Effect of Storage Conditions on Shelf Stability of Undiluted Neutral Electrolyzed Water

ABSTRACT

Neutral electrolyzed water (NEW) is an oxidizing sanitizer that can be made locally on-site; it is often stored in a ready-to-use format to accumulate the large volumes required for periodic or seasonal use. The shelf stability of NEW sanitizer was, therefore, assessed under various storage conditions to guide the development of protocols for its industrial application. To that end, fresh NEW with an available chlorine concentration (ACC) of 480 mg/L, pH 6.96, and oxidation reduction potential (ORP) of 916 mV was stored under different conditions. These were open or sealed polypropylene bottles, three different surface area–to–volume (SA:V) ratios (0.9, 1.7, and 8.7), and two temperatures (4 and 25°C). NEW stored at 4°C was significantly more stable than NEW stored at 25°C; ACC and pH decreased by 137 mg/L and 0.7, respectively, whereas ORP increased by 23 mV, after 101 days of storage. At 25°C, ACC decreased to <0.01 mg/L after 52 days in bottles with a SA:V ratio of 8.7, with a similar decrease after 101 days in bottles with a SA:V ratio of 1.7. However, pH decreased by up to 3.7 pH units, and ORP increased by up to 208 mV. The antimicrobial efficacy of “aged” electrolyzed oxidizing (EO) water with different ACC and ORP, but the same pH (i.e., 3.4 ± 0.2), was evaluated against Escherichia coli and Listeria innocua to determine any differences in residual antimicrobial activity. EO water with an ACC of ≥7 mg/L and an ORP of 1,094 mV caused a reduction of at least 4.7 log, whereas EO water with nondetectable ACC and considerably high ORP (716 mV) had little antimicrobial effect (<1-log reduction). Results from this study indicate that the efficacy of NEW as a sanitizer for large-scale applications such as horticulture can be maintained for at least 3 months when it is stored in closed containers with low SA:V ratio at low temperatures.

HIGHLIGHTS

• NEW sanitizer (480 mg/mL) was more stable when stored at 4 than at 25°C.
• Containers with a lower SA:V ratio improved the stability of NEW.
• Antimicrobial effects of NEW were significant at chlorine concentration >7 ppm.

Keywords

Antimicrobial

Chlorine

Escherichia coli

Listeria

Physicochemical properties

Electrolyzed water, referred to as electrolyzed oxidizing (EO) water, is a nonspecific broad-spectrum sanitizer (28). It is typically generated by the electrolysis of pure water with added hydrochloric acid (HCl) and/or NaCl in an electrolytic cell (3). Water molecules and chloride ions can then be electrolytically reacted to form different chlorine species, such as hypochlorous acid (HOCl), hypochlorite ions (ClO−), and chlorine (Cl2). The proportion of these species in EO water is mainly dependent upon the pH of the EO water generated (28). Accordingly, different types of EO water exist and can be produced by changing the composition of the feed stream, the reaction conditions, and the arrangements of the electrolytic cell. These include acidic, slightly acidic, neutral, and alkaline EO water. Among all types, neutral EO water (NEW) has gained increasing attention. The neutral pH minimizes corrosion potential and is physiologically compatible. Whereas the pH of NEW minimizes safety issues from Cl2 off-gassing, it maximizes the availability of the hypochlorous acid species and, therefore, maximizes antimicrobial efficacy (22).

EO water has notable biocidal activity, which is mainly due to its physicochemical properties, such as available chlorine concentration (ACC), pH, and oxidation reduction potential (ORP) (2,22). There are numerous studies demonstrating that EO water could be used as a surface disinfectant of different materials commonly found in food processing facilities, including cutting boards (17), stainless steel, vitreous china, ceramic tiles, and glass (21). Other studies have also reported EO water potential as an effective sanitizer for a range of fresh fruits and vegetables (12., 13., 14.), barley grains (25), and a variety of meat and produce (6,9,21). Reviews by Huang et al. (10) and Rahman et al. (22) indicated that the antimicrobial efficacy of EO water can range from no effects to reductions of at least 6 log in viable count. This variation has been suggested to be related to both intrinsic and extrinsic factors, including the type of EO water used, temperature, ACC, food composition and surface characteristics, water hardness, organic load, and bacterial type (10). Therefore, understanding these factors, their interactions, and how they affect the antimicrobial effects of NEW would aid its development as an effective and reliable antimicrobial application in the food industry.

In addition to the need to better characterize the factors affecting EO efficacy, improved studies to determine the best storage conditions for EO water are important. In horticulture, produce that is eaten raw requires sanitation after harvest and possibly in the field preharvest to moderate the presence of pathogens in harvested produce and/or control phytopathogens that can reduce crop yield. These large-scale applications require large volumes of sanitizer, which may be mixed or made on-site or may be made in a ready-to-use format and transported to the point of application. EO water is typically produced in an electrolytic cell and accumulated in a holding vessel until use. EO water, once produced, needs to be stored under the conditions that best preserve its stability as a sanitizer.

Previous studies have identified a range of storage factors, including material type, light conditions, temperature, agitation level, and open or closed container to influence the shelf stability of EO water (3,11,19,23,24,32,33). However, these studies examined EO water with a low ACC (i.e., ≤100 mg/L). Such low levels of ACC do not reflect the large-scale application of NEW as a sanitizer in commercial settings, in which ACC >400 mg/L is commonly produced (7,15,18). Arguably, freshly made NEW with a higher initial ACC may result in longer shelf stability, but this remains unclear.

The aim of this study was, therefore, to assess the changes in physicochemical properties (i.e., ACC, pH, and ORP), of NEW at a high level of ACC (i.e., that is relevant to commercial use, initial ACC 480 mg/L, pH 6.96, ORP 916 mV) stored under various conditions. Another aspect of this study was to specifically characterize the antimicrobial efficacy of “aged” EO water (with different ACC and ORP values at the same pH) to provide an insight into its potential mode of action and an assurance that degradation mechanisms or products do not alter the relative efficacy of the antimicrobial activity.

MATERIALS AND METHODS

Preparation of electrolyzed water solution

NEW was generated by the electrolysis of a saturated sodium chloride solution using an Envirolyte ELA-400 (Envirolyte Industries International OÜ, Tallinn, Estonia) (4,22). ACC (i.e., free chlorine) was analyzed colorimetrically, using a Compact CLO2+ meter (Palintest, Peakhurst, New South Wales, Australia) with a detection limit of 0.01 mg/L, which determines ACC by a colorimetric reaction of free chlorine with di-ethyl-p-phenyl diamine. pH and ORP were analyzed with an Orion 250A pH meter (Orion, Beverly, MA) and an MW 500 ORP meter (Milwaukee Instruments, Rocky Mount, NC), respectively. The anolyte produced had an initial ACC, pH, and ORP of 480 mg/L, 6.96, and 916 mV, respectively.

Effect of storage conditions on stability of NEW

The changes in physicochemical properties were measured in NEW stored under two different conditions. These included (i) small-volume bottles (50, 250, and 500 mL) with container surface area–to–volume ratios (SA:V ratios) of 8.7, 1.7, and 0.9, stored at 4 and 25°C; and (ii) open or closed bottles (500 mL) stored at 25°C (with SA:V of 0.9). The surface area–to–area volume ratio was determined by the surface area of a cylindrical bottle (436.4 cm2) divided by the volume of each respective solution (Supplemental Fig. S1). All experiments were undertaken in the dark. This is because light is well known to decompose HClO and hypochlorite anions, which absorb energy in the region of 292 to 380 nm (1,19,20,31).

For experiment 1, NEW was produced and dispensed into 25-L polyethylene containers and stored sealed at 4°C for 1 day. NEW was transferred into wide-mouth polypropylene screw-cap bottles (18 by 500 mL; LP1403PP-500, Wiltronics, Alfredton, Victoria, Australia), which were tightly capped and stored at 4 or 25°C in the dark (three replicates per treatment of 50, 250, and 500 mL with SA:V ratios of 8.7, 1.7, and 0.9 respectively, stored at either 4 or 25°C). For each treatment, the ACC, pH, and ORP of the NEW was measured periodically from the same bottles over a storage period up to 101 days.

For experiment 2, NEW was produced and dispensed into 25-L polyethylene containers and transferred into polypropylene screw-cap bottles (42 by 500 mL) (three replicates per treatment per time point). In the first treatment, “closed” bottles were filled with NEW such that there was no headspace and were tightly capped. For the second treatment, “open” bottles were filled, and the mouths of the bottles were covered loosely with aluminum foil. All bottles were stored in the dark at 25°C. For each treatment, ACC, pH, and ORP were measured periodically from different bottles over a storage period up to 62 days.

Evaluating antimicrobial efficacy of aged EO water

E. coli K-12 MG1655 and L. innocua ATCC 33090 were used to assess the antimicrobial efficacy of the EO water after storage. Cultures were obtained from the culture collection of the Tasmanian Institute of Agriculture, University of Tasmania, Australia.

Preparation of bacterial inocula

Bacterial cultures, previously maintained at −80°C, were resuscitated by streaking onto brain heart infusion agar (AM11, Amyl Media, Dandenong, Victoria, Australia) and were incubated at 37°C for 24 h. A well-isolated single colony from each culture was aseptically transferred into 10 mL of brain heart infusion broth and incubated at 37°C for 24 h to achieve a cell density of approximately 109 CFU/mL. The cultures were stored at 4°C and used as “stock” cultures within a week.

To prepare the inocula, stock cultures of E. coli and L. innocua were diluted 1:103 in 10 mL of brain heart infusion broth to prepare working cultures. These cultures were incubated at 37°C for 20 h to achieve a cell density of approximately 109 CFU/mL. A 1-mL aliquot of each of these working cultures was then harvested by centrifugation at 5,000 × g for 5 min (Eppendorf 5417R centrifuge, Eppendorf, Hamburg, Germany) at 20°C. The pellet was resuspended and washed twice with 1 mL of 0.1% peptone water (LP0037, Oxoid Ltd., Basingstoke, Hampshire, England) with subsequent centrifugation at 5,000 × g for 5 min at 20°C. The final washed pellet was resuspended in 1 mL of 0.1% peptone water and used as the inoculum (approximately 109 CFU/mL) for trials to assess antimicrobial efficacy.

Antimicrobial evaluation

EO water with different SA:V ratios that had been stored at 25°C for 87 and 101 days in experiment 1 was used to specifically study the antimicrobial effects of “aged” EO water. After measurement of ACC, ORP, and pH, an aliquot (0.9 mL) of the aged EO water was transferred into a sterile microcentrifuge tube and combined with 0.1 mL of either an E. coli or L. innocua suspension, outlined above. Bacterial numbers, before and after exposure to treatments for 1, 5, and 10 min at approximately 20°C, were determined by spread plating 0.1-mL aliquots of appropriately diluted samples on brain heart infusion agar. After incubation at 37°C for 18 h, bacterial colonies were enumerated.

Statistical analysis

All experiments were set up in a completely randomized design, with three replications per treatment. The effects of temperature, open or closed storage, volume, time, and their interactions were analyzed by two-way analysis of variance (ANOVA) within a repeated measures framework using a Kenward-Roger degrees of freedom adjustment. For the antimicrobial study, treatment effects were analyzed by one-way ANOVA for each exposure time. The assumptions of ANOVA, such as homogeneity of variance and the Gaussian distribution, were confirmed by the use of quantile–quantile plots and residual plots for all variables. Significant differences were established at a level of P < 0.05. All statistical analyses were done using SAS version 9.3 (SAS Institute, Cary, NC).

RESULTS

Effect of SA:V ratio and temperature on the stability of NEW

pH and ORP, but not ACC, were significantly influenced by the interaction among volume, temperature, and time (Table S1). When NEW was stored at 4°C for 101 days, the ACC decreased for all three SA:V ratios (Fig. 1A). The observed decrease also occurred faster with an increase in SA:V. The three treatments (SA:V ratios of 8.7, 1.7, and 0.9) had a final ACC of 343, 400, and 420 mg/L, respectively (Fig. 1A). The initial pH of 7.0 remained relatively stable, decreasing to 6.5, 6.4, and 6.3 for treatments with SA:V ratios of 8.7, 1.7, and 0.9, respectively (Fig. 1B). The ORP also remained relatively stable throughout the duration of the experiment. From an initial ORP of 916 mV, final ORP levels were 955, 961, and 939 mV for SA:V ratios of 8.7, 1.7, and 0.9, respectively (Fig. 1C).

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FIGURE 1. Effect of temperature, 4°C (black) and 25°C (gray), on (A) concentration of available chlorine (ACC), (B) pH, and (C) ORP of NEW with SA:V ratios of 8.7 (square), 1.7 (circle), and 0.9 (triangle) stored for up to 101 days. Error bars equal to 1 SD.

When NEW was stored at 25°C, the ACC decreased from 480 to <0.01 mg/L for bottles with a SA:V ratio of 8.7 and 1.7 by day 53 and 101, respectively (Fig. 1A). This was in contrast to bottles with a SA:V ratio of 0.9, in which the ACC decreased from 480 to 59 mg/L over 101 days (Fig. 1A), reinforcing the influence of the SA:V ratio on retention of ACC. The pH of all NEW treatments declined over the first 65 days of storage and then remained relatively stable. Bottles with SA:V ratios of 8.7, 1.7, and 0.9 had final pH values of 3.6, 3.2, and 3.3, respectively (Fig. 1B). The ORP of all treatments initially increased from 916 to 1,130 mV within the first 52 days (Fig. 1C). This was followed by a period in which the ORP of all bottles, except for the bottles with a SA:V ratio of 8.7, remained constant above 1,100 mV. On days 31 and 80, the ORP of NEW in bottles with a SA:V ratio of 8.7 and 1.7 decreased from above 1,000 mV to 493 and 716 mV, respectively.

Effect of open or closed containers on the stability of NEW at 25°C

In these experiments, ACC, pH, and ORP were significantly influenced by the interaction between treatment and time (Table S2). The ACC declined more quickly in open bottles compared with closed bottles (Fig. 2A). The initial ACC of 480 mg/L for both open and closed treatments decreased to 170 and 253 mg/L over the course of 62 days of storage, respectively. The pH of NEW with both open and closed treatments decreased from 7.0 to 4.8 and 3.6, respectively (Fig. 2B). As for ORP, it increased for both treatments within the first 50 days of storage (from 916 mV to approximately 1,100 mV; Fig. 2C), followed by a period in which ORP appeared to be unchanged.

FIGURE 2

FIGURE 2. Effect of “open” (black) or “closed” (gray) containers on the rate of change of the (A) concentration of available chlorine, (B) pH, and (C) ORP of NEW stored at 25°C over time. Error bars equal to 1 SD.

Antimicrobial efficacy of aged EO water

Aged EO water with different ACC and ORP, but at the same pH (pH 3.4 ± 0.2), was found to significantly influence the mean log reduction of E. coli and L. innocua for each exposure time (Table 1; P < 0.05). The initial inoculum level of E. coli and L. innocua was 9.09 and 9.21 log CFU/mL, respectively. EO water with an ACC of 63 mg/L and an ORP of 1,121 mV reduced E. coli and L. innocua to below the detection limit following 1 min of exposure. EO water with an ACC of 7 mg/L and an ORP of 1,094 mV, however, reduced E. coli only by 4.76 ± 0.09 log CFU/mL within 1 min. Thereafter, no further effects were observed with longer exposure times. For EO water with nondetectable ACC (<0.01 mg/L) and ORP of 716 mV, E. coli and L. innocua were reduced by 0.98 ± 0.08 and 1.02 ± 0.02 log CFU/mL, respectively, after 1 min, with no further effects observed after 10 min. This was similar to our observation for EO water with much lower ORP (364 mV) but with the same ACC (i.e., <0.01 mg/L) and comparable pH (i.e., 3.66). It was found that E. coli and L. innocua populations were reduced by 0.97 ± 0.07 and 1.05 ± 0.03 log CFU/mL, after 1 min, respectively.

TABLE 1. Antimicrobial effects of stored EO water on Escherichia coli and Listeria innocuaa

a Different letters (a, b, c) denote a significant treatment (α = 0.05) effect within each exposure time. n = 3 for each EO water treatment within each exposure time. ACC, available chlorine concentration; ORP, oxidation reduction potential.

b Mean Log reduction = initial counts − final count at each sampling time point (data not shown).

c Below the detection limit of the Compact ClO2+ meter (0.01 mg/L).

d Below the detection limit of the enumeration method (<1 log CFU/mL).

DISCUSSION

Our study showed that the physicochemical properties of NEW with an ACC of 480 mg/mL were more stable at lower temperature (4 versus 25°C) in closed (cf. open) containers, and with lower SA:V ratios (0.9 > 1.7 > 8.7) over a storage period of 101 days (Fig. 1). The observed increase in stability of NEW at 4°C is consistent with previous studies. For instance, Meireles et al. (17) reported that the ACC of NEW (with an initial ACC of 100 ppm) was more stable at 5°C than at 25 and 30°C after 200 days. A similar observation was also made by Xin et al. (32) when NEW (with an initial ACC of 98 ppm, ORP of 875 mV, and pH 6.5) was stored at 4°C, compared with 20 and 35°C, over a period of 3 weeks.

The observed effects of temperature on NEW also agree well with those on acidic EO water. Previous studies showed that acidic EO water (pH < 3) stored in glass bottles for up to 398 days was more stable at 4°C than at ≥20°C (5,24). However, comparison of the observed shelf stability of NEW with that previously observed for acidic EO water revealed that NEW might have a longer shelf stability than acidic EO water (16,24,27), particularly at low storage temperatures and in sealed containers with low SA:V ratios. Of particular note was a long stability study (i.e., 398 days) that showed acidic EO water to be unstable at both 4 and 25°C, with ACC decreasing to <1 mg/L within the first 65 days (24). The longer shelf stability of NEW versus acidic EO water also agrees well with the idea that the loss of ACC is due to volatilization of the chlorine gas (Cl2) into the headspace. This reaction would most likely occur faster at higher temperatures and lower pH values and would potentially increase degradation of HClO under near-neutral pH conditions at the higher temperatures via a chlorine dioxide intermediate with release of a proton (1). This might explain the observed changes in pH and ORP (Fig. 1B and 1C).

The effects of the SA:V ratio on the ACC of NEW were more noticeable at 25 than at 4°C (Fig. 1A). The ACC decreased faster in bottles with an increase in the SA:V ratio (0.9 < 1.7 < 8.7). To our knowledge, the effect of container shape on shelf stability has not been examined previously, but our results suggest that the size of the headspace of containers may potentially affect shelf stability of NEW, rather than the SA:V ratio per se. In practical terms, shelf stability of NEW could be improved by storing it in containers that are filled to maximum capacity to minimize headspace. Similarly, the physicochemical properties of NEW were more stable in closed bottles than in open bottles (Fig. 2). This is consistent with a previous study in which little change was seen in the ACC and ORP of NEW, acidic, or slightly acidic EO water in closed bottles and not in open bottles when stored at 20°C (3,8,33). Taken together, the higher stability of EO water observed in bottles with lower SA:V ratio and in closed bottles might be because the evaporation rate of the dissolved Cl2 increases with an increase in the SA:V ratio (1), thereby driving the decomposition of HClO in the EO water. Furthermore, the decomposition of HClO leads to hydrochloric acid and oxygen, resulting in a decrease in the pH (29,31).

In addition, at different ACC and ORP levels, aged EO water at pH 3.4 ± 0.2 exhibited different antimicrobial effects on E. coli and L. innocua (Table 1). EO at an ACC of 63 mg/L and ORP of 1,121 mV caused a >9-log CFU/mL reduction of both test organisms within 1 min at ∼20°C. In contrast, EO water with low ACC (7 ppm) but similar ORP (1,094 mV) was able to reduce E. coli by approximately half (∼4.8-log reduction). EO water with a much lower ACC and ORP (ACC of <0.01 mg/L and ORP of either 364 or 716 mV) produced the least antimicrobial effects, reducing both bacterial species by only ∼1 log CFU/mL within 1 min. These results, taken together, indicate that ACC plays a major role in the antimicrobial effects of EO water, whereas pH and ORP contribute much less. Similarly, Waters and Hung (30) showed that EO water with pH of 2.8 and ACC of 0 mg/L had little effect on E. coli (<0.3-log reduction), whereas treatment with EO water with pH of 7.0 but higher ACC (34 mg/L) caused a 4.8-log reduction of E. coli. Note also that the lack of efficacy of EO water with nondetectable ACC, but considerably high ORP (716 mV), was inconsistent with previously published data, in which ORP values ≥650 mV were effective in eliminating bacteria within a few seconds, although ACC was not reported (26,34).

We determined the effects of various storage conditions on the stability of NEW in the context of commercial use (i.e., with high ACC) as a sanitizer. Our data revealed that the stability of the physicochemical properties of NEW (i.e., ACC, pH, and ORP) was higher at low temperature (4 versus 25°C) in a closed container and with a low SA:V ratio (0.9 > 1.7 > 8.7). Results from this study indicate that the antimicrobial effects of aged EO water depend primarily upon ACC rather than ORP and pH. The results of this study are useful for the food industry: NEW should be accumulated and stored under the conditions that best preserve its stability as a sanitizer (i.e., maintaining ACC). However, further experiments to measure the stability of NEW when it is produced and stored on an industrial scale (e.g., 1,000- to 10,000-L closed polyethylene holding tanks of NEW, at 500 mg/L ACC) for use as a sanitizer should be considered. A predictive model for NEW stability should also be developed to provide a tool for industry to better manage EO water.

ACKNOWLEDGMENTS

This work was supported by Meat and Livestock Australia (MLA, project no. G.MFS.0289), the Australian Research Council's Industrial Transformation Training Centres scheme under grant IC140100024, and the Australian Postgraduate Award scheme. We thank three anonymous reviewers for valuable comments on an earlier draft of the manuscript.

SUPPLEMENTAL MATERIAL

Supplemental material associated with this article can be found online at: https://doi.org/10.4315/JFP-20-104.s1

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