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Stability of low concentration electrolyzed water and its sanitization potential against foodborne pathogens
Journal of Food Engineering 113 (2012) 548–553
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Stability of low concentration electrolyzed water and its sanitization potential against foodborne pathogens S.M.E. Rahman a,b, Joong Hyun Park a, Jun Wang a, Deog-Hwan Oh a,⇑ a b
Department of Food Science and Biotechnology, Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea Department of Animal Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
a r t i c l e
i n f o
Article history: Received 9 January 2012 Received in revised form 30 June 2012 Accepted 5 July 2012 Available online 16 July 2012 Keywords: Current Electrolysis time Salt concentration Stability of LcEW Sanitizing effect
a b s t r a c t Low concentration electrolyzed water (LcEW) has been proved to be an effective sanitizer against pathogens in cell suspensions as well as pathogens and spoilage organisms attached to vegetables, poultry and meat. In this study, effect of current, electrolysis time and salt concentration on physical properties (pH, ORP and ACC) and inactivation efficacy of LcEW was monitored. Pure cultures of Escherichia coli O157:H7 and Listeria monocytogenes were prepared and exposure treatment was performed for bacteria inactivation study in cell suspensions at room temperature (23 ± 2 °C). Our results showed increased reduction of both pathogens with the increase in current. Changes of current also affected the ACC, pH and ORP values of the tested solution. Values of ACC, pH and ORP were increased with the increase in current. Log reduction of 4.9–5.6 log CFU/mL for both pathogens was achieved when the current was increased from 1.15 to 1.45 A. Electrolysis time and percent of salt concentration also influenced the physical properties of LcEW. Stability of LcEW was also investigated under different conditions and it was observed that LcEW produced with increased electrical current was more stable during storage. Therefore, current might influence the properties and sanitizing effect of LcEW. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In the recent years, people in Korea as well as other nations are concerned about the safety of foods that are consumed fresh or undercooked. Thus, food industries are thriving their effort for the safety of food products (Issa-Zacharia et al., 2010). Escherichia coli, Staphylococcus aureus, Campylobacter jejuni, Salmonella spp. and Listeria monocytogens are considered as the important pathogens of major public health concern and were reported to be the common foodborne pathogens causing illness and death (Issa-Zacharia et al., 2010; Mead et al., 1999) with outbreaks reported in Canada, USA, UK, Japan and some other parts of the world. Therefore, developing effective methods for reducing or eliminating pathogens in food and agricultural products is an important step for the hazard analysis and critical control points (HACCP) of the food industry (Issa-Zacharia et al., 2010). So far, several chemical solutions such as sodium hypochlorite, acidified sodium chlorite, chlorine dioxide, hydrogen peroxide, organic acids and ozone have been used as sanitizers in the food industry (Liao, 2009; Zhang and Farber, 1996; Kim et al., 2009; Nagashima and Kamoi, 1997). Chlorine and chlorine-containing compounds have been the most commonly used sanitizers for a long time as antimicrobial agents in food ⇑ Corresponding author. Tel./fax: +82 33 250 6457. E-mail address: [email protected] (D.-H. Oh). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.07.011
processing (Beuchat et al., 1998) due to their availability, relative low cost and efficacy. The strength of sanitizing should be the range of 50–200 mg/L of available chlorine or its equivalent (USDA, 2001). High levels of chlorine can be detrimental to the quality of food (Bialka et al., 2004) and have not been completely acceptable because of the chemical residues, limited effectiveness and adverse environmental impacts (Cao et al., 2009). Therefore, developing an effective method to reduce or eliminate foodborne pathogens on food is crucial to the food safety and human health. Thus, the use of acidic electrolyzed water (AEW) with low chlorine concentration has been introduced as an alternative and a novel sanitizer in agriculture and food industry (Huang et al., 2008). AEW has shown good efficacy against cell suspensions of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes (Venkitanarayanan et al., 1999), spoilage organisms associated with vegetables (Izumi, 1999) and pathogens in solution (Fabrizio and Cutter, 2003). However, the potential application of AEW is limited because of its low pH values (62.7). At this low pH, dissolved Cl2 gas can be rapidly lost due to volatilization, resulting in decreased bactericidal activity of the solution with time (Len et al., 2002). Also its negative effect on human health and environment is umbrage. Moreover, high acidity of AEW may cause the corrosion of equipments and consequently limit its practical application (Cao et al., 2009). In contrast, a solution at slightly acidic or neutral pH generated by electrolysis was reported as an effective antimicrobial agent with low
S.M.E. Rahman et al. / Journal of Food Engineering 113 (2012) 548–553
available chlorine concentration (Koide et al., 2009; Cao et al., 2009; Gómez-López et al., 2007), and recently attention is being paid to LcEW for disinfection (Rahman et al., 2010a,b). So far, limited studies have been reported about freshly produced electrolyzed water at slightly acidic or neutral pH with low ACC (510 mg/L) (Rahman et al., 2010a,b). The present study was designed to determine the stability of LcEW over time and to investigate the effect of current, electrolysis time and salt concentration (%) on physical properties and bactericidal effectiveness of LcEW. 2. Materials and methods
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amount of salt solution (0.1%) and tap water through an electrolytic cell, within which the anode and cathode were separated by a membrane at a setting of 12 A to give a ACC of about 50 mg/L. 2.3. Determination of pH, ORP and ACC The pH, ORP and ACC of the treatment solutions (LcEW and SAEW) were measured immediately before treatment with a dual-scale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ) bearing pH and ORP electrodes. The ACC was determined by a colorimetric method using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan).
2.1. Bacteria and culture 2.4. Stability of LcEW E. coli O157:H7 (ATCC 43894) and L. monocytogenes (ATCC 19115) stock cultures were transferred into tryptic soy broth (TSB, Sparks, MD, USA) and incubated for 24 h at 35 °C. Following incubation, 10 mL of each culture was sedimented by centrifugation (3000g for 10 min), washed and resuspended in 10 mL of 0.85% sodium chloride solution to obtain a final cell concentration of 109 CFU/mL. The bacterial population in each culture was confirmed by plating 0.1 mL portions of appropriately diluted cultures on tryptic soy agar (TSA) (Difco Laboratories) plates and incubation at 35 °C for 24 h.
Changes in properties and bactericidal activity of LcEW stored in open or closed glass bottles at room temperature for 4 weeks in a dark room were evaluated. ORP, pH and ACC were measured on days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28. Bactericidal activities of LcEW against E. coli O157:H7 and L. monocytogenes were also measured until storage period of day 7. Electrolysis time and percent of salt concentration were also studied to investigate the changes of physical properties in produced LcEW. 2.5. Inactivation test
2.2. LcEW preparation Low concentration electrolyzed water (LcEW) was generated (Fig. 1) by passing a diluted salt (NaCl) solution (0.9%) through an electrolytic cell equipped with a platinum electrode, within which no separating membrane between anode and cathode using an electrolysis device (model OH-3000) at a setting of 3 V and 1.45 A. The LcEW used in this study had a pH of 6.8–7.4, an oxidation reduction potential (ORP) of 660–700 mV and an available chlorine concentration (ACC) of 510 mg/L. For comparison with LcEW, strong acid electrolyzed water (SAEW) with a pH of 2.54 and an ORP of 1100–1120 mV was generated using an EO generator (A2-1000, Korean E&S Fist Inc., Seoul, Korea) with a small
An in vitro inactivation of target pathogens in suspension was designed as illustrated in Fig. 2. For each replicate, a 1 mL aliquot of prepared inoculum (approximately 109 CFU/mL) was added to a sterile test tube. Once the test material (LcEW/SAEW) was generated, 9 mL of the prepared test agent was added to the tube containing 1 mL of the prepared inoculums, a timer was started and the tube was mixed immediately at room temperature. After each contact time (30, 60, and 90 s), a 1 mL sample was transferred to a tube containing 9 mL of neutralizer (0.85% NaCl + 0.5% sodium thiosulphate) followed by Serial 10-fold dilution in 0.85% saline solution dilution blanks. Distilled water (DW) washing was used as control. All tests were carried out in triplicate.
Fig. 1. A low concentration electrolyzed water (LcEW) generating system. An elaboration of a systematic mixing up of tap water and diluted NaCl during its production.
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Fig. 2. Flow diagram showing in vitro inactivation experiment. Sodium thiosulphate solution (0.5%) was used to stop the inactivation reaction after EW treatment.
2.6. Incubation and enumeration Upon completion of the test, the surviving population of bacteria was determined by plating 0.1 mL of each dilution in duplicate on TSA plates. Colonies of the pathogen were enumerated on TSA plates after incubation at 35 °C for 18–24 h. The colonies were counted and the CFU/mL at each contact time was determined. All tests were carried out in triplicate. 2.7. Statistical analysis For each occasion, the data from the independent replicate trials were pooled and the mean value and standard deviation were determined. Data were subjected to one way analysis of variance (ANOVA) and Tukey HSD multiple range test was used to determine the differences at p 6 0.05 using SPSS 13.0 (SPSS software for Windows, release 13.0, SPSS, Inc., USA). 3. Results and discussion 3.1. Properties of LcEW under different conditions The ACC, pH, and ORP of LcEW used in this study were 5–10 mg/L, 6.8–7.4, and 660–700 mV, respectively (Table 1). From our previous LcEW producing system (1.15–1.17 A) it is revealed that ACC reduces with an increase in exposure time (Rahman et al., 2010a) dur-
ing inactivation test which could have resulted in lower reductions at increased exposure times. In order to make the system more stable with enhanced antimicrobial activity the effect of current was examined. After increasing the current up to 1.45–1.47 A, we observed that the produced LcEW with increased current was more stable during storage and changes of ACC with an increase in exposure time were minimized (data not shown) compared to the previous system. Table 1 shows us how the current affects properties and inactivation efficacy of LcEW against E. coli O157:H7 and L. monocytogenes. From the results of current study it was found that increased reduction (4.9–5.6 log CFU/mL) for both pathogens was gained with the increase in current. Changes of current also affected the pH and ORP values of the tested solution. The values of pH and ORP increased with the increase in the current. Log reduction of 4.9 and 5.2 log CFU/mL for E. coli O157:H7 and L. monocytogenes, respectively was found when the current of 1.15 A was used. On the other hand, the new system with 1.45 A reduced E. coli O157:H7 and L. monocytogenes by 5.30 and 5.58 log CFU/mL, respectively. Compared to the previous system, new system showed enhanced bactericidal effect. ACC increased with the increase in the current. Park et al. (2001) suggested that the concentration of chlorine reactants in AEW is influenced by the amperage of the water generator, but other reports contend that the amount of HOCl produced during electrolysis is positively correlated with the amount of NaCl added (Anonymous, 1997; Al-Haq et al., 2002). Few reports are available on the effects of chlorine, pH, and ORP values of EO water on the
Table 1 Effect of current on physical properties and inactivation efficacy of low concentration electrolyzed water (LcEW) against E. coli O157:H7 and L. monocytogenes in 1 min dipping. Voltage (V)
Current (A)
ACC (mg/L)
pH
ORP (mV)
3
1.15–1.17 1.45–1.47
5 ± 0.2 10 ± 0.5
6.2 ± 0.06 6.8 ± 0.04
520 ± 10 700 ± 20
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Initial count for Listeria monocytogenes: 9.18 log CFU/mL. (1) Log reductions (log CFU/mL) reported as means of triplicate determinations ± standard deviation.
Log reduction (CFU/mL)(1) E. coli O157:H7
L. monocytogenes
4.90 ± 0.06 5.30 ± 0.12
5.20 ± 0.04 5.58 ± 0.10
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S.M.E. Rahman et al. / Journal of Food Engineering 113 (2012) 548–553 Table 2 Effect of electrolysis time on physical properties and inactivation efficacy of LcEW against E. coli O157:H7. Current (A)
Electrolysis time (s)
ACC (mg/L)
pH
ORP (mV)
Reduction of E. coli (log CFU/mL)
1.45–1.47
27 41 50
5 ± 0.5 7 ± 0.5 10 ± 0.5
7.34 ± 0.02 7.12 ± 0.04 6.80 ± 0.05
660 ± 5 680 ± 8 700 ± 10
5.3 ± 0.21 5.5 ± 0.18 5.9 ± 0.08
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Values are means of triplicate determinations ± standard deviation.
inactivation of pathogens. Kim et al. (2000) suggested that the ORP of EO water might be the primary factor responsible for its bactericidal effect. However, Koseki et al. (2004) noted that ORP could not be the main factor in antimicrobial activity, because the higher ORP of ozonated water did not show a higher disinfectant effect than that of EO water with lower ORP. They further explained that the free chlorine in EO water (mainly HOCl) produces hydroxyl radicals ( OH), which then act on microorganisms. Len et al. (2000) reported that the relative concentrations of aqueous molecular chlorine, HOCl, hypochlorite ions (OCl ), and chlorine gas (Cl2) were also factors that accounted for the bactericidal potency of EO water. Theoretically, at pH values of 6.0 and 9.0, the predominant chlorine species in a solution is not dissolved Cl2 gas but HOCl and OCl (White, 1999). The pH value of AEW also plays a role in restricting microbial growth. Iwasawa et al. (2002) discussed the effect of pH on the bactericidal properties of AEW; Len et al. (2000) discussed the influence of amperage and pH on these properties. Electrolysis times also affect the physical properties and influence the inactivation efficacy of LcEW (Table 2). Total residual chlorine increased with increases of salt concentration (Table 3) whereas, pH decreased and ORP increased. Similar result has been reported by Hsu (2005). Kiura et al. (2002) also found that the concentrations of free chlorine increased with the time of electrolysis and concentration of NaCl.
Table 4 Sanitization potency of LcEW against pure cultures of E. coli O157:H7 at different ACC and exposure times. Treatment solution
DW (control) LcEW – 5 mg/L LcEW – 7 mg/L LcEW – 10 mg/L SAEW – 50 mg/L
Table 3 Effect of salt concentration on physical properties of LcEW. Current (A)
Salt concentration (%)
ACC (mg/L)
pH
ORP
1.45–1.47
0.1 0.3 0.5 0.7 0.9
2 ± 0.5 3 ± 0.5 5 ± 1.0 7 ± 0.5 10 ± 0.5
7.50 ± 0.03 7.40 ± 0.05 7.30 ± 0.01 7.12 ± 0.04 6.84 ± 0.02
620 ± 15 640 ± 12 650 ± 10 670 ± 10 700 ± 10
Values are means of triplicate determinations ± standard deviation.
30 s
60 s
90 s
0.70 ± 0.02aA 5.02 ± 0.08aB 5.30 ± 0.04aBC 5.54 ± 0.12aC 5.18 ± 0.05aB
0.82 ± 0.04aA 5.32 ± 0.05abB 5.58 ± 0.10abBC 5.94 ± 0.07bC 5.44 ± 0.08abB
0.78 ± 0.05aA 5.88 ± 0.10bB 6.14 ± 0.12bBC 6.49 ± 0.06cC 6.02 ± 0.04bB
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Log reductions (log CFU/mL) reported as means of triplicate determinations ± standard deviation. Different lowercase letters within same row differed significantly (p < 0.05) and different uppercase letters within same column differed significantly (p < 0.05). (1)
Table 5 Sanitization potency of LcEW against pure cultures of L. monocytogenes at different ACC and exposure times. Treatment solution
Bacterial log reduction at different exposure time (log CFU/mL)(1) 30 s
DW (control) LcEW – 5 mg/L LcEW – 7 mg/L LcEW – 10 mg/L SAEW – 50 mg/L
3.2. Bactericidal activity of LcEW at different ACC and exposure times Based on the results of current study, 1.45 A was chosen for subsequent experiments to find out the effect of available chlorine concentration and exposure time on inactivation of E. coli O157:H7 and L. monocytogenes in cell suspensions (Tables 4 and 5). Table 4 indicates that both exposure time and ACC have significant effect (p < 0.05) on the reduction of E. coli O157:H7. Ten parts per million showed the highest reduction of 6.49 log CFU/mL at 90 s exposure, whereas, distilled water (DW) showed the lowest reduction of E. coli by 0.70 log CFU/mL. Regardless of exposure time, treatment solutions (5/7/10/50 ppm) showed significant reduction (p < 0.05) compared to distilled water control. Inactivation effect of strong acidic electrolyzed water containing 50 ppm ACC did not show significant difference (p < 0.05) with 5 and 7 ppm LcEW. But 50 ppm SAEW was significantly different with 10 ppm LcEW in reduction of E. coli cell. Similar trend was observed in case of L. monocytogenes inactivation (Table 5) and resulted in reduction of 6.79 log CFU/ mL at 90 s dipping with 10 ppm LcEW. Kim et al. (2000) reported that a 60-s treatment with AEW containing 10 mg/L of residual
Bacterial log reduction at different exposure time (log CFU/mL)(1)
60 s a
0.84 ± 0.05 A 5.32 ± 0.18aB 5.61 ± 0.07aBC 5.94 ± 0.11aC 5.41 ± 0.08aB
90 s a
1.02 ± 0.06 A 5.60 ± 0.08abB 5.90 ± 0.12abBC 6.28 ± 0.05abC 5.72 ± 0.09abB
1.08 ± 0.10aA 6.14 ± 0.08bB 6.44 ± 0.11bBC 6.79 ± 0.09bC 6.25 ± 0.07bB
Initial count for L. monocytogenes: 9.18 log CFU/mL. Log reductions (log CFU/mL) reported as means of triplicate determinations ± standard deviation. Different lowercase letters within same row differed significantly (p < 0.05) and different uppercase letters within same column differed significantly (p < 0.05). (1)
chlorine was very effective in reducing the populations of E. coli O157:H7, L. monocytogenes, and B. cereus vegetative cells to undetectable levels. Park et al. (2004) investigated the effects of chlorine and pH on the efficacy of EO water to inactivate E. coli O157:H7 and L. monocytogenes. They demonstrated that EO water effectively inactivated E. coli O157:H7 and L. monocytogenes in a wide pH range (between 2.6 and 7.0) if sufficient free chlorine (greater than 2 mg/L) was present. For a given chlorine content, bactericidal activity and ORP increased with decrease in pH. On the other hand, the 60 s treatment of E. coli pure culture using slightly acidic electrolyzed water (pH 5.6, 23 mg/L ACC and 940 mV ORP), strong acidic electrolyzed water (50 mg/L ACC) and sodium hypochlorite solution (NaOCl, 120 mg/L ACC) achieved a log reduction of 5.07, 6.02 and 5.13 log CFU/mL of its population (Issa-Zacharia et al., 2010). 3.3. Change of properties and sanitization potency of LcEW under storage conditions Changes of ACC, pH, and ORP of LcEW under open and closed storage conditions for 28 days at room temperature in the dark
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S.M.E. Rahman et al. / Journal of Food Engineering 113 (2012) 548–553 Table 6 Inactivation efficacy of LcEW against E. coli O157:H7 and L. monocytogenes reduction (log CFU/mL) during storage under open and closed condition. Storage period (d)
0 1 2 3 4 5 6 7 14 21 28
Log reductions (CFU/mL)(1) Open condition
Close condition
E. coli O157:H7
L. monocytogenes
E. coli O157:H7
L. monocytogenes
5.98 ± 0.07a 5.69 ± 0.14b 5.44 ± 0.21bc 5.20 ± 0.04c 4.95 ± 0.11cd 4.41 ± 0.07d 4.20 ± 0.18de – – – –
6.77 ± 0.12a 6.52 ± 0.19ab 6.18 ± 0.06b 5.78 ± 0.13c 5.32 ± 0.21d 4.89 ± 0.25e 4.54 ± 0.14f – – – –
5.98 ± 0.05a 5.95 ± 0.06a 5.84 ± 0.11ab 5.82 ± 0.15ab 5.68 ± 0.22b 5.68 ± 0.24b 5.52 ± 0.29bc 5.24 ± 0.14c 3.47 ± 0.06d – –
6.97 ± 0.10a 6.98 ± 0.12a 6.77 ± 0.04ab 6.77 ± 0.07ab 6.50 ± 0.15b 6.49 ± 0.17b 6.32 ± 0.23bc 5.96 ± 0.14c 3.92 ± 0.08d – –
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Initial count for L. monocytogenes: 9.18 log CFU/mL. (1) Log reductions (CFU/mL) reported as means of triplicate determinations ± standard deviation. Different letters within same column differed significantly (p < 0.05). –, not measured.
open, agitated, and diffused light condition, the EO water had the highest chlorine loss rate. With open storage, chlorine loss through evaporation followed first-order kinetics. The rate of chlorine loss was increased by approximately 5-fold with agitation, but it was not significantly affected by diffused light (Len et al., 2002). EO water exposed to the atmosphere showed more reduction in chlorine and oxygen than that kept in closed systems for a longer time (Hsu and Kao, 2004). Fabrizio and Cutter (2003) reported that EO water stored at 4 °C was more stable than that stored at 25 °C. In conclusion, LcEW has the advantage of possessing antimicrobial activity with low available chlorine. Also, this study demonstrated that LcEW has a relatively stable shelf life in closed storage condition and showed strong bactericidal activity against E. coli O157:H7 and L. monocytogenes in cell suspensions with the increase in current. In addition, this study provides the foundation for further development of dynamic LcEW producing system for commercial use in the food industry. Fig. 3. Changes of LcEW properties during storage under open and closed condition.
References are shown in Fig. 3. Under open storage condition, the ACC of LcEW decreased gradually from 10 to 0 mg/L after 7 days. However, ACC was slightly reduced from 10 to 5 mg/L after 7 days under the closed storage condition. The pH of LcEW increased during the 28 days storage period in both storage conditions with the decrease in ORP values. The pH of LcEW increased from 6.8 to 7.7 in open storage and from 6.8 to 7.4 in closed storage after 28 days. In contrast, Quan et al. (2010) reported that the pH of weakly acidic electrolyzed water (pH 5.9; ACC 35 mg/L) decreased in open and closed storage. The ORP of LcEW decreased from 700 to 400 mV in open storage and from 700 to 500 mV in closed storage after 28 days. When LcEW characteristics were monitored over time, it was determined that LcEW stored at closed condition was more stable than that stored at open condition. Bactericidal activities of LcEW against E. coli O157:H7 and L. monocytogenes in open and closed storage conditions are shown in Table 6. LcEW maintained bactericidal activities against cell suspensions of the two strains up to 6 days in open and 14 days in closed storage condition. Storage conditions can affect the chemical and physical properties of EO water. When stored under an
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Stability of low concentration electrolyzed water and its sanitization potential against foodborne pathogens S.M.E. Rahman a,b, Joong Hyun Park a, Jun Wang a, Deog-Hwan Oh a,⇑ a b
Department of Food Science and Biotechnology, Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea Department of Animal Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
a r t i c l e
i n f o
Article history: Received 9 January 2012 Received in revised form 30 June 2012 Accepted 5 July 2012 Available online 16 July 2012 Keywords: Current Electrolysis time Salt concentration Stability of LcEW Sanitizing effect
a b s t r a c t Low concentration electrolyzed water (LcEW) has been proved to be an effective sanitizer against pathogens in cell suspensions as well as pathogens and spoilage organisms attached to vegetables, poultry and meat. In this study, effect of current, electrolysis time and salt concentration on physical properties (pH, ORP and ACC) and inactivation efficacy of LcEW was monitored. Pure cultures of Escherichia coli O157:H7 and Listeria monocytogenes were prepared and exposure treatment was performed for bacteria inactivation study in cell suspensions at room temperature (23 ± 2 °C). Our results showed increased reduction of both pathogens with the increase in current. Changes of current also affected the ACC, pH and ORP values of the tested solution. Values of ACC, pH and ORP were increased with the increase in current. Log reduction of 4.9–5.6 log CFU/mL for both pathogens was achieved when the current was increased from 1.15 to 1.45 A. Electrolysis time and percent of salt concentration also influenced the physical properties of LcEW. Stability of LcEW was also investigated under different conditions and it was observed that LcEW produced with increased electrical current was more stable during storage. Therefore, current might influence the properties and sanitizing effect of LcEW. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In the recent years, people in Korea as well as other nations are concerned about the safety of foods that are consumed fresh or undercooked. Thus, food industries are thriving their effort for the safety of food products (Issa-Zacharia et al., 2010). Escherichia coli, Staphylococcus aureus, Campylobacter jejuni, Salmonella spp. and Listeria monocytogens are considered as the important pathogens of major public health concern and were reported to be the common foodborne pathogens causing illness and death (Issa-Zacharia et al., 2010; Mead et al., 1999) with outbreaks reported in Canada, USA, UK, Japan and some other parts of the world. Therefore, developing effective methods for reducing or eliminating pathogens in food and agricultural products is an important step for the hazard analysis and critical control points (HACCP) of the food industry (Issa-Zacharia et al., 2010). So far, several chemical solutions such as sodium hypochlorite, acidified sodium chlorite, chlorine dioxide, hydrogen peroxide, organic acids and ozone have been used as sanitizers in the food industry (Liao, 2009; Zhang and Farber, 1996; Kim et al., 2009; Nagashima and Kamoi, 1997). Chlorine and chlorine-containing compounds have been the most commonly used sanitizers for a long time as antimicrobial agents in food ⇑ Corresponding author. Tel./fax: +82 33 250 6457. E-mail address: [email protected] (D.-H. Oh). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.07.011
processing (Beuchat et al., 1998) due to their availability, relative low cost and efficacy. The strength of sanitizing should be the range of 50–200 mg/L of available chlorine or its equivalent (USDA, 2001). High levels of chlorine can be detrimental to the quality of food (Bialka et al., 2004) and have not been completely acceptable because of the chemical residues, limited effectiveness and adverse environmental impacts (Cao et al., 2009). Therefore, developing an effective method to reduce or eliminate foodborne pathogens on food is crucial to the food safety and human health. Thus, the use of acidic electrolyzed water (AEW) with low chlorine concentration has been introduced as an alternative and a novel sanitizer in agriculture and food industry (Huang et al., 2008). AEW has shown good efficacy against cell suspensions of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes (Venkitanarayanan et al., 1999), spoilage organisms associated with vegetables (Izumi, 1999) and pathogens in solution (Fabrizio and Cutter, 2003). However, the potential application of AEW is limited because of its low pH values (62.7). At this low pH, dissolved Cl2 gas can be rapidly lost due to volatilization, resulting in decreased bactericidal activity of the solution with time (Len et al., 2002). Also its negative effect on human health and environment is umbrage. Moreover, high acidity of AEW may cause the corrosion of equipments and consequently limit its practical application (Cao et al., 2009). In contrast, a solution at slightly acidic or neutral pH generated by electrolysis was reported as an effective antimicrobial agent with low
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available chlorine concentration (Koide et al., 2009; Cao et al., 2009; Gómez-López et al., 2007), and recently attention is being paid to LcEW for disinfection (Rahman et al., 2010a,b). So far, limited studies have been reported about freshly produced electrolyzed water at slightly acidic or neutral pH with low ACC (510 mg/L) (Rahman et al., 2010a,b). The present study was designed to determine the stability of LcEW over time and to investigate the effect of current, electrolysis time and salt concentration (%) on physical properties and bactericidal effectiveness of LcEW. 2. Materials and methods
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amount of salt solution (0.1%) and tap water through an electrolytic cell, within which the anode and cathode were separated by a membrane at a setting of 12 A to give a ACC of about 50 mg/L. 2.3. Determination of pH, ORP and ACC The pH, ORP and ACC of the treatment solutions (LcEW and SAEW) were measured immediately before treatment with a dual-scale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ) bearing pH and ORP electrodes. The ACC was determined by a colorimetric method using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan).
2.1. Bacteria and culture 2.4. Stability of LcEW E. coli O157:H7 (ATCC 43894) and L. monocytogenes (ATCC 19115) stock cultures were transferred into tryptic soy broth (TSB, Sparks, MD, USA) and incubated for 24 h at 35 °C. Following incubation, 10 mL of each culture was sedimented by centrifugation (3000g for 10 min), washed and resuspended in 10 mL of 0.85% sodium chloride solution to obtain a final cell concentration of 109 CFU/mL. The bacterial population in each culture was confirmed by plating 0.1 mL portions of appropriately diluted cultures on tryptic soy agar (TSA) (Difco Laboratories) plates and incubation at 35 °C for 24 h.
Changes in properties and bactericidal activity of LcEW stored in open or closed glass bottles at room temperature for 4 weeks in a dark room were evaluated. ORP, pH and ACC were measured on days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28. Bactericidal activities of LcEW against E. coli O157:H7 and L. monocytogenes were also measured until storage period of day 7. Electrolysis time and percent of salt concentration were also studied to investigate the changes of physical properties in produced LcEW. 2.5. Inactivation test
2.2. LcEW preparation Low concentration electrolyzed water (LcEW) was generated (Fig. 1) by passing a diluted salt (NaCl) solution (0.9%) through an electrolytic cell equipped with a platinum electrode, within which no separating membrane between anode and cathode using an electrolysis device (model OH-3000) at a setting of 3 V and 1.45 A. The LcEW used in this study had a pH of 6.8–7.4, an oxidation reduction potential (ORP) of 660–700 mV and an available chlorine concentration (ACC) of 510 mg/L. For comparison with LcEW, strong acid electrolyzed water (SAEW) with a pH of 2.54 and an ORP of 1100–1120 mV was generated using an EO generator (A2-1000, Korean E&S Fist Inc., Seoul, Korea) with a small
An in vitro inactivation of target pathogens in suspension was designed as illustrated in Fig. 2. For each replicate, a 1 mL aliquot of prepared inoculum (approximately 109 CFU/mL) was added to a sterile test tube. Once the test material (LcEW/SAEW) was generated, 9 mL of the prepared test agent was added to the tube containing 1 mL of the prepared inoculums, a timer was started and the tube was mixed immediately at room temperature. After each contact time (30, 60, and 90 s), a 1 mL sample was transferred to a tube containing 9 mL of neutralizer (0.85% NaCl + 0.5% sodium thiosulphate) followed by Serial 10-fold dilution in 0.85% saline solution dilution blanks. Distilled water (DW) washing was used as control. All tests were carried out in triplicate.
Fig. 1. A low concentration electrolyzed water (LcEW) generating system. An elaboration of a systematic mixing up of tap water and diluted NaCl during its production.
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Fig. 2. Flow diagram showing in vitro inactivation experiment. Sodium thiosulphate solution (0.5%) was used to stop the inactivation reaction after EW treatment.
2.6. Incubation and enumeration Upon completion of the test, the surviving population of bacteria was determined by plating 0.1 mL of each dilution in duplicate on TSA plates. Colonies of the pathogen were enumerated on TSA plates after incubation at 35 °C for 18–24 h. The colonies were counted and the CFU/mL at each contact time was determined. All tests were carried out in triplicate. 2.7. Statistical analysis For each occasion, the data from the independent replicate trials were pooled and the mean value and standard deviation were determined. Data were subjected to one way analysis of variance (ANOVA) and Tukey HSD multiple range test was used to determine the differences at p 6 0.05 using SPSS 13.0 (SPSS software for Windows, release 13.0, SPSS, Inc., USA). 3. Results and discussion 3.1. Properties of LcEW under different conditions The ACC, pH, and ORP of LcEW used in this study were 5–10 mg/L, 6.8–7.4, and 660–700 mV, respectively (Table 1). From our previous LcEW producing system (1.15–1.17 A) it is revealed that ACC reduces with an increase in exposure time (Rahman et al., 2010a) dur-
ing inactivation test which could have resulted in lower reductions at increased exposure times. In order to make the system more stable with enhanced antimicrobial activity the effect of current was examined. After increasing the current up to 1.45–1.47 A, we observed that the produced LcEW with increased current was more stable during storage and changes of ACC with an increase in exposure time were minimized (data not shown) compared to the previous system. Table 1 shows us how the current affects properties and inactivation efficacy of LcEW against E. coli O157:H7 and L. monocytogenes. From the results of current study it was found that increased reduction (4.9–5.6 log CFU/mL) for both pathogens was gained with the increase in current. Changes of current also affected the pH and ORP values of the tested solution. The values of pH and ORP increased with the increase in the current. Log reduction of 4.9 and 5.2 log CFU/mL for E. coli O157:H7 and L. monocytogenes, respectively was found when the current of 1.15 A was used. On the other hand, the new system with 1.45 A reduced E. coli O157:H7 and L. monocytogenes by 5.30 and 5.58 log CFU/mL, respectively. Compared to the previous system, new system showed enhanced bactericidal effect. ACC increased with the increase in the current. Park et al. (2001) suggested that the concentration of chlorine reactants in AEW is influenced by the amperage of the water generator, but other reports contend that the amount of HOCl produced during electrolysis is positively correlated with the amount of NaCl added (Anonymous, 1997; Al-Haq et al., 2002). Few reports are available on the effects of chlorine, pH, and ORP values of EO water on the
Table 1 Effect of current on physical properties and inactivation efficacy of low concentration electrolyzed water (LcEW) against E. coli O157:H7 and L. monocytogenes in 1 min dipping. Voltage (V)
Current (A)
ACC (mg/L)
pH
ORP (mV)
3
1.15–1.17 1.45–1.47
5 ± 0.2 10 ± 0.5
6.2 ± 0.06 6.8 ± 0.04
520 ± 10 700 ± 20
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Initial count for Listeria monocytogenes: 9.18 log CFU/mL. (1) Log reductions (log CFU/mL) reported as means of triplicate determinations ± standard deviation.
Log reduction (CFU/mL)(1) E. coli O157:H7
L. monocytogenes
4.90 ± 0.06 5.30 ± 0.12
5.20 ± 0.04 5.58 ± 0.10
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S.M.E. Rahman et al. / Journal of Food Engineering 113 (2012) 548–553 Table 2 Effect of electrolysis time on physical properties and inactivation efficacy of LcEW against E. coli O157:H7. Current (A)
Electrolysis time (s)
ACC (mg/L)
pH
ORP (mV)
Reduction of E. coli (log CFU/mL)
1.45–1.47
27 41 50
5 ± 0.5 7 ± 0.5 10 ± 0.5
7.34 ± 0.02 7.12 ± 0.04 6.80 ± 0.05
660 ± 5 680 ± 8 700 ± 10
5.3 ± 0.21 5.5 ± 0.18 5.9 ± 0.08
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Values are means of triplicate determinations ± standard deviation.
inactivation of pathogens. Kim et al. (2000) suggested that the ORP of EO water might be the primary factor responsible for its bactericidal effect. However, Koseki et al. (2004) noted that ORP could not be the main factor in antimicrobial activity, because the higher ORP of ozonated water did not show a higher disinfectant effect than that of EO water with lower ORP. They further explained that the free chlorine in EO water (mainly HOCl) produces hydroxyl radicals ( OH), which then act on microorganisms. Len et al. (2000) reported that the relative concentrations of aqueous molecular chlorine, HOCl, hypochlorite ions (OCl ), and chlorine gas (Cl2) were also factors that accounted for the bactericidal potency of EO water. Theoretically, at pH values of 6.0 and 9.0, the predominant chlorine species in a solution is not dissolved Cl2 gas but HOCl and OCl (White, 1999). The pH value of AEW also plays a role in restricting microbial growth. Iwasawa et al. (2002) discussed the effect of pH on the bactericidal properties of AEW; Len et al. (2000) discussed the influence of amperage and pH on these properties. Electrolysis times also affect the physical properties and influence the inactivation efficacy of LcEW (Table 2). Total residual chlorine increased with increases of salt concentration (Table 3) whereas, pH decreased and ORP increased. Similar result has been reported by Hsu (2005). Kiura et al. (2002) also found that the concentrations of free chlorine increased with the time of electrolysis and concentration of NaCl.
Table 4 Sanitization potency of LcEW against pure cultures of E. coli O157:H7 at different ACC and exposure times. Treatment solution
DW (control) LcEW – 5 mg/L LcEW – 7 mg/L LcEW – 10 mg/L SAEW – 50 mg/L
Table 3 Effect of salt concentration on physical properties of LcEW. Current (A)
Salt concentration (%)
ACC (mg/L)
pH
ORP
1.45–1.47
0.1 0.3 0.5 0.7 0.9
2 ± 0.5 3 ± 0.5 5 ± 1.0 7 ± 0.5 10 ± 0.5
7.50 ± 0.03 7.40 ± 0.05 7.30 ± 0.01 7.12 ± 0.04 6.84 ± 0.02
620 ± 15 640 ± 12 650 ± 10 670 ± 10 700 ± 10
Values are means of triplicate determinations ± standard deviation.
30 s
60 s
90 s
0.70 ± 0.02aA 5.02 ± 0.08aB 5.30 ± 0.04aBC 5.54 ± 0.12aC 5.18 ± 0.05aB
0.82 ± 0.04aA 5.32 ± 0.05abB 5.58 ± 0.10abBC 5.94 ± 0.07bC 5.44 ± 0.08abB
0.78 ± 0.05aA 5.88 ± 0.10bB 6.14 ± 0.12bBC 6.49 ± 0.06cC 6.02 ± 0.04bB
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Log reductions (log CFU/mL) reported as means of triplicate determinations ± standard deviation. Different lowercase letters within same row differed significantly (p < 0.05) and different uppercase letters within same column differed significantly (p < 0.05). (1)
Table 5 Sanitization potency of LcEW against pure cultures of L. monocytogenes at different ACC and exposure times. Treatment solution
Bacterial log reduction at different exposure time (log CFU/mL)(1) 30 s
DW (control) LcEW – 5 mg/L LcEW – 7 mg/L LcEW – 10 mg/L SAEW – 50 mg/L
3.2. Bactericidal activity of LcEW at different ACC and exposure times Based on the results of current study, 1.45 A was chosen for subsequent experiments to find out the effect of available chlorine concentration and exposure time on inactivation of E. coli O157:H7 and L. monocytogenes in cell suspensions (Tables 4 and 5). Table 4 indicates that both exposure time and ACC have significant effect (p < 0.05) on the reduction of E. coli O157:H7. Ten parts per million showed the highest reduction of 6.49 log CFU/mL at 90 s exposure, whereas, distilled water (DW) showed the lowest reduction of E. coli by 0.70 log CFU/mL. Regardless of exposure time, treatment solutions (5/7/10/50 ppm) showed significant reduction (p < 0.05) compared to distilled water control. Inactivation effect of strong acidic electrolyzed water containing 50 ppm ACC did not show significant difference (p < 0.05) with 5 and 7 ppm LcEW. But 50 ppm SAEW was significantly different with 10 ppm LcEW in reduction of E. coli cell. Similar trend was observed in case of L. monocytogenes inactivation (Table 5) and resulted in reduction of 6.79 log CFU/ mL at 90 s dipping with 10 ppm LcEW. Kim et al. (2000) reported that a 60-s treatment with AEW containing 10 mg/L of residual
Bacterial log reduction at different exposure time (log CFU/mL)(1)
60 s a
0.84 ± 0.05 A 5.32 ± 0.18aB 5.61 ± 0.07aBC 5.94 ± 0.11aC 5.41 ± 0.08aB
90 s a
1.02 ± 0.06 A 5.60 ± 0.08abB 5.90 ± 0.12abBC 6.28 ± 0.05abC 5.72 ± 0.09abB
1.08 ± 0.10aA 6.14 ± 0.08bB 6.44 ± 0.11bBC 6.79 ± 0.09bC 6.25 ± 0.07bB
Initial count for L. monocytogenes: 9.18 log CFU/mL. Log reductions (log CFU/mL) reported as means of triplicate determinations ± standard deviation. Different lowercase letters within same row differed significantly (p < 0.05) and different uppercase letters within same column differed significantly (p < 0.05). (1)
chlorine was very effective in reducing the populations of E. coli O157:H7, L. monocytogenes, and B. cereus vegetative cells to undetectable levels. Park et al. (2004) investigated the effects of chlorine and pH on the efficacy of EO water to inactivate E. coli O157:H7 and L. monocytogenes. They demonstrated that EO water effectively inactivated E. coli O157:H7 and L. monocytogenes in a wide pH range (between 2.6 and 7.0) if sufficient free chlorine (greater than 2 mg/L) was present. For a given chlorine content, bactericidal activity and ORP increased with decrease in pH. On the other hand, the 60 s treatment of E. coli pure culture using slightly acidic electrolyzed water (pH 5.6, 23 mg/L ACC and 940 mV ORP), strong acidic electrolyzed water (50 mg/L ACC) and sodium hypochlorite solution (NaOCl, 120 mg/L ACC) achieved a log reduction of 5.07, 6.02 and 5.13 log CFU/mL of its population (Issa-Zacharia et al., 2010). 3.3. Change of properties and sanitization potency of LcEW under storage conditions Changes of ACC, pH, and ORP of LcEW under open and closed storage conditions for 28 days at room temperature in the dark
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S.M.E. Rahman et al. / Journal of Food Engineering 113 (2012) 548–553 Table 6 Inactivation efficacy of LcEW against E. coli O157:H7 and L. monocytogenes reduction (log CFU/mL) during storage under open and closed condition. Storage period (d)
0 1 2 3 4 5 6 7 14 21 28
Log reductions (CFU/mL)(1) Open condition
Close condition
E. coli O157:H7
L. monocytogenes
E. coli O157:H7
L. monocytogenes
5.98 ± 0.07a 5.69 ± 0.14b 5.44 ± 0.21bc 5.20 ± 0.04c 4.95 ± 0.11cd 4.41 ± 0.07d 4.20 ± 0.18de – – – –
6.77 ± 0.12a 6.52 ± 0.19ab 6.18 ± 0.06b 5.78 ± 0.13c 5.32 ± 0.21d 4.89 ± 0.25e 4.54 ± 0.14f – – – –
5.98 ± 0.05a 5.95 ± 0.06a 5.84 ± 0.11ab 5.82 ± 0.15ab 5.68 ± 0.22b 5.68 ± 0.24b 5.52 ± 0.29bc 5.24 ± 0.14c 3.47 ± 0.06d – –
6.97 ± 0.10a 6.98 ± 0.12a 6.77 ± 0.04ab 6.77 ± 0.07ab 6.50 ± 0.15b 6.49 ± 0.17b 6.32 ± 0.23bc 5.96 ± 0.14c 3.92 ± 0.08d – –
Initial count for E. coli O157:H7: 9.30 log CFU/mL. Initial count for L. monocytogenes: 9.18 log CFU/mL. (1) Log reductions (CFU/mL) reported as means of triplicate determinations ± standard deviation. Different letters within same column differed significantly (p < 0.05). –, not measured.
open, agitated, and diffused light condition, the EO water had the highest chlorine loss rate. With open storage, chlorine loss through evaporation followed first-order kinetics. The rate of chlorine loss was increased by approximately 5-fold with agitation, but it was not significantly affected by diffused light (Len et al., 2002). EO water exposed to the atmosphere showed more reduction in chlorine and oxygen than that kept in closed systems for a longer time (Hsu and Kao, 2004). Fabrizio and Cutter (2003) reported that EO water stored at 4 °C was more stable than that stored at 25 °C. In conclusion, LcEW has the advantage of possessing antimicrobial activity with low available chlorine. Also, this study demonstrated that LcEW has a relatively stable shelf life in closed storage condition and showed strong bactericidal activity against E. coli O157:H7 and L. monocytogenes in cell suspensions with the increase in current. In addition, this study provides the foundation for further development of dynamic LcEW producing system for commercial use in the food industry. Fig. 3. Changes of LcEW properties during storage under open and closed condition.
References are shown in Fig. 3. Under open storage condition, the ACC of LcEW decreased gradually from 10 to 0 mg/L after 7 days. However, ACC was slightly reduced from 10 to 5 mg/L after 7 days under the closed storage condition. The pH of LcEW increased during the 28 days storage period in both storage conditions with the decrease in ORP values. The pH of LcEW increased from 6.8 to 7.7 in open storage and from 6.8 to 7.4 in closed storage after 28 days. In contrast, Quan et al. (2010) reported that the pH of weakly acidic electrolyzed water (pH 5.9; ACC 35 mg/L) decreased in open and closed storage. The ORP of LcEW decreased from 700 to 400 mV in open storage and from 700 to 500 mV in closed storage after 28 days. When LcEW characteristics were monitored over time, it was determined that LcEW stored at closed condition was more stable than that stored at open condition. Bactericidal activities of LcEW against E. coli O157:H7 and L. monocytogenes in open and closed storage conditions are shown in Table 6. LcEW maintained bactericidal activities against cell suspensions of the two strains up to 6 days in open and 14 days in closed storage condition. Storage conditions can affect the chemical and physical properties of EO water. When stored under an
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