jejuni suspension (in 250

0.1 M of phosphate buffer saline solution). They reported complete inactivation of C. jejuni (7.5 251

log10 CFU/mL) after 0.2 min using EW (50 mg/L ACC) and chlorinated water (50 mg/L ACC). 252

In this work, we applied strong alkaline (SALEW) and weak alkaline (WALEW) 253

electrolyzed waters to inactivate bacteria in cell suspensions. V. parahaemolyticus SALEW and 254

WALEW can both significantly decrease bacteria populations by 2.10 and 1.80 log10 within 2 255

min (P < 0.05). Our results show that no significant differences were observed for both 256

treatments between 2 min and 4 min (P > 0.05). However, the lowest bacteria population was 257

observed within 6 min for both treatments (P < 0.05) (Table 2). For L. monocytogenes (Table 3) 258

and A. hydrophila (Table 4) SALEW and WALEW could decrease the bacteria population 259

significantly within 2 min (P < 0.05). However, we found no significant differences among 260

treatment times (P > 0.05). 261

Our results also show that C. jejuni (Table 5) and E. coli O104:H4 (Table 6) populations 262

significantly decreased by applying the ALEW (P < 0.05) by up to 3 and 1.5 log10 CFU/mL, 263

respectively. Alkaline electrolyzed water is a by-product of acidic electrolyzed water production. 264

It is produced in a cathode compartment, and has not yet been fully characterized. Only a few 265

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studies have investigated the antimicrobial properties of alkaline electrolyzed water (Ayebah et 266

al., 2005; Koseki et al., 2004; Park et al., 2008; Rahman et al., 2011; Xie et al., 2012). 267

Park, Hung, Lin, and Brackett (2005) reported that 1 min treatment with ALEW water 268

followed by a 1 min treatment with ACEW water containing 41 mg/L of chlorine reduced 269

Salmonella and Listeria in inoculated egg shells by amounts similar to a 1 min treatment with 270

chlorinated water containing 200 mg/L of chlorine. Koseki et al. (2004) found that washing 271

lettuce in ALEW for 1 min and then treating it with ACEW for another minute significantly 272

reduced aerobic bacteria, molds, and yeasts. Ayebah et al. (2005) reported that ALEW alone 273

could not reduce the L. monocytogenes biofilm on stainless steel. However, they found that the 274

most effective treatment for decreasing the L. monocytogenes population is ACEW, or ALEW 275

followed by ACEW. In addition, applying ALEW to sanitize carrots significantly reduced 276

bacteria, mold and yeast within 1 min (Rahman et al., 2011). However, Park et al. (2008) 277

reported that ALEW alone cannot decrease the L. monocytogenes, E. coli O157:H7 and 278

Salmonella Typhimurium in lettuce and spinach after a 5 min treatment. Similar results were 279

found by Fabrizio and Cutter (2003). 280

ALEW alone is not sufficient to decrease bacterial loads (Ayebah et al., 2005; Park et al., 281

2008; Rahman et al., 2011). ALEW has been effectively used in combination with either mild 282

heat treatments or in conjunction with ACEW. However, we found that applying ALEW in cell 283

suspensions produced a 1 to 3 log reduction, depending on bacterial strain and treatment time 284

which might be because of using ALEW for cell suspension in media free condition and the ratio 285

of cell suspension and ALEW. 286

Although the use of ACEW has been studied as an antimicrobial agent in food industry, 287

the use of ALEW water has received little attention. The effectiveness of ACEW water may be 288

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due to the ORP, rather than pH or ACC (Kim et al., 2000). ACEW with high ORP (>1100 mV) 289

has been found to sequester electrons from the cellular membrane, rendering it unstable. This 290

allows the antimicrobial ACC to oxidize and ultimately inactivate the cell (Kim et al., 2000). 291

Other studies show that ACC in ACEW water is the source of its antimicrobial activity (Oomori, 292

Oka, Inuta, & Arata, 2000). Since sodium hydroxide saponifies fat and reacts with proteins, 293

ALEW water may destabilize or dissolve the extracellular polymeric substances surrounding 294

bacterial cells. This facilitates penetration of active components or destroys the cell wall. In 295

addition, when bacteria are subjected to extreme ORP conditions, either with alkaline or acidic 296

electrolyzed water, the cellular membrane is altered (Ayebah et al., 2005; Fabrizio and Cutter, 297

2003). 298

Our results show that ALEW can reduce bacteria by 1 to 3 log10 in a cell suspension. 299

Technically, the optimum ORP condition for growth of aerobic bacteria is +200 to +800 mV. 300

Hence, our results may be due to extremely low ORP in ALEW (-715 and -850 mV) or high 301

concentrations of electrolyzed water applied. We used a ratio of 19 mL electrolyzed water 302

(acidic and alkaline) for the 1 mL cell suspension. We then removed the bacterial growth media 303

completely before conducting the electrolyzed water treatment. However, other studies applied 304

organic matter to contact surfaces, decreasing the efficacy of electrolyzed waters (Quan et al., 305

2010). Quan et al. (2010) compared the efficacy of acidic electrolyzed water on V. 306

parahaemolyticus in cell suspensions and cell cultures with different ratios of ACEW treatment 307

solution, including a 1:19, 1:49, and 1:99 cell suspension or cell culture to ACEW (v:v). They 308

found that electrolyzed water lost effectiveness with higher concentrations of organic material, 309

requiring higher quantities of ACEW for bacterial inactivation. They also reported that the 310

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concentration ratio of bacteria and ACEW is important when the ACEW is applied for cell 311

cultures. This confirms that organic matter can decrease the efficacy of ACEW. 312

ALEW has been used by many researchers in Japan for food processing to modify 313

texture. For example, ALEW is used in the production of bread with a softer texture, higher 314

quality aged rice, and modified tofu and noodles (Hara, Matsuda, & Arai, 2003a; Hara, 315

Watanuki, & Arai, 2003b; Onishi, Hara, & Arai, 1999). However, due to its mild bactericidal 316

activity, applications of ER in the food industry can reduce the need to add preservative 317

chemicals, reduce heating temperature or times for heat-sensitive foods, such as tofu or seafood 318

products. 319

320

4. Conclusions 321

This study found that strong and weak acidic electrolyzed waters (SACEW, WACEW) can 322

completely inactivate the populations of V. parahaemolyticus, L. monocytogenes, A. hydrophila, 323

and C. jejuni within 2 min at 20oC (107 to 109 CFU/mL) in a 0.85% salt suspension. E. coli 324

O104:H4 can be completely inactivated in 4 min (SACEW) or 6 min (WACEW). This study also 325

found a 1 to 3 log reduction in strong and weak alkaline electrolyzed water (SALEW, WALEW) 326

treatments. 327

328

Acknowledgments 329

This study was funded by USDA-NIFA 2011-68003-20096, the Agricultural Research Center at 330

Washington State University and the University of Jordan. 331

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Table Captions 485

Table 1 Properties (ORP, pH, Free Chlorine) of ACEW and ALEW Generated Using Different 486

NaCl Concentrations. 487

Table 2 Mean viable counts of Vibrio parahaemolyticus (log10 CFU/mL) treated with 488

electrolyzed water. 489

Table 3 Mean viable counts of Listeria monocytogenes (log10 CFU/mL) treated with electrolyzed 490

water. 491

Table 4 Mean viable counts of Aeromonas hydrophila (log10 CFU/mL) treated with electrolyzed 492

water. 493

Table 5 Mean viable counts of Campylobacter jejuni (log10 CFU/mL) treated with electrolyzed 494

water. 495

Table 6 Mean viable counts of Escherichia coli O104:H4 (log10 CFU/mL) treated with 496

electrolyzed water. 497

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Table 1 Properties (ORP, pH, Free Chlorine) of ACEW and ALEW Generated Using Different 507

NaCl Concentrations.1,2 508

NaCl concentration

Acidic Alkaline

Free chlorine (mg/L)

ORP (mV)

pH Free Chlorine (mg/L)

ORP (mV)

pH

5000 mg/L 250±3 1180±0 2.3±0.3 6±1 -890±10 11.8±0.2

1000 mg/L 60±2 1180±0 2.3±0.3 ND -885±5 11.7±0.1

200 mg/L3 20±1 1150±5 3.1±0.4 ND -840±6 11.1±0.1

40 mg/L4 10±1 950±5 3.55±0.4 ND -715±5 10.47±0.2

1Measurements were conducted at 23oC. 509

2Values are the means of two replicates measurement (Mean±SD). 510

3Strongly acidic electrolyzed water (SACEW), and strongly alkaline electrolyzed water 511

(SALEW). 512

4Weakly acidic electrolyzed water (WACEW), and weakly alkaline electrolyzed water 513

(WALEW). 514

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526

Table 2 Mean viable counts of Vibrio parahaemolyticus (log10 CFU/mL) treated with 527

electrolyzed water.1,2,3,4 528

Treatment

Exposure time (min)

2 4 6

Nonselective media

Selective media

Nonselective media

Selective media

Nonselective media

Selective media

SACEW ND b ND ND b ND ND b ND

WACEW ND b ND ND b ND ND b ND

SALEW 4.97±0.15 c (2.10)

3.93±0.04 (3.13)

4.83±0.15 c (2.24)

3.73±0.05 (3.32)

3.85±0.08 d (3.22)

3.39±0.09 (3.67)

WALEW 5.28±0.04 e (1.80)

4.43±0.14 (2.66)

5.02±0.12 e (2.06)

4.02±0.05 (3.07)

4.01±0.06 d (3.10)

3.74±0.06 (3.35)

1Values are the mean of two independent replicate experiments ± standard deviation, with log10 529

reductions (expressed in CFU/mL) presented in parentheses. 530 2ND, not detected due to lethal injury. 531 3Average initial count (control) = 7.08 log10 CFU/mL which selected as “a” in statistical analysis. 532 4Pairwise comparison of treatment means were done for only those with nonselective media. 533

Treatment means without shared superscripts are different from each other (Tukey HSD, P < 534

0.05). 535

536

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Table 3 Mean viable counts of Listeria monocytogenes (log10 CFU/mL) treated with electrolyzed 546

water.1,2,3,4 547

Treatment

Exposure time (min)

2 4 6

Nonselective media

Selective media

Nonselective media

Selective media

Nonselective media

Selective media



SALEW 6.28±0.05 c (1.84)

6.08±0.04 (1.97)

6.25±0.06 c (1.87)

6.03±0.04 (2.02)

6.24±0.04 c (1.88)

6.03±0.04 (2.02)

WALEW 6.31±0.03 c (1.77)

6.16±0.04 (1.94)

6.27±0.03 c (1.80)

6.13±0.03 (1.97)

6.24±0.02 c (1.83)

6.11±0.03 (2.00)




0.05). 554

555

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Table 4 Mean viable counts of Aeromonas hydrophila (log10 CFU/mL) treated with electrolyzed 567

water.1,2,3,4 568

Treatment

Exposure time (min)

2 4 6

Nonselective media

Selective media

Nonselective media

Selective media

Nonselective media

Selective media


WACEW ND c ND ND c ND ND c ND

SALEW 6.06±0.05 d (1.51)

4.83±0.14 (2.81)

5.93±0.11 d (1.65)

4.42±0.2 (3.22)

5.95±0.13 d (1.62)

4.41±0.26 (3.23)

WALEW 6.15±0.08 d (1.38)

4.99±0.2 (2.48)

6.00±0.1 d (1.53)

4.91±0.17 (2.56)

6.02±0.16 d (1.50)

4.77±0.14 (2.71)




0.05). 575

576

577

578

579

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Table 5 Mean viable counts of Campylobacter jejuni (log10 CFU/mL) treated with electrolyzed 585

water.1,2,3,4 586

Treatment

Exposure time (min)

2 4 6

Nonselective media

Selective media

Nonselective media

Selective media

Nonselective media

Selective media



SALEW 5.81±0.11 c (2.59)

5.77±0.07 (2.6)

5.45±0.05 d (2.95)

5.25±0.06 (3.11)

5.37±0.07 d (3.02)

5.14±0.04 (3.22)

WALEW 5.84±0.07 c (2.6)

5.80±0.03 (2.52)

5.58±0.04 e (2.85)

5.30±0.04 (3.02)

5.51±0.11 d,

e (2.92) 5.19±0.06

(3.14)




0.05). 593

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Table 6 Mean viable counts of Escherichia coli O104:H4 (log10 CFU/mL) treated with 605

electrolyzed water.1,2,3,4 606

Treatment

Exposure time (min)

2 4 6

Nonselective media

Selective media Nonselective media

Selective media

Nonselective media

Selective media

SACEW 5.18±0.06 b (3.90)

4.00±0.2 (5.10) ND c ND ND c ND

WACEW 5.28±0.02 b (3.77)

4.86±0.03 (4.21)

3.40±0.06 d (5.65)

2.18±0.05 (6.88)

ND c ND

SALEW 7.71±0.07 e, f (1.42)

7.51±0.03 (1.53)

7.65±0.05 e, g (1.48)

7.48±0.02 (1.56)

7.63±0.03 e, h (1.49)

7.47±0.06 (1.57)

WALEW 7.81±0.04 f (1.22)

7.57±0.03(1.50) 7.74±0.05 f, g (1.30)

7.53±0.02 (1.54)

7.72±0.07 f, h (1.32)

7.48±0.03 (1.58)




0.05) 613

614

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Highlights

• Strong and weak acidic electrolyzed waters completely inactivated tested pathogenic bacteria cell suspensions within 2 min.

• E. coli O104:H4 was the most resistant among tested bacteria • Strong and weak acidic electrolyzed waters inactivated E. coli O104:H4 in 4 and 6

min, respectively. • Alkaline electrolyzed water treatments showed 1 to 3 log reduction in tested bacteria

Efficacy of Acidic and Alkaline Electrolyzed Water for Inactivating Escherichia coli O104:H4,...

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